Natural Killer express β-AR, which are usually inhibitory. Monocytes express β-AR and their activation is usually antiinflammatory. On murine Dentritic cells (DC), β-AR mediate sympathetic influence on DC-T cells interactions. In human DC β2-AR may affect Th1/2 differentiation of CD4+ T cells. In microglia and in astrocytes, β2-AR dysregulation may contribute to neuroinflammation in autoimmune and neurodegenerative disease. In conclusion, extensive evidence supports a critical role for adrenergic mechanisms in the regulation of innate immunity, in peripheral tissues as well as in the CNS. Sympathoadrenergic pathways in the innate immune system may represent novel antiinflammatory and immunomodulating targets with significant therapeutic potential.The sympathetic nervous system has a major role in the brain-immune cross-talk, but few information exist on the sympathoadrenergic regulation of innate immune system. The aim of this review is to summarize available knowledge regarding the sympathetic modulation of the innate immune response, providing a rational background for the possible repurposing of adrenergic drugs as immunomodulating agents. The cells of immune system express adrenoceptors (AR), which represent the target for noradrenaline and adrenaline. In human neutrophils, adrenaline and noradrenaline inhibit migration, CD11b/CD18 expression, and oxidative metabolism, possibly through β-AR, although the role of α The first step in the synthesis of Noradrenaline is the transformation of the aminoacid tyrosine in Levodopa through the enzyme tyrosine hydroxilase that is the key rate-limiting enzyme in the biosynthetic pathway of Noradrenaline.Levodopa is decaborxlated into dopamine and finally noradrenaline is synthesized from dopamine by dopamine β-hydroxylase and is converted to adrenaline by phenylethanolamine N-methyltransferase , and their axons project to hippocampus, septum, hypothalamus and thalamus, cortex and amygdala, to cerebellum, as well as to spinal cord. Brain adrenergic pathways control attention, arousal and vigilance, and regulate hunger and feeding behavior. Some central nervous system (CNS) neurons, mainly located in the medullary reticular formation, utilize adrenaline as the main neurotransmitter, possibly contributing to the modulation of eating behavior and to blood pressure regulation. In the periphery, noradrenaline is the main transmitter of sympathetic postganglionic fibers. Main direct effects of noradrenaline and adrenaline on peripheral tissues include: smooth muscle contraction in blood vessels supplying skin, kidney, and mucous membranes, stimulation of exocrine glands, smooth muscle relaxation in the gut wall, bronchi, and blood vessels supplying skeletal muscle, increases of heart rate and force of contraction, increased glycogenolysis in liver and muscle, lipolysis in adipose tissue, thermogenesis in the brown adipose tissue, modulation of the secretion of insulin and rennin  and noradrenaline (~20%). In the brain, noradrenergic neurons are located mainly in the 1, α2, and β—each further divided into three subtypes, which are widely expressed throughout the CNS and in virtually all peripheral tissues  and classified in three major types—α1- and α2-AR and adrenaline > noradrenaline for the β-AR.The order of potency in the activation of these receptors by physiological ligands is noradrenaline > adrenaline for the α1-AR by adrenergic agonists induces the stimulation of a Gq and consequent phospholipase C (PLC) activation that promotes hydrolysis of phosphatidylinositol bisphosphate producing inositol trisphosphate and diacylglycerol. The results of this activation is the release of Ca++ as second messenger from non-mitochondrial pools or protein kinase C (PKC) and mediating intracellular release  and secondary (spleen and lymph nodes) lymphoid organs, where noradrenaline and adrenaline are released from nerve varicosities and or diffuse from the bloodstream to act on AR expressed on immune cells  containing 15–45 aminoacid residues, which are both potent antibiotics usually targeting bacterial membranes as well as effective modulators of the immune response , monocytes/macrophages, dendritic cells (DC), natural killer (NK) cells, γδ T lymphocytes, as well as the recently described innate lymphoid cells (ILC). Immune responses in the CNS are mediated by resident microglia and astrocytes, which are innate immune cells without direct counterparts in the periphery.The innate immune system consists of effector molecules such as complement and antibacterial peptides as well as of effector cells. e Boman, . Innate pattern recognition receptors (PRR), which can be activated by both exogenous pathogen-associated molecular patterns (PAMP) and endogenous danger (or damage)-associated molecular patterns (DAMP). PRR includes families of toll-like receptors , NOD-like receptors , C-type lectin receptors , RIG-I-like receptors , and AIM2-like receptors . Formyl peptide receptors  and scavenger receptors (binding oxidized or acetylated low-density lipoprotein) could be also included among PRR , possibly through the activation of α1-AR -13 attenuates such effects, decreasing SPLUNC1 and β-AR expression  (but not β-defensin-2/hBD-2) in normal and asthma airway epithelial cells, resulting in reduced Antibacterial peptides may also affect the response of tissues to adrenergic stimulation. Recently, it has been reported that α-defensin-1/HNP1 has relaxing effects on adrenergic contractions of rat detrusor muscles, possibly through NF-κB pathways   are named after the presence of intracellular granules. Granulocytes are currently divided into different types, based on their staining characteristics: neutrophils , eosinophils, basophils, and mast cells.  Mócsai, . Eosinops) Kita, . BasophiAdrenergic modulation of granulocytes has been examined mainly in neutrophil, nonetheless available evidence suggests that adrenergic agents may profoundly affect all granulocyte subtypes.2-AR  and β-AR, in the following order: β3 > β2 > α1A > α1B ~ α2A ~ β1 = α1D = α2C and that exposure of cells to IL-8, a potent proinflammatory CXC-chemokine that promotes neutrophil chemotaxis and degranulation, increases mRNA levels of all AR, and that adrenaline, probably through the involvement of β-AR, profoundly affects neutrophil function  lung inflammation in rodent models of acute lung injury. Adrenalectomized animals show even enhanced catecholamine release from phagocytes as well as enhanced expression of catecholamine-synthesizing enzymes in these cells. Such results have been explained suggesting that in rodent phagocytes noradrenaline and adrenaline possibly activate NFκB resulting in enhanced release of tumor necrosis factor (TNF)-α, IL-1β and IL-6, and in the subsequent amplification of acute inflammation via α2-AR  have been identified human neutrophils . This is a relevant issue, since many effects exerted by β2-AR agonists may actually occur through β-AR-independent mechanisms  release in purified human peripheral blood eosinophils . Microglial cells are resident macrophages in the CNS. The specific origins and functions of all these subsets however still await thorough investigation  secretion from the murine macrophage-like cell line RAW264.7   than in PMA-differentiated U937 (macrophages)  and B (fibroblast-like) synoviocytes produced an increase and a decrease in the respective cell number, probably through Gi-coupled PLC activation and the resulting stimulation of the PKC betaII/MAP kinase -mediated proinflammatory responses  TNF-α production. In the same study, the presence of intracellular noradrenaline was also reported and interpreted as an evidence supporting the existence of an adrenergic autocrine loop, possibly even more pronounced in macrophages obtained from rats with streptococcal-cell-wall-induced arthritis  that induces a profound suppression of the production of proinflammatory cytokines  1 priming is impaired. Such observations may explain how reduced noradrenaline activity in the skin may promote contact sensitizers-induced Th1 responses  protein. Inhibition of cross-presentation was neither due to reduced costimulatory molecule expression nor antigen uptake, but rather to impaired phagosomal antigen degradation. A crosstalk between the TLR4 and β2-AR transduction pathways at the NF-κB level was also described, and in vivo treatment with a β2-AR agonist resulted in inhibition of antigen protein cross-presentation to CD8+ T cells, however with preservation of their exogenous major histocompatibility complex (MHC) class I peptide presentation capability  and CD86, and suppressing Th-cell proliferation. Dexmedetomidine also suppressed DC migration, and vaccination of animals with dexmedetomidine-treated DC significantly suppressed the contact hypersensitivity reaction in vivo  that regulate their activation. NK cells also express activating receptors, and their complex interplay with inhibitory receptors is a matter of intense investigation. NK cells circulate through the blood, lymphatics and tissues, patrolling the body for the presence of transformed or pathogen-infected cells   were identified, and infusion of adrenaline  significantly decreased all AR on NK cells  T lymphocytes are unconventional T cells that, like NK cells, functionally and phenotypically belong to both the innate and the adaptive immune system and represent a connection between the two. They represent about 1–10% of circulating T cells , and are involved in the defense against infectious diseases as well as in the inhibition of tumor development and progression -AR, although α-AR may occur on selected cell types and under specific conditions. Further studies however are needed to define the functional significance of α-AR-mediated influence on the innate immune response.Consistent evidence however indicates that adrenergic mechanisms play a significant role even in immune cells. In particular, in human neutrophils migration, CD11b/CD18 expression, and oxidative metabolism are inhibited possibly through β-AR, although a contribution by α2-AR are currently used as bronchodilating agents in asthma, however the relative contribution of any eventual immunomodulating activity of these drugs to their overall therapeutic effects remains to be established. In addition, their usefulness in different inflammatory conditions such as atherosclerosis, where neutrophils are emerging key players . In particular, no information is yet available on innate immune cells which have been recently discovered and characterized, such as ILC  forms compared to wild-type (arg16 and gln27) forms (Chong et al., Circumstantial evidence also suggests the opportunity to apply a pharmacogenetic approach to better understand adrenergic modulation of the immune response and in particular of innate immunity. For instance, individuals who were homozygous for β2-AR antagonists in acute lung injury (Flierl et al., Nonetheless, the present knowledge about the adrenergic modulation of innate immunity already supports relevant therapeutic applications, such as the use of αThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline  Despih medium . In HeLanduction . Hydrogeod cells . Endoplaod cells . ROS andod cells . To dateEPR-spin trapping is an analytical technique employed in the detection and identification of short-lived free radicals. Spin trapping involves the addition of radicals to nitrone spin traps to form a spin adduct, a nitroxide-based radical of relatively longer half-life to allow its detection using an EPR spectrometer. Depending on the radical, each spin adduct usually yields a distinctive EPR spectrum characteristic to that radical. The spin adduct is usually identified based on the basis of the measurement of hyperfine-coupling constants of relevant nuclei. The relative amount of spin adducts can also be obtained by comparing their spectra with that of a stable nitroxide. In this study, EPR-spin trapping using a number nitrone spin traps was utilized to identify and quantify free radicals in aqueous solutions exposed to Ar-CAP. The obtained EPR spectra were compared with the spectra from radiolysis of air-saturated water. Other radicals that might arise following exposure to Ar-CAP were detected using the nitrone spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) ,28. DBNB4PO; Sigma Aldrich Chem. Co., MO), phenyl N-t-butylnitrone , 3,5-dibromo-4-nitrosobenzene sulfonate , and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide  were used to detect the presence of different free radical species in ultrapure water following exposure to CAP by EPR spin trapping.The inert gases helium (He), neon (Ne), argon (Ar), krypton (Kr), and xenon (Xe) were bought from Hokusan Co., Ltd. . All gases were of pure grade (≥ 99.9%). The spin traps 5,5-dimethyl-1-pyrroline N-oxide , 3,3,5,5-tetramethyl-1-pyrroline-N-oxide , aminophenyl fluorescein (APF), and diaminofluorescein-2 (DAF-2DA) were from Sekisui Medical Co., Ltd. . Hydroethidine (HE) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were from Molecular Probes Inc. .2.Human myelomonocytic lymphoma U937 cells were obtained from Human Sciences Research Resource Bank . They were maintained in RPMI-1640 medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in humidified air with 5% COA jet of Ar-plasma was produced in a quartz tube of 2.0 mm inner diameter and about 150 mm length. Two electrodes connected to a high-voltage power supply unit with low frequency  were fixed on the quartz tube surface. Discharge voltage was measured with a high-voltage probe . The Ar-gas flow rate was controlled with a gas flow controller . The Ar gas flow was at 2 L/min. The length of the plasma jet was approximately 10 mm. The operational parameters for typical experiments were the irradiation distance between the tip of the quartz tube and the solution surface, and irradiation time. The system and the experimental setup are illustrated in The temperature changes of the medium during the Ar-CAP irradiation was carried out by a four channel fluoroptic temperature measurement system. . The temperature measurement probe is a 0.5 mm in diameter optical fiber, totally insensitive for electromagnetic field. The sample to be measured was 1ml tap water in a well of a 24 well plate. All the four probes of the temperature measurement system were immersed into the water to be irradiated with Ar-Cap, on a bottom of the well, exactly in the way of the plasma plume.-1; ANDOR, Belfast, UK) and an intensified charge-coupled-device (ICCD) camera .Optical emissions Ar-CAP were collected by an optic fiber and a lens directed to a position at 8 mm in the remote region of Ar-CAP. The emission were observed using a spectrometer  and N (4So) atoms were measured by vacuum ultraviolet absorption spectroscopy (VUVAS) with a micro discharge hollow cathode lamp (MHCL) as a light source operated at a fixed pressure of 1 atm [3So-2p4 3P2 at 130.217 nm, 3s 3So-2p4 3P1 at 130.487 nm, and 3s 3So-2p4 3P0 at 130.604 nm. Those used for measuring N atoms were 4P5/2-4So3/2 at 119.96 nm, 4P3/2 -4So3/2 at 120.02 nm, and 4P1/2-4So3/2 at 120.07 nm. The VUV light emitted from the MHCL was transmitted through a measured region with an absorption length of 7 mm set using two MgF2 windows attached to the light-guide pipes made of ceramics. The transmitted VUV light was dispersed by a VUV monochromator and detected by a photomultiplier tube. The total absorption intensity for three sublevels of O and N atoms was measured. Ar-CAP was located 8 mm above the measured region. To remove background absorption due to atmospheric gases, N2 gas was used as a purge gas to maintain the pressure of the chamber at atmospheric pressure. The absorption intensity of the O2 gas feed was measured using the transition lines of H2 molecules emitted from MHCL at approximately 128 nm [The absolute densities of O  dosimeter. Changes in absorbance of the chemical system with exposure time were determined with a spectrophotometer at 304 nm.X-ray irradiation was carried out at room temperature using an X-ray generator  operated at 150 kV and 20 mA with a 1 mm Al filter at a dose rate of 5 Gy/min determined by Fricke’s chemical dosimetry. The air-saturated sample solutions (1 ml) in plastic tubes were irradiated on a rotating table at 6 rpm. Immediately after X-irradiation, samples were measured with EPR and UV-spectrophotometer. Final doses were 5, 7.5 and 10 Gy.4PO. An aqueous solution containing a spin trap at a concentration of 10 mM was treated with CAP at increasing durations and distances. Immediately after irradiation, the samples were transferred to a glass capillary tube  and inserted into a special quartz tube in an EPR spectrometer . In general, EPR setting were microwave power; 4 mW, frequency; 9.425 GHz, center magnetic field; 329.5 mT, and modulation width; 0.1 mT. The nitroso spin trap DBNBS was used at a concentration of 5 mM for the detection of radicals derived from thymine (20 mM), thymidine (100 mM), uracil (20 mM), uridine (20 mM), L-alanine (1 M), and sodium acetate (3 M) aqueous solutions. The EPR spectra of the treated samples were recorded at room temperature. The yields of spin adducts were determined using the stable nitroxide radical 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrroline-1-oxide as a standard. The peak heights of EPR signals were expressed in relative units compared with that of the Mn2+ internal standard, one unit was calculated to approximately equal 7.7 x 106 M nitroxide radicals.The detection of ·OH radicals and H atoms induced by Ar-CAP was carried out using the spin traps DMPO, PBN, and M2·-), HPF (2.5 μM) for ·OH, and peroxy nitrite (ONOO-); APF (2.5 μM) for ·OH, ONOO-and hypochlorite (OCl-), DAF-2 DA (10 μM) for nitric oxide (NO).Intracellular oxidative stress following CAP treatment was assessed flow cytometrically using a number of fluorescent probes with different affinities to detect a wide range of free radical species. One million cells were preloaded with a probe in phosphate-buffered saline (PBS) for 15 min at 37°C and then exposed to Ar-CAP. Immediately after the exposure, the cells were injected into the flow cytometer to analyze at least 10,000 events. The probes included were DCFH-DA (10 μM) for overall oxidative stress, HE (10 μM) for superoxide anion . The cells were then stained with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) at 4°C for 20 min in the dark according to the manufacturer’s instructions. Finally, samples were injected into the flow cytometer where at least 10,000 events were counted. The dot plots display the percentages of cells externalizing phosphatidylserine bound to FITC-annexin V (early apoptosis), cells with damaged membranes internalizing PI (necrosis), or both (secondary necrosis). The total apoptosis fraction is expressed as the sum of early apoptotic and secondary necrotic fractions.2/NO3 assay was performed using a NO2/NO3 Assay Kit-CII , the optical density was measured at 570 nm by an automatic microplate reader .Briefly, 1 ml milli-Q water was added to 24-well tissue culture plate, and Ar-CAP irradiation was performed for 1 min. After treatment, 80 μL sample was taken out to 96-well plate, and a NOp < 0.05 considered to indicate a statistically significant difference. Pearson’s correlation coefficient was used to test the correlation among obtained data. All tests were performed using GraphPad Prism 5 .Experiments were performed in independent triplicates. All results are shown as mean ± standard deviation (SD). Tests of significance were performed using unpaired t-student test (two-tailed) with 5 4s→3p5 4p) were observed at 696.5, 706.7, 727.3, 738.4, 750.4, 751.5, 763.5, 772.4, 794.8, 800.6, 801.5, 810.4, 811.5, 826.5, 840.8, 842.5, and 852.1 nm. In addition, between 300 and 450 nm, the emission bands from excited N2 were observed at approximately 316, 337, 358, 381, 406, and 416 nm for the N2 2nd positive system (C 3Πu→B 3Πg). The emission band signals for the N2+ 1st negative system (B 2Σu+→X 2Σg+) appeared relatively weak in the range between 391 and 428 nm. An emission band from ·OH (A 2Σg→ X 2Π) occurred with a relatively low signal intensity at approximately 308 nm. Because the emission bands of N2 and ·OH were also detected, these findings indicate that the effluent Ar gas mixed with atmospheric air at remote regions beyond the exit of the glass tube. Optical emission spectroscopy is essentially more sensitive for measuring electron temperature than for measuring the densities of the observed species. Thus, the optical emission data do not enable the quantitative estimation of the density of any species.Optical emission spectra were measured to obtain the spectral lines emitted from excited-state species in Ar-CAP. 13 cm-3 at 8 mm distance, 4.5×1013 cm-3 at 13 mm, and 2.3×1013 cm-3 at 18 mm. The density of O atoms thus correlates indirectly with the distance from the plasma tube nozzle and the O atoms fed into the samples at remote regions can be calculated using the first-order kinetics within the distance range tested. Assuming that the gas is at equilibrium at room temperature, then the O atom fluxes would be in the order of 1017 cm-2 s-1, and the total O atom dose for 60s would be approximately 6×1018 cm-2 in a 6 ml-volume sample, i.e., 1018 atoms/ml.Furthermore, the absolute densities of atomic species were measured by VUVAS. The average numbers of O atoms were 6.3×104PO, and PBN. To measure temperature rise, Ar-CAP irradiation in aqueous solution was performed under continuous real time temperature data recording. The temperature rise after 2 and 5 min exposure was 1.7°C ± 0.2°C (n = 4) and 2.3 °C ± 0.2°C (n = 4), respectively. When 1 ml of 10 mM DMPO aqueous solution was exposed to Ar-CAP for 2 min, an EPR spectrum consisting of a 1:2:2:1 quartet as well as other small peaks was observed. The equal nitrogen and hydrogen hyperfine coupling constants  are characteristic of the hydroxyl spin adduct of DMPO (DMPO-OH) as shown in N = 1.65 mT) that is further split by two secondary hydrogens (aβH = 2.25 mT). An EPR spectrum of DMPO-OH and DMPO-H generated by X-irradiation is shown in Plasma irradiation produces immense quantities of free radicals. The chemical activity of Ar-CAP was confirmed by Fricke dosimeter where the oxidation of ferrous ions to ferric ions increased linearly with increasing exposure time . However4PO and PBN. For M4PO, the resulting EPR spectra consisted of a primary triplet due to nitrogen (aN = 1.53 mT), with each line split into a doublet by a secondary proton (aH = 1.69 mT). These hyperfine coupling constants are in agreement with those reported previously for M4PO-OH [N = 1.65 mT) further split by two secondary protons (aβH = 2.17 mT), consistent with the M4PO spin adduct of H atoms. The EPR spectrum obtained from PBN solution (10mM) showed a primary nitrogen triplet (aN = 1.67 mT) further split by two secondary protons (aβH = 1.06 mT), consistent with the PBN spin adduct of H atoms [The formation of ·OH radicals and H atoms by Ar-CAP was further confirmed using M M4PO-OH ,33 4PO and P3CHOH adduct increased with ethanol concentration.Moreover, the use of ethanol as ·OH radical scavenger further confirmed the formation of ·OH by Ar-CAP irradiation. As shown in 2) of hydrogen peroxide (H2O2) solution (40 mM), and the decay in the EPR spectra of DMPO-OH adducts was investigated. The half-lives of the first-order decay of DMPO-OH adducts induced by Ar-CAP irradiation and UV-photolysis of H2O2 were calculated as 6.1 ± 0.7 min and 19.2 ± 2.6 min, respectively. The rapid decay of the DMPO-OH adduct formed after Ar-CAP irradiation indicates a change in pH and/or the generation of nitric acid in the plasma-irradiated solution [Compared with 5 Gy X-ray irradiation, Ar-CAP irradiation yielded huge amounts of ·OH radicals but smaller amounts of H atoms . The amosolution –37.When the extent of ·OH radical production was assessed at graded distances from the plasma tube nozzle, the amount of DMPO-OH adducts was found to decrease with increasing distance . With th2O2, owing to the rapid recombination of ·OH radicals, small amounts of H atoms, and nitrate/nitrite were also detected.The effect of different rare gases on CAP-induced ·OH radical production was investigated. The yield of DMPO-OH adducts was in the order Kr > Ar = Ne > He. No plasma discharge was observed when Xe was used. When the yield of DMPO-OH adducts from different rare gas plasmas under similar conditions was plotted against the ionization energies of the rare gases used, an inverse correlation was observed . The NO N = 1.30 mT) which can be attributed to the spin-trapped radicals at the C-5 position most likely formed by ·OH addition (and H addition) to the C-6 position of the thymine ring (N = 1.30 mT) was also observed for the similarly irradiated thymidine aqueous solution (100 mM)  and DBNBS (5 mM) irradiated with Ar-CAP for 2 min showed an EPR spectrum of a triplet (aine ring . The sam(100 mM) . Analysi(100 mM) . It is sN = 1.37 mT) further split by two secondary protons (aβH = 1.0 mT) (2COO-) radical. Unlike the case of sonication of 3.3 M sodium acetate solution for 10 min in the presence of Ar, there was no evidence of methyl (·CH3) radicals in Ar-CAP [N = 1.36 mT) further split by two secondary protons (aβH = 1.36 mT) which indicates ·CH2-CH(NH3+)-COO- radical formation as a result of H abstraction from the methyl group of alanine. A primary nitrogen triplet (aN = 1.37 mT) further split by a secondary proton (aβH = 0.65 mT) was also observed  . The spen Ar-CAP . L-alaniobserved . This ismolecule . No evid-; APF for ·OH, ONOO-, and OCl-; HE for O2·-; and DAF-2 for NO·. When HPF and APF were utilized to measure the intracellular oxidative stress due to Ar-CAP irradiation, the shift of distribution of fluorescence intensity was observed  was almost similar to that of X-irradiation at a dose of 7.5 Gy. Thus, although Ar-CAP (1min) would create about 30 times more ·OH radicals than that formed by X-irradiation (7.5 Gy), the apoptosis-inducing efficacy of both treatments was similar reflecting the superiority of X-rays in inducing cell killing.In this study, we have shown that Ar-CAP irradiation can generate enormous amounts of ·OH radicals and H- were detected in large amounts. The latter may have resulted from the reaction between ·OH radicals and chloride anions (Cl-) that are abundant in cells. Unlike OCl-, it is not clear whether ·OH radicals were formed intracellularly or traversed from the extracellular milieu through the cell membrane. The elucidation of the mechanism by which reactive oxygen and nitrogen species are formed, and the consequent effects on cellular responses remain for future plasma research to unveil. All data are available in Our EPR-spin trapping study revealed that no pyrolysis or direct degradation radicals were formed in aqueous solutions containing non volatile solutes. Intracellularly, ·OH radicals and OClS1 FileBiological effects were determined based on apoptosis induction in U937 cells assessed flow cytometrically. Formation of free radicals was traced by different methods. In extracellular milieu, different spin adducts were detected by EPR spin trapping method. In presence of cells, various intracellular fluorescent probes were used with flow cytometry. Moreover, the overall chemical activity of Ar-CAP was detected with Fricke dosimeter. Data are given as mean ± SD.(XLSX)Click here for additional data file.


Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as Regulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.The online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users. Since the 1940s advanced prostate cancer has been treated with surgical or chemical castration in order to reduce systemic androgen levels , and casAR transcriptional induction in normal prostate epithelial homeostasis and to which extent these mechanisms are retained in putative prostate cancer stem cells (CSCs) are not understood. One hypothesis that could explain that prostate cancer invariably escapes from ADT and androgen targeted therapy (ATT) would be the existence of a subpopulation of prostate CSCs that are AR negative and therefore insensitive to androgen deprivation. Evidence has been found to support the paradoxical possibility that ADT and ATT could lead to expansion of the pool of prostate CSCs [The molecular mechanisms that underlie ate CSCs  hypothetate CSCs , 5.The understanding of essential molecular mechanisms of putative prostate CSCs is hampered by the low number of these cells in patient materials. If those cells are AR negative and AR non-responsive and give rise to AR positive and AR-dependent cells it is possible that some features of normal prostate cells are retained, although with loss of abilities to terminal differentiation and apoptosis induction. Better understanding of normal differentiation is likely to offer new insights into tumor initiation and may help explain the functional significance of common genetic alterations seen in prostate cancer . Utilizi3)2, 10 % FCS or grown in RPMI-1640 with 10 % FCS. To study epigenetic restriction cells were grown in standard medium with 10 μM 5-Aza-2′-deoxycytidine (5-Aza-dC)  for five days with addition of 250 nM trichostatin A (TSA)  the last two days. Medium was changed each day. DNA microsatellite validation of progeny identity of EP156T, EPT1, EPT2, EPT3-PT1 and EPT3-M1 cells has been published previously [2 at 37 °C. Primary antibodies; AR , actin (Cat# ab8226), GAPDH (Cat# ab181602) and PSA (Cat# ab53774) were purchased from Abcam .Primary Prostate Epithelial Cells ; Cat# ATCC-PCS-440-010) and prostate cancer cell lines LNCaP (ATCC-CRL-1740), VCaP (ATCC-CRL-2876) and 22Rv1 cells (ATCC-CRL-2505) were bought from LGC Standards GmbH . The prostate cell lines EP156T, EPT1, EPT2 and PrECs were grown in MCDB153 medium  with 1 % for EP156T and PrECs, and 5 % fetal calf serum (FCS) for EPT1 and EPT2 cells, and supplemented with growth factors and antibiotics as described elsewhere , 15. EPTThe pLenti6.3/V5-DEST-AR expression clone was generated by LR recombination reaction between the entry clone pDONR-AR , and the destination vector pLenti6.3/V5-DEST. Correct insertion of the AR gene was verified by sequencing with CMV forward primer and V5(C-term) reverse primer, according to the manufacturer’s protocol .lacZ control vector .The pLenti6.3/V5-DEST-AR and ViraPower™ Packaging Mix were co-transfected in the 293FT producer cell line, according to the manufacturer’s protocol . EP156T and EPT3-PT1 cells were seeded in six-well plates and infected with the viral supernatant. After 48 h incubation the supernatant was removed and cells were maintained in androgen-free MCDB medium with 2 μg/ml blasticidine for the selection of stably transduced EP156T-AR and EPT3-PT1-AR cells. Negative control cells were made for each cell type using the pLenti6.3/V5-GW/For IF, cells were grown on 12 mm glass coverslips  in 24 well plates, then washed with PBS, fixed , permeabilized (0.5 % Triton X-100 for 10 min.), blocked (100 mM glycin for 10 min) and stored (in PBS at 4 °C) with PBS washes between each step. Following blocking with 0.5 % BSA/PBS for 15 min. primary antibodies were added at room temperature for 1 hour at indicated dilutions in 0.5 % BSA/PBS. The FITC-labelled secondary anti-rabbit or mouse IgG  was added for 30 minutes at room temperature in 0.5 % BSA/PBS. Coverslips were mounted in Prolong Gold with DAPI  on glass slides and analyzed using Leica DM IRBE fluorescence microscopy.For Wb analysis cells were lysed in RIPA-buffer with 1:100 Protease Inhibitor Cocktail Set I . Protein concentrations were measured using the Pierce BCA Protein Assay Kit , and 5 μg protein lysates were separated by SDS electrophoresis in NuPAGE® 10 % Bis-Tris Gels  followed by blotting to PVDF membranes  using Pierce 1-Step Transfer Buffer  and Pierce G2 Fast Blotter (ThermoFisher). Membranes were blocked for one hour in PBS 0.1 % Tween and 5 % Skim milk powder . Primary antibodies were incubated for 1 hour in blocking buffer at RT, and HRP-labelled secondary antibodies , were incubated as the primary antibodies 1/10000. Pierce ECL Western Blotting Substrate  or SuperSignal West Femto Maximum Sensitity Substrate  was used for detection with Chemidoc XRS using Quantity One 4.6.5 (Bio-Rad). Molecular weight marker used was MagicMark XP .Cell culture supernatants were centrifuged in an Eppendorf centrifuge at 14 000 x g for 2 minutes at room temperature, and 0.5 ml of the supernatants were analyzed using the Elecsys total PSA immunoassay (#04641655 190) in a Cobas analyzer  according to the kit manual and according to the accredited routines of the Laboratory of Clinical Biochemistry (LKB) Haukeland University Hospital. The lower detection limit is 0.003 ng/ml total PSA. Values above 100 ng/ml are considered above the measuring range.ACTB (Hs99999903_m1), AR (Hs00171172_m1), KLK3 (Hs02576345_m1), NKX3-1 (Hs00171834_m1), TMPRSS2 (Hs00237175_m1). The Agilent Human Whole Genome (4x44 k) Oligo Microarray with Sure Print Technology , was used to analyze samples in the present study. Total RNA purification, cDNA labeling, hybridization and normalization have been described previously [http://www.molmine.com) [Total RNA was extracted using the miRNeasy kit from Qiagen . The total RNA was DNase treated, ss-cDNA was synthesized and the RT-qPCR was run and analyzed as previously described [-1 Hs001734_m1, TMine.com)  was usedine.com)  and for TMS2  kits and instrumentation according to instructions. cDNAs were tagged with barcoded adapters for multiplexing. Paired-end sequencing with read length 150 base pairs and 100 million reads per sample were chosen for raw sequence data acquisition. Raw data were formatted in BAM files and mapped to the December 2013 build of the UCSC Human genome browser. The following module versions were used in the TopHat and Cufflinks analyses for alignment and to estimate expression levels: TopHat2 v2.0.7, Bowtie 0.12.9, Cufflinks 2.1.1, Isaac Variant Caller 2.0.5, Picard tools 1.72. RNA-seq data is available at Gene Expression Omnibus (ID: GSE71797).Total RNAs were included for RNA-seq if RIN (RNA Integrity Number) was above 9 and total RNA was at least 500 ng according to the Agilent 2100 Bioanalyzer™. Illumina HiSeq™ 2000 (Illumina) RNA-Seq was performed according to manufacturer’s instructions and according to StarSeq™  protocols. Prior to cDNA synthesis rRNA depletion of total RNA was done. The Qubit™/Bioanalyzer™ instruments were used for concentration and quality control and fragmentation and sizing was achieved using the CovarisResults from real-time RT-qPCR were analyzed using the RQ Manager v1.2 software and DataAssist v3.01 . Error bars show 95 % confidence intervals. 95 % confidence intervals were analyzed for secreted PSA values using Microsoft Excel 2011 .AR mRNA could not be detected in either of these conditions using Agilent oligonucleotide microarray analyses  and the established prostate cancer cell lines LNCaP, VCaP and 22Rv1. LNCaP cells are widely used as an approximation to androgen sensitive cancer and 22Rv1 cells are considered one model of AR positive CRPC. PrECs reach senescence and die following a limited number of cell divisions. A low level of AR mRNA was detectable in PrECs according to sensitive RT-qPCR assays. But addition of androgen did not lead to increased expression of AR target genes as exemplified for the KLK3, NKX3-1 and TMPRSS2 mRNA  and the histone deacetylase inhibitor trichostatin A (TSA). We found that even if imprinted genes were robustly activated as assessed by RT-qPCR .As exemplified in Fig. In order to obtain a genome-wide perspective on androgen-dependent AR target genes in EP156T-AR cells, total RNA of cells that were grown either with or without androgen in monolayer cultures or grown in the presence of androgen in Matrigel cultures for 14 days were profiled using the Agilent 44 k microarrays. In monolayer culture 1836 genes were differentially regulated by a factor of at least 2 and a FDR < 10 in cells expressing exogenous AR, 924 genes were upregulated by androgens and 912 downregulated. In Matrigel culture 1673 genes were differentially regulated, 894 genes were upregulated by androgen and 779 downregulated. 855 genes were differentially expressed following androgen addition both in 2D- and 3D-culture.AR, KLK3, TP63 and TMPRSS2  of the AR mRNA. This sequence is absent in the AR mRNA that is transcribed from the AR open reading frame of the expression vector. When the TaqMan real-time RT-qPCR assay is used to detect AR exon sequences in parallel, a distinction can be made between endogenous and exogenous AR mRNAs of the same cell cultures. It was of considerable interest to examine the possibility if basal AR expression might have a positive feedback effect on endogenous AR transcription. Expression levels of AR in the absence and presence of androgen were examined in cells with or without exogenous AR expression, but endogenous AR was not detectable. These experiments showed that in EP156T-AR cells the restriction of endogenous AR expression persisted even if the classical AR target genes were activated by exogenous AR and androgen.One advantage of the gene expression analysis is that the −) prostate epithelial stem cells divide asymmetrically to self-renew and to differentiate into either non-proliferating AR− neuroendocrine cells or TP63+/AR− transient amplifying (TA) cells in the normal adult prostate. The basally located AR− TA cells undergo a limited number of amplifying rounds of proliferation before maturing into TP63+/PSCA+ intermediate cells [AR negative . Add. Add− anere (see , 62–67.− prostate CSC with basal cell features could help to explain the recurrence of transdifferentiated neuroendocrine cancers following highly potent ATT of CRPC [The existence of AR of CRPC , 69. It  of CRPC , 70, 71  of CRPC –74. In tKLK3 mRNA translation was observed and PSA was detected in confluent monolayers and in Matrigel cultures. However exogenous AR with or without androgen did not induce endogenous AR mRNA transcription. Low-level AR and high-level exogenous AR were unable to induce KLK3 mRNA or other classical AR target genes in mesenchymal type prostate cells. In summary these results demonstrate the context dependent function of the AR, and that epigenetics and/or availability of cofactors greatly influence the AR transcriptome and ultimately if AR acts in a tumor suppressive or oncogenic manner. It also demonstrates that PSA might not be a good biomarker in cancers with high cellular plasticity, particularly for cancers that are induced towards a mesenchymal phenotype. However more knowledge is required to understand the specific conditions that govern AR-regulated phenotypes including its role in differentiation.Androgen receptor expression and classical target gene expression were restricted and androgen non-responsive in PrECs and immortalized EP156T cells both in monolayer and Matrigel cultures. Expression of exogenous AR in EP156T-AR cells induced an extensive androgen-dependent trancriptome including classical target genes. No restriction of ADT, androgen deprivation therapy; ATT, highly active androgen targeted therapy; ChIP-chip, chromatin immunoprecipitation with DNA microarray chip; CRPC, castration - resistant prostate cancer; CSC, cancer stem cell; E, epithelial; EMT, epithelial to mesenchymal transition; FDR, false discovery rate; FPKM, fragments per kilobase of exon per million reads mapped; IF, indirect immunofluorescence; M, mesenchymal; PrECs, human primary prostate transit amplifying cells with basal cell features; RNA-seq, RNA-sequencing; RT-qPCR, reverse transcription quantitative polymerase chain reaction


With advances in molecular biologic and genomic technology, detailed molecular mechanisms for development of castration-resistant prostate cancer (CRPC) have surfaced. Metastatic prostate cancer (PCa) no longer represents an end stage, with many emerging therapeutic agents approved as effective in prolonging survival of patients from either pre- or post-docetaxel stage. Given tumor heterogeneity in patients, a one-size-fits-all theory for curative therapy remains questionable. With the support of evidence from continuing clinical trials, each treatment modality has gradually been found suitable for selective best-fit patients: e.g., new androgen synthesis inhibitor arbiraterone, androgen receptor signaling inhibitor enzalutamide, sipuleucel-T immunotherapy, new taxane carbazitaxel, calcium-mimetic radium-223 radiopharmaceutical agent. Moreover, several emerging immunomodulating agents and circulating tumor cell enumeration and analysis showed promise in animal or early phase clinical trials. While the era of personalized therapy for CRPC patients is still in infancy, optimal therapeutic agents and their sequencing loom not far in the future. Prostate cancer (PCa) is the lead malignancy among males in Western countries, accounting for 28% 238,590) of newly diagnosed cancers in United States in 2013 8,590 of Figure In the case of advanced PCa, ADT is standard treatment, which initially reduced tumor burden and prostate-specific antigen (PSA) level to low or undetectable level. Most PCa ultimately recurs despite of ADT, presenting with progressively rising of PSA level, termed CRPC. Docetaxel was regarded as the only reasonable option before 2010. Additionally, there is no therapeutic agent for patients who experience progression after first-line docetaxel. Recent years have seen a number of novel anticancer drugs for CRPC clinics. The past three years can be considered exceptional due to positive outcomes in Phase III trials. Key antitumor agents showing positive results include taxane cabazitaxel , 9, vaccCabazitaxel, a second-generation taxane, demonstrably improves overall survival when added to prednisone versus mitoxantrone plus prednisone in TROPIC (treatment of hormone-refractory metastatic PCa previously treated with docetaxel-containing regimen) trial: median overall survival is 15.1 months versus 12.7 months in CRPC patients with progression after docetaxel treatment . Progresex vivo with a recombinant fusion protein (PA2024) [Sipuleucel-T, an active cellular immunotherapy, is a type of therapeutic cancer vaccine consisting of autologous peripheral-blood mononuclear cells (PBMCs), including antigen-presenting cells (APCs) activated (PA2024) . PA2024 (PA2024) . In concAbiraterone acetate blocks androgen biosynthesis by inhibiting 17α-hydroxylase/C17,20-lyase (CYP17). The COU-AA-301 and COU-AA-302 trials established the role of abiraterone in mCRPC patients with or without previous docetaxel chemotherapy. In COU-AA-301 trial, overall survival as primary endpoint was longer with abiraterone acetate-prednisone than with placebo-prednisone  . In COU-Enzalutamide, a novel androgen receptor signaling inhibitor, competitively inhibits binding of androgens to the androgen receptor (AR), inhibits AR nuclear translocation, and inhibits association of the AR with DNA . The AFFRadium-223 , calcium-mimetic radiopharmaceutical, has high bone affinity. Alsympca (ALpharadin in SYMptomatic Prostate Cancer) Phase III trial shows improved overall survival: median duration 14 months . Time toCabozantinib (XL184), an orally bioavailable tyrosine kinase inhibitor, shows potent activity against MET and VEGF Receptor 2 (VEGFR2). It suppresses MET and VEGFR2 signaling, rapidly inducing apoptosis of endothelial and tumor cells, resulting in tumor regression. It can also block progression of osteolytic and osteoblastic lesions , 21It is well documented that response to standard therapy differs among patients diagnosed with the same cancer. Obviously, a one-size-fits-all concept is not expected to achieve identical outcome; individualized approach is needed. Progress in understanding intricate molecular mechanisms for transformation of normal cells into cancer, plus aberrant control of complementary pathways, leads us into a more complex world for diagnosis and treatment. Oncology has entered an era with treatment individualized or customized, therapy based on molecular and genetic traits of a tumor and its microenvironment, tailored to improve outcomes and decrease both toxicity and health-care costs. Personalized cancer therapy targets aberrations that drive tumor progression, administering the right therapy for the right person at the right time. Success requires identification of novel validated markers for prognosis, treatment response, resistance and toxicity. Chief task in practice is modifying therapy for diverse tumor nature with inadequate, limited prognostic tools , 24.The American Urological Association (AUA) announced clinical guidelines for CRPC in May, 2013 . With seLarge-scale cancer genomic characterization projects offer critical new insights into molecular classification of cancers and have potential to identify new therapeutic targets . PCa exhTMPRSS2-ERG fusion (around 50%) [AR (Xq12) and MYC (8q24), and homozygous focal deletions of PTEN (10q23) and NKX3.1 (8p21) are frequently identified in PCa [NCOA2 gene, which encodes an AR coactivator [RB1 on 13q14.2, TP53 on 17p31.1, interstitial 21q22.2-3 deletion spanning ERG and TMPRSS2 [ETV6 and DUSP16 as well as CDKN1B [MYC on 8q24.21 and NCOA2 on 8q13.3. Focal amplification of AR (Xq12) is likewise common but restricted to metastatic tumors. Among mutated genes, the most common is androgen receptor (AR); other oncogenes like IDH1, IDH2, PIK3CA, KRAS, and BRAF do not commonly mutate in PCa [PTEN alteration is considered with INPP4B and PHLPP phosphatase alterations recently implicated in PI3K regulation, the PIK3CA gene itself, and regulatory subunits PIK3R1 and PIK3R3 [Copy number alterations (CNAs) can result in amplification of oncogenes or deletion of tumor suppressor genes; these changes contribute significantly to cancer etiology. Consistent and common findings from global analyses of CNAs within PCa include und 50%) , 28, 8p und 50%) , 30. Focd in PCa , 32. Recs CDKN1B  were repe in PCa . Integrae in PCa . PTEN ned PIK3R3 . Explorid PIK3R3 , 36.TMPRSS2 and members of the ETS transcription factor family  has been identified in a majority of prostate cancers [TMPRSS2-ERG have shown modest evidence of oncogenic activity with cooperating transforming events [TMPRSS2-ERG fusion as the single most established PCa molecular lesion [TMPRSS2 androgen-responsive promoter [TMPRSS2-ERG fusion: spanning tumor suppressors PTEN and TP53, plus another spanning 3p14 multigenic region. The 3p14 deletion, whose association with TMPRSS2-ERG loomed predominant, appeared only in PCa [TMPRSS2-ERG fusion in 19% of high-grade prostatic intraepithelial neoplasia (PIN) lesions and in 50% of localized PCa suggests this fusion as either occurring after onset or associated with early events predisposing to clinical progression [TMPRSS2-ERG is technically feasible nowadays; in PSA-screened cohorts it shows sensitivity of 30-50% and specificity >90%. Examination for TMPRSS2-ERG may detect 15-20% of men harboring PCa but with normal DRE  and PSA levels, including a substantial proportion of those who harbor high-grade Gleason disease [TMPRSS2-ERG fusions, inducing regression in >50% of CRPC cases [TMPRSS2-ERG tumors manifested a subgroup of PCa remaining exquisitely sensitive to CYP17 blockade [A recent rearrangement involving the androgen-regulated  cancers , 37. Furg events , 28: TMPr lesion , meaningpromoter . Signifiy in PCa , 39. Homgression . Recent gression . Populat disease . Most 5’PC cases . Hormoneblockade . Also, Eblockade . As dereNCOA2 [NCOA2 gain in primary tumors plus a known role as AR coactivator [NCOA2 functions as a driver oncogene in primary tumors by increasing AR signaling; in contrast, AR amplification is largely restricted to mCRPC and likely a mechanism of drug resistance rather than a natural step in tumor progression.AR signaling is essential for growth and differentiation of a normal prostate and is responsible for treatment failure in CRPC or metastatic PCa. The contribution of AR to prostate tumorigenesis and disease progression is incontrovertible. The exclusive requirement of PCa cells for AR activity is illuminated at clinic, wherein therapeutic suppression of AR signaling, typically achieved through ligand depletion and direct AR antagonists, results in PSA decline and objective tumor regressions. Conventional therapy currently focuses on androgen-dependent activation of AR via its C-terminal ligand-binding domain (LBD). Mechanisms of therapeutic failure include AR amplification and/or overexpression, gain-of-function AR mutations, intracrine androgen production; overexpression of AR coactivators, expression of constitutively active splice variants of AR, and ligand-independent AR activation through growth factors, cytokines, or aberrant AR phosphorylation . Among ANCOA2 . High frctivator  lends inRecently developed androgen-ablative and AR antagonist strategies that achieve complete androgen ablation and sufficient suppression of AR signaling in the prostate improve efficacy of AR targeting and subsequent therapeutic outcome. A new means to deplete androgens is a selective CYP17 inhibitor, which inhibits both testicular-derived androgen production and tumor-derived androgen synthesis, meaning a great advance toward durable androgen depletion and suppression of AR activity. Despite strong rationale for aiming at CYP17, this target remains largely unexploited, with relatively few candidate agents progressing to clinical trials and only ketoconazole, an unspecific CYP17 inhibitor, in widespread clinical use . PromisiDirect AR antagonists are often combined with orchiectomy or GnRH agonists/antagonists, to inhibit AR signaling further. Docking of AR antagonists into the AR C-terminal LBD results in both passive AR inhibition, via competition for agonists, and active mechanism of AR inhibition: e.g., prevention of coactivator binding and inducement of corepressor recruitment. AR can be alternatively spliced so that the C-terminal domain is deleted, rendering AR constitutively active . Splice Epigenetics is defined as heritable changes in gene expression caused by mechanisms other than altered DNA sequence. Unlike many other genetic changes, epigenetic processes are reversible and do not change DNA sequence or quantity, though they enhance genomic instability that might lead to oncogenic activation and inactivation of tumor suppressors . Among t. SeveralCTCs appear in the bloodstream, having detached from their tumor of origin. A major cause of cancer-associated mortality is tumor metastasis, which depends on successful dissemination to the whole body, mainly through blood. Therefore, CTCs shed into vasculature and possibly on the way to potential metastatic sites arouse obvious interest. Studies in past years have shown CTCs as markers predicting cancer progression and survival in metastatic -57 or evAlthough CTC counts are of prognostic relevance, CTC enumeration is not yet validated as a surrogate of clinical benefit. Technical challenge in this field consists of finding tenuous tumor cells (a few CTCs mixed with approximately 10 million leukocytes and 5 billion erythrocytes in 1 ml of blood) and distinguishing them from epithelial non-tumor cells and leukocytes. It should be feasible with advanced technology that allows automated and high-throughput separation, visualization and quantification of cancer cells from blood . Abilityde novo antitumor response through vaccination. Both pilimumab (MDX-010) and tremelimumab (CP-675206) are clinical applications of checkpoint inhibitors, antibodies specific for cytotoxic T lymphocyte antigen 4 (CTLA-4). Ipilimumab is an antagonistic mAbs that recognizes CTLA-4, an immunomodulatory molecule expressed by activated T cells, and to CD80 on APCs. It was proven active both in PSA response and clinical improvement, with or without radiotherapy in mCRPC patients [The concept of immune modulation, which aims at generating a meaningful antitumor immune response, has been extensively evaluated in melanoma and renal cell carcinoma. This principle has been extended to PCa, known as slow-growing and more indolent, which can allow sufficient time for generating effective antitumor immune response. Moreover, recent studies indicated that PCa is more immunogenic than considered earlier, with evidence of PCa-specific autoantibodies in blood samples of patients. Sipuleucel-T is the first immunotherapy approved by the US FDA in April 2010 -69. The patients . BecauseMost treatment regimens for advanced cancer highlight a combination of chemotherapy drugs, or concurrent radio-chemotherapy, raising a possibility that immunotherapy may need combination with conventional therapy to achieve maximal effect. Fortunately, conventional cancer treatments have immunological benefits , making In the past decade, cancer therapy has slowly but steadily transformed from a one-size-fits-all to a more personalized approach, each patient treated according to specific genetic defects of his/her own tumor. Appearance of genomic technologies has now provided the means to develop data that address complexity of biologic states. Practice of cancer therapy continually faces the challenge of matching the right therapeutic regimen with the right patient at the right time, balancing relative benefit with risk to attain optimal outcome. Cases with CRPC may represent myriad heterogeneity in terms of performance, comorbidity, and underlying molecular mechanisms. Prior to 2010, the sole agent for CRPC was docetaxel; positive results now available from clinical trials of cabazitaxel, sipuleucel-T, abiraterone, and enzalutamide mean we now have a plethora of agents to choose from. New AUA guidelines for CRPC treatment in 2013 represent a prototype of personalized therapy. Despite these recent advances, efforts in molecular therapeutics should continue and bring further changes in the PCa treatment paradigm. Moreover, many emerging personalized therapies are under scrutiny: e.g., immunotherapy and CTC-targeted therapy. Though personalized therapy for CRPC is still in its infancy, ideal therapy tailored for individual CRPC patients continues to advance.We thank Dr. Ming-Chei Maa and Dr. Yuan-Man Hsu for valuable suggestions and editorial assistance. The authors thank Dr. Ganesh Raj  for permission to use Figure


Vegfa gene gives rise to three major isoforms termed VEGF121, VEGF165 and VEGF189. VEGF165 binds the transmembrane protein neuropilin 1 (NRP1) and promotes the migration, survival and axon guidance of subsets of neurons, whereas VEGF121 cannot activate NRP1-dependent neuronal responses. By contrast, the role of VEGF189 in NRP1-mediated signalling pathways has not yet been examined. Here, we have combined expression studies and in situ ligand-binding assays with the analysis of genetically altered mice and in vitro models to demonstrate that VEGF189 can bind NRP1 and promote NRP1-dependent neuronal responses.The vascular endothelial growth factor  regulates neurovascular patterning. Alternative splicing of the  Summary: Although VEGF165 was thought to be the sole VEGF isoform acting through neuropilin 1 (NRP1), VEGF189 also binds to and signals through NRP1 in several types of developing mouse neurons. VEGFA) of eight exons that is alternatively spliced into isoforms, the major ones containing 121, 165 and 189 amino acid residues and therefore termed VEGF121, VEGF165 and VEGF189, respectively  is a potent inducer of blood vessel growth, but also has essential roles in neurodevelopment . In humaectively . The alt VEGF121 . Unexpec VEGF121 . By cont VEGF121 , promoti VEGF121  and enha VEGF121 .120/120Vegfa mice, which express VEGF120, the murine equivalent of VEGF121, but lack VEGF164 and VEGF188, corresponding to human VEGF165 and VEGF189, respectively -conjugated VEGF isoforms for g assays  to examiVegfa isoforms were expressed in these developmental contexts. For this experiment, we designed isoform-specific primers that can distinguish the Vegfa120, Vegfa164 and Vegfa188 mRNA species by reverse transcription (RT)-PCR . This analysis demonstrated that all three isoforms were co-expressed during relevant periods of VEGF/NRP1-dependent neurodevelopment in mice . Nrp1−/− hindbrains showed some defasciculation of these dorsolateral axons, but they were still clearly present in the mutant, suggesting that this are a suitable model to examine VEGFA isoform binding to NRP1.Because prior studies of VEGF binding to NRP1 have not examined whether VEGFfor NRP1  to confi121, VEGF165 and VEGF189, we fused each isoform to AP and performed in situ ligand binding assays on E12.5 hindbrains. As expected, all three isoforms bound vessels . These observations suggest that VEGF188, similar to VEGF164, can promote GnRH neuron survival. The ineffectiveness of VEGF120 agreed with the previously observed NRP1-dependent neuroprotection of GN11 cells and the fact that 120/120Vegfa mice have fewer GnRH neurons  and the following oligonucleotide pairs: 120-F 5′-GTAACGATGAAGCCCTGGAG-3′ and 120-R 5′-CCTTGGCTTGTCACATTTTTC-3′; 164-F 5′-AGCCAGAAAATCACTGTGAGC-3′ and 164-R 5′-GCCTTGGCTTGTCACATCT-3′; 188-F 5′-AGTTCGAGGAAAGGGAAAGG-3′ and 188-R 5′-GCCTTGGCTTGTCACATCT-3′.Total RNA was reverse transcribed using Superscript III (Life Technologies) and Open reading frames for the VEGF isoforms were amplified by PCR with the oligonucleotides 5′-AATAATGGATCCGCACCCATGGCAGAAGG-AG-3′ and 5′-TATATGCTCGAGCTCACCGCCTCGGCTTGTC-3′. The PCR products were cloned into pAG3-AP containing an upstream in-frame AP cassette. Binding assay were performed as described previously .in situ hybridisation, we used a digoxigenin-labelled Isl1 probe , goat anti-rat NRP1 , rabbit anti-mouse TUJ1  and mouse anti-rat ISL1 . Secondary antibodies used were: Alexa594-conjugated rabbit anti-goat Fab , Alexa488-conjugated donkey anti-rabbit Fab , Alexa488-conjugated goat anti-mouse  and biotinylated goat anti-rabbit . To detect blood vessels, we used biotinylated IB4 (Sigma) followed by Alexa633-conjugated streptavidin (Life Technologies). For l1 probe .164 in PBS (Preprotech) or VEGF188 (Reliatech). FBM neuron migration was measured with ImageJ (NIH) as the distance travelled from r5 to the leading group of cells in r6 in each hindbrain and normalised to the control side of each hindbrain.Hindbrain explants were cultured as previously described . Affi-GeDiI labelling was performed with fixed tissues as described previously . Briefly120, VEGF164 or VEGF188.Immunolabelled GnRH-positive cells were quantitated and GN11 survival assays performed as described previously . For sur


The 2014 Ebola virus disease (EVD) outbreak is the largest in history, affecting multiple countries across West Africa, Europe, and the United States. Sierra Leone is among the predominant three nations affected, alongside Guinea and Liberia. As of 14 December 2014, there have been 8,356 cases of Ebola in Sierra Leone, with 2,417 deaths  recognised that global EVD response in itself was not enough, that past efforts have neglected rebuilding of health systems, and that ‘Ebola has destroyed whatever hopes these three countries might have had for even moderate prosperity and human security’ . NationaVageesh Jain King's College London London, UK Email: vageesh.jain@kcl.ac.ukColin Stewart Brown King's Centre for Global Health, King's Health Partners King's College London London, UKOliver Johnson King's Centre for Global Health, King's Health Partners King's College London London, UK


We retrospectively analyzed 4756 cases of infertility patients treated with conventional-IVF(c-IVF) or ICSI, which were divided into three groups according to NSMR: ≥14%, 4%–14% and <4%. Here, we demonstrate that, with decrease in NSMR, in the c-IVF group, the rate of fertilization, normal fertilization, high-quality embryo, multi-pregnancy and birth weight of twins gradually decreased significantly (P<0.05), while the miscarriage rate was significantly increased (p<0.01) and implantation rate, clinical pregnancy rate, ectopic pregnancy rate, preterm birth rate, live birth rate, sex ratio, and birth weight(Singleton) showed no significant change. In the ICSI group, with decrease in NSMR , high-quality embryo rate, multi-pregnancy rate and birth weight of twins were gradually decreased significantly (p<0.05), while other parameters had no significant difference. Considering the clinical assisted methods selection, in the NFMR ≥14% group, normal fertilization rate of c-IVF was significantly higher than the ICSI group (P<0.05), in the 4%–14% group, birth weight (twins) of c-IVF were significantly higher than the ICSI group, in the <4% group, miscarriage of IVF was significantly higher than the ICSI group. Therefore, we conclude that NSMR is positively related to embryo reproductive potential, and when NSMR<4% (5th edition), ICSI should be considered first, while the NSMR≥4%, c-IVF assisted reproduction might be preferred.Sperm morphology is the best predictor of fertilization potential, and the critical predictive information for supporting assisted reproductive methods selection. Given its important predictive value and the declining reality of semen quality in recent years, the threshold of normal sperm morphology rate (NSMR) is being constantly corrected and controversial, from the 4 Fifteen percent of couples are subfertile, and in one-half of these cases subfertility can be attributed to the male partner It is often unclear whether conventional IVF or ICSI would be the most appropriate reproductive to apply, a din the case of isolated teratozoospermia, there are no widely accepted criteria governing this decision. As a result coupled with isolated teratozoospermia often pursue ICSI unnecessarily or experience complete fertilization failure following conventional IVF (c-IVF). To date, most researches on sperm morphology have focused on the correlation between NSMR and DNA integrity and chromosomal aneuploidy So, this study selected 4,765 pairs of infertile couples  and recorded male partner sperm morphology. Patients were further divided into three groups of NSMR ≥14%, 4%–14%, <4%. Thirteen indicators of embryo preimplantation development, clinical pregnancy outcomes, neonatal conditions were recorded in order to clarify the predictive value of NSMR. The predictive value of NSMR in c-IVF and ICSI were compared in order to provide a guidance for selecting an appropriate assisted reproductive methods for patients with teratozoospermia.4th), 4%–14% and <4% (the threshold was recommend by WHO5th). The outcome of c-IVF or ICSI - pre-implantation development, clinical pregnancy outcome and neonatal conditions – was retrospectively analyzed. There was no significant difference in male age, female age, endometrial thickness, number of oocytes retrieved, and number of embryos transferred between the patient subgroups (p>0.05)  were enrolled at the Tangdu Reproductive Medical Center between January 2011 and March 2013. Patients were divided into three groups of NSMR ≥14% (the threshold was recommend by WHO(p>0.05) .During preimplantation development, we retrospectively analyzed four indicators . Results showed that in the c-IVF treatment group, with the decrease in NSMR , fertilization rate, normal fertilization rate and high quality embryo rate gradually decreased, and the decrease reached a statistical significance or very significance . However, ICSI reproduction had lower birth weight of twins than c-IVF group, with statistically significant difference . Therefore, in couples with 4%≤NSMR<14%, c-IVF assisted reproductive method is preferred.In the NSMR <4% subgroup, ICSI assisted reproduction had miscarriage rate of 6.32%, while c-IVF assisted reproduction had miscarriage rate of 13.6%. The two had statistically significant difference (p<0.01). Therefore, for infertile couples with NSMR<4%, ICSI assisted reproductive method is preferred to reduce abortion rate.To the best of our knowledge, this is a first study to analyze the relationship between the sperm morphology and the clinical selection of assisted reproductive methods based on comprehensive parameters, from the preimplantation to newborn stage. And, a preliminary view is that for patients with NSMR <4%, ICSI assisted reproductive method should be given in priority, and for patients with NSMR≥4%, c-IVF assisted reproductive method should be chosen in priority.Some studies have demonstrated that fertilization potential appears to decline with advancing paternal age. Ford et al. In our sample, in patients that received c-IVF assisted conception the embryo fertilization rate and normal fertilization rate gradually declined with NSMR, however, these measured were not correlated with NSMR in patients who received ICSI assisted conception. The most likely explanation for this is that during ICSI the embryologist can microscopically select individual sperm that appear morphologically “normal,” from even the most impaired specimens. Thus, fertilization occurs with sperm that may not be representative of the sperm population within the entire sample, making the initial semen morphology assessment irrelevant. This concept has been taken to the subcellular level in new techniques such as intracytoplasmic morphologically selected sperm injection and motile sperm organellar morphology examination (MSOME). In these techniques, investigators select sperm for ICSI using a high power×63,000 objective to magnify and allow more critical assessment of sperm morphology. Sperm nucleus morphology by the MSOME method has positively correlated with fertilization and pregnancy rates In clinical practice, we select the best quality embryos for transplantation. This step of evaluation largely offsets the impact of paternal abnormal sperm on outcome.. Therefore, regardless of the degree of sperm normal morphology, and method of reproductive technology employed, the embryo implantation rate, clinical pregnancy rate, ectopic pregnancy rate, and premature birth rate did not differ between patients subdivided by NSMR.However, in contrast, we did find that the rate of multiple births decreased gradually with the decrease in NSMR, regardless of the method of insemination, reflecting the important contribution of male factors on embryonic development potential. We were also surprised to find that, when using c-IVF, in patients with NSMR <4%, miscarriage rate increased, and live birth rate decreased. Reasons may be that, although c-IVF retained the natural property of sperm-egg recognition, combination and penetration and is closer to a physiological insemination, compared to the normal process of fertilization, it lacks the screening mechanisms of PH value, antibody killing, and liquid concentration gradient on sperms in the “trek” process from the cervical to the oviduct belly. Therefore, when semen with a lower NSMR is used for c-IVF insemination, it means that there is a great probability for abnormal sperms to combine with eggs and fertilize, while in the normal fertilization process, this part of abnormal sperms may not be able to reach the ampulla.Previous studies have indicated that, in comparison to other parameters of sperm, sperm morphology is a more reliable predictor of clinical outcomes. Lewis-Jones et al Male and female preimplantation embryos differ not only in their chromosomal complement, but in their proteome and subsequent metabolome. Studies have reported With the widespread application of assisted reproductive technology more potential parents express concerns about potential birth defects. Most studies In conclusion, our study suggests that NSMR is closely related to the potential of embryonic development and cycle outcomes, and can serve as a powerful reference in clinical choice of assisted reproductive methods in patients with teratozoospermia. The molecular mechanisms governing these observations remaind to be determined, and we recommend genetic and epigenetic analysis of the embryo and placenta in teratozoospermia patients.The c-IVF and ICSI cycles were performed at the Tangdu reproductive medical center from January 2011 to March 2013 and retrospectively analyzed. The inclusion criteria of infertile couples were as follows: male, age<38 years old; female, age <38 years old, infertility due to tubal factor or unexplained infertility. The exclusion criteria: patients with endometrial fibroids, endometriosis, uterine adhesion due to previous uterine surgery, or thyroid function abnormality th WHO 6/ml, total count ≥39×106, total motility ≥40% or progressive motility ≥32%).Semen samples were collected by masturbation for semen analysis after an abstinence of 3–5 d. In all the occasions, semen volume, concentration, and motility were determined according to 5th WHO manual. The criterion used for assessment of sperm morphology: (1) The head should be smooth, regularly contoured and generally oval in shape. There should be a well-defined acrosomal region comprising 40–70% of the head area. The acrosomal region should contain no large vacuoles, and not more than two small vacuoles, which should not occupy more than 20% of the sperm head. The post-acrosomal region should not contain any vacuoles. (2) The midpiece should be slender, regular and about the same length as the sperm head. The major axis of the midpiece should be aligned with the major axis of the sperm head. Residual cytoplasm is considered an anomaly only when in excess, i.e. when it exceeds one third of the sperm head size. (3) The principal piece should have a uniform calibre along its length, be thinner than the midpiece, and be approximately 45um long (about 10 times the head length). It may be looped back on itself, provided there is no sharp angle indicative of a flagellar break. Basic Procedure is as follow: Briefly, 5–10 µl of semen (depending on the sperm concentration) was placed on a previously cleaned slide and stained using the Papanicolaou staining protocol. Two technicians, who have gotten the national docimaster qualification and have been trained for documenting teratospermia with the standard of WHO-5, analyzed at least 200 sperm cells independently under oil immersion microscope with 1000× magnification. Mean values of their reports were recorded. Quality control mechanism for morphology assessment included a weekly calculation of inter-observer coefficient of variation as obtained by their concurrent evaluation of the same discarded semen sample. An inter-observer variation of <5% was considered to be acceptable. The dyes were checked daily for cross contamination and were changed weekly.Sperm morphology was assessed using the Kruger/Tygerberg Strict Criteria as outlined by the 52. Oocytes were placed in 15,000 sperm per 100-ul drop or fertilized by ICSI 3–4 h after the retrieval. Fertilization check was performed 18–20 h after the c-IVF/ICSI procedure. Normally fertilized zygotes were subsequently moved to microdrops containing HTF medium with 10% synthetic serum substitute and cultured for an additional 2 days. Embryo transfer was performed on day 3 under ultrasound guidance using a Wallace catheter. Embryo selection for day 3 transfer was based on morphologic parameters. Embryos were graded on the basis of cell number, regularity of blastomeres, good blastomere expansion, fragmentation level, and signs of embryonic compaction. Pregnancy testing was performed 15 days after the embryo transfer. Clinical pregnancy was confirmed by the presence of a fetal heart on ultrasonic examination at 6–8 weeks of pregnancy.Down-regulation with leuprolide acetate  followed by ovulation induction with gonadotropins was the primary stimulation protocol used. Oocytes were recovered by transvaginal aspiration of follicles under ultrasound guidance. Oocytes were cultured in microdrops of human tubal fluid(HTF) medium supplemented with 10 mg/ml human serum albumin under an oil overlay. Culture dishes were incubated at 37°C with 6% COThe rates of fertilization, normal fertilization, cleavage, high quality embryo, implantation, pregnancy, multiple pregnancy, ectopic pregnancy, miscarriage, preterm birth, live birth and sex ratio were compared between groups using chi-square analyses. Female age, male age, endometrial thickness, number of oocytes retrieved, number of embryos transferred and birth weight were compared between groups using Student's t test. P values of <0.05 were considered to be statistically significant.Figure S1The basic information of patient couple treated by c-IVF.(XLSX)Click here for additional data file.Figure S2The basic information of patient couple treated by ICSI.(XLSX)Click here for additional data file.


In osteosarcoma cell lines, U2OS and SaOS2, the knock down of DBC1 and AR with siRNA significantly reduced cellular proliferation and inhibited proliferation-related signaling. In addition, the knock down of DBC1 and AR decreased the invasion activity and inhibited invasion-related signaling of osteosarcoma cells. Interestingly, DBC1 affects the stabilization of AR protein via a mechanism involving the ubiquitination of AR. Proteosome-mediated degradation and poly-ubiquitination of AR were increased with the knock-down of DBC1. In conclusion, this study has shown that DBC1 is involved in the stabilization of AR protein and DBC1-AR pathways might be involved in the progression of osteosarcoma.Deleted in breast cancer 1 (DBC1/CCAR2) is a protein of interest because of its diverse roles in tumorigenesis and its possible role as an androgen receptor (AR) co-activator. However, there are limited studies on the role of DBC1 in osteosarcoma. Therefore, we investigated the role of DBC1 and AR and their relationship in osteosarcoma. Immunohistochemical expression of DBC1 and AR was significantly associated with higher clinical stage and higher histologic grade, and predicted shorter survival. Especially, DBC1 expression was an independent prognostic indicator of overall survival ( Osteosarcoma is the most common primary malignant bone tumor3DBC1/CCAR2) gene was named based on its deletion in breast cancer68910111213141515171819121421910Deleted in breast cancer 1 . The immunostaining for AR was considered positive when the IHC score was equal or greater than 11  (p = 0.028), higher tumor stage (p = 0.035), and higher histologic grade (p = 0.015). AR-positivity was significantly associated with higher tumor stage (p = 0.005) and higher histologic grade (p = 0.002). In addition, there was a significant association between DBC1-positivity and AR-positivity (p = 0.001). When we evaluated the correlation between the IHC scores of DBC1 expression and AR expression, there was a significant correlation .In human osteosarcoma tissues, the expression of DBC1 was mainly localized in the nuclei of tumor cells and AR was expressed both in the nuclei and cytoplasm . The cut2–0.938) . In ostep = 0.005) greater risk of death and a 6.657-fold  greater risk of relapse or death compared with the DBC1-negative group. The expression of AR also predicted a 5.551-fold  greater risk of death and a 4.201-fold  greater risk of relapse or death. Multivariate analysis was performed with the inclusion of the factors significantly associated with OS or RFS by univariate analysis. Multivariate analysis revealed DBC1-positivity as an independent prognostic indicator of OS and RFS. The expression of DBC1 predicted an 8.639-fold  greater risk of death and a 6.555-fold  greater risk of relapse or death. The age of the patients was a possible prognostic indicator of RFS (p = 0.057) by multivariate analysis (p = 0.007) and RFS (p = 0.025)  and relapse-free survival (RFS) . The ostanalysis  Model 1.= 0.025)  Model 2.Because clinical data suggests that the expression of DBC1 and AR may be involved in the progression of osteosarcomas despite the limited number of cases, we evaluated the effects of a knock-down of DBC1 and AR in osteosarcoma cells. The knock-down of DBC1 and AR with two sets of siRNA significantly inhibited the proliferation of both U2OS (wild-type P53) and SaOS2 (P53-null) osteosarcoma cells demonstrated by MTT and colony forming assays . In addiBecause the expression of DBC1 and AR were associated with the proliferation and invasion activity of osteosarcoma cells, we evaluated the expression of DBC1, AR, and the molecules related with the proliferation and invasiveness of cells . As prevThere was significant correlation between the expression of DBC1 and AR protein levels as shown in immunohistochemical staining in human osteosarcoma tissue samples and Western blotting in osteosarcoma cells . HoweverIn this study, the expression of DBC1 was significantly associated with advanced clinicopathologic factors of osteosarcoma such as larger tumor size, higher tumor stage, and higher histologic grade. Moreover, DBC1 expression in osteosarcoma was an independent prognostic indicator of OS despite the limited number of cases of osteosarcoma patients. In line with these results, DBC1 expression was significantly associated with higher tumor stage, higher histologic grade, and distant metastasis in soft-tissue sarcomas126101112cell cycle activator and apoptosis regulator 2)14151618In addition to the role of DBC1 as a negative regulator of SIRT16One of the interesting findings from this study and a possible explanation of the tumorigenic role of DBC1 is that DBC1 is involved in the regulation of hormone receptors, especially for the regulation of ARin vitro that DBC1 is important in the post-translational stabilization of AR. Moreover, the inhibition of the proliferation and invasion activity of osteosarcoma cells with the knock-down of DBC1 or AR suggested that the DBC1-AR pathway could be usable as a new therapeutic target in the treatment of osteosarcoma patients. However, due to conflicting reports for the oncogenic role of DBC1 further study is needed.In conclusion, this study demonstrated that the expression of DBC1 and AR could be usable as prognostic indicators of osteosarcoma. However, the limitation of this study is that we evaluated only 35 cases of osteosarcomas due to its rarity; however despite the relatively small sample size, we observed statistically significant differences in these cases of osteosarcoma. Therefore, additional study with a large number of cases is needed to confirm the clinical significance of the expression of DBC1 and AR in osteosarcoma patients. However, this study found 40 cases of primary osteosarcoma patients who underwent surgical resection for the primary lesion between January 1998 and December 2012 were evaluated in this study. Among them, two cases of extra-skeletal osteosarcoma were excluded in this study, and the original histologic slides or tissue blocks were not available in three cases of osteosarcoma of the bone. Finally, we evaluated 35 cases of primary osteosarcoma of the bone, which were reviewed according to the 2013 World Health Organization classification of tumors of soft tissue and boneTo establish a tissue microarray (TMA), we reviewed original H&E slides and took two 3.0 mm cores from each case at the most intact solid areas, mainly composed of tumor cells with the highest histologic grade and no necrosis or degenerative change. The 4 μm thick sections of TMA blocks were incubated with primary antibodies for DBC1  and AR  after microwave antigen retrieval. Immunohistochemical scoring was performed with investigators blinded to the clinicopathological information. Two pathologists (KYJ and KMK) evaluated immunostaining slides for the nuclear staining of DBC1 or AR with consensus under a multi-viewing microscope . The nuclear staining for DBC1 and AR were evaluated by the sum of the staining intensity score  and staining area score 10112.The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank  and cultured in DMEM medium supplemented with 10% fetal bovine serum , penicillin and streptomycin (100 U/ml), and fungizone  at 37 °C in humidified incubator with 5% CO5) and SaOS2 (4 × 105) cells were seeded in 60 mm culture dish in DMEM medium without antibiotic to until they reached 60% confluence and then treated with an siRNA duplex for DBC1 and AR or a plasmid for wild-type DBC1 with Lipofectamine® RNAimax  optimized for a 60 mm culture dish following the manufacturer’s protocol.The short interfering RNA (siRNA) for DBC1 and AR and negative control siRNA duplexes were purchased from Bioneer Corporation . The first DBC1 duplex (siDBC1 #1) had the sense and antisense sequences 5′-GUGAUGACUAUGACUCCAA-3′ and 5′-UUGGAGUCAUAGUUCAUCAC-3′, respectively. The second DBC1 duplex (siDBC1 #2) had the sense and antisense sequences 5′-GUGUUCUUUGAUGCCAACU-3′ and 5′-AGUUGGCAUCAAAGAACAC-3′, respectively, and the negative control duplex specific had the sense and antisense sequences 5′-CUCAUGGACGGUUGGAUCA-3′ and 5′-UGAUCCAACCGUCCAUGAG-3′, respectively. The first AR duplex (siAR #1) had the sense and antisense sequences 5′-CACAAGUCCCGGAUGUACA-3′ and 5′-UGUACAUCCGGGACUUGUG -3′, respectively. The second AR duplex (siAR #2) had the sense and antisense sequences 5′-CUGAUCUGGUUUUCAAUGA-3′ and 5′-UCAUUGAAAACCAGAUCAG-3′, and the negative control duplex specific had the sense and antisense sequences 5′-CCUACGCCACCAAUUUCGU-3′ and 5′-ACGAAAUUGGUGGCGUAGG-3′, respectively. The plasmid for wild-type DBC1 was kindly provided by L Zannini 3 U2OS cells and 2 × 103 SaOS2 cells were seeded in 96-well plates and the absorbance at 560 nm was measured. Colony forming assays were performed by seeding U2OS (1 × 103 cells/well) and SaOS2 (2 × 103 cells/well) cells in 24 well plates for seven days.The proliferation of osteosarcoma cell lines was evaluated by an MTT  assay and a colony forming assay. For the MTT assay, 1 × 104) and SaOS2 (1 × 105) cells were seeded in the migration chamber in which the bottom chamber consisted of 1% FBS. For the invasion assay, the same numbers of cells were seeded in the upper chamber containing an 8 μm-pore sized Matrigel Invasion Chamber . DMEM with 10% FBS was added in the lower chamber in the invasion assay. The migration or invasion chambers were incubated at 37 °C for 24 hours. Thereafter, the lower surface of the filter was stained with DIFF-Quik staining solutions , and the number of cells which had migrated or invaded the filter were counted in five microscopic fields (magnification x100) per well.The migration assay was performed with 24-transwell migration chambers with 8 μm-pore sized filters . U2OS . Thereafter, cell cycle analysis was performed with a FACStar flow cytometer  and analyzed by CellQuest Pro software .The cells were lysed with PRO-PREP Protein Extraction Solution  and the following antibodies were used for western blot analysis: DBC1 , AR , P53 , acetylated-P53 , P21 , P27 , BAX , BCL-2 , TGFβ , NFκB , PCNA , Rho A , Ubiquitin , PARP1 , and actin . Western blot images quantified with ImageJ software. For IP, polyclonal anti-DBC1 or anti-AR antibodies were cross-linked to Dynabeads-protein A . DBC1 or AR antibodies were immobilized with Dynabeads-protein A for 20 min and then incubated with cell lysates for 1 h at 4 °C. DBC1- or AR-IP complexes were washed three times and the proteins were eluted. Thereafter, we performed western blotting.® Green Realtime PCR Master Mix . All the experiments were performed in triplicate, and the results were normalized to GAPDH expression.Total RNA was extracted from cells using an RNeasy Mini Kit . The cDNA was synthesized by using Random 9-mer primers provided with the cDNA synthesis kit . Specific primers for DBC1, AR, and GAPDH were designed and checked against the Genbank database (NCBI). The primer sequences were as follows: DBC1/CCAR2; 5′-AAGGGAGACGCCAGAGCAT-3′ and 5′-CATCCAGGGAAGGAGACCAT-3′, AR; 5′-CTGGACACGACAACAACCAG-3′ and 5′-CAGATCAGGGGCGAAGTAGA-3′, and GAPDH; 5′-AACAGCGACACCCACTCCTC-3′ and 5′-GGAGGGGAGATTCAGTGTGG-3′. Quantitative reverse-transcrition polymerase chain reaction was performed in 96 well plates using the Applied Biosystems Prism 7500 Real Time PCR System and SYBR® 488 anti-mouse IgG or Alexa Fluor® 594 anti-rabbit IgG . The slides were counterstained with DAPI and observed under a Zeiss LSM 510 confocal microscope .To evaluate the localization of DBC1 and AR in osteosarcoma cells, immunofluorescence staining for DBC1 and AR was performed in U2OS and SaOS2 cells. The cells were fixed with methanol and incubated with antibodies against DBC1  and AR  antibody and then incubated with Alexa FluorThe U2OS cells were transfected with DBC1 siRNA as described above. 48 hours after transfection, the cells were treated with 20 μg/ml cycloheximide  or 20 μM MG132  for 0.5 to 8 hours. The cell lysates were blotted with anti-AR antibody  and anti-actin antibodies. In addition, U2OS cells transfected with control-siRNA or DBC1 siRNA for 24 hours were treated with MG-132 (20 μM) for 4 hours and total lysates of cells were immnunoprecipitated with anti-Androgen receptor antibodies and blotted with anti-Ubiquitin antibodies.t-test. IBM SPSS software (version 20.0) was used in statistical analysis. The p values less than 0.05 were considered statistically significant.The immunohistochemical staining for DBC1 and AR were grouped as negative or positive with a receiver operating characteristic curve analysis. The end points of interest were OS and RFS. The endpoint of follow-up was the date of last contact or the death of a patient through February 2013. OS was calculated as the period from diagnosis till death from osteosarcoma or last contact. Patients alive at the last contact were censored for OS. RFS was calculated as the time from diagnosis to the date of recurrence, death from osteosarcoma, or last contact of the patient. Those patients who were alive at last contact and had not recurred were censored for RFS. Survival analysis was performed by univariate and multivariate Cox proportional hazards regression analysis and Kaplan-Meier survival analysis with log-rank test. The comparison or relationships between the factors interested in this study were performed by Pearson’s chi-square test or student’s How to cite this article: Wagle, S. et al. DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma. Sci. Rep.5, 13144; doi: 10.1038/srep13144 (2015).