Receptor-independent Mechanism Research Articles

A trial design to maximize knowledge of the effects of rodatristat ethyl in the treatment of pulmonary arterial hypertension (ELEVATE 2).

Serotonin plays a key role in the development and maintenance of the pathobiology associated with pulmonary arterial hypertension (PAH). Platelet‐driven and locally produced serotonin from lung tissue and arterial endothelial cells induce excessive growth of pulmonary artery smooth muscle cells. The unchecked growth of these cells is a major driver of PAH including the remodeling of pulmonary arteries that dramatically reduces the diameter and flexibility of the arterial lumen. Tryptophan hydroxylase 1 (TPH1) is the rate‐limiting enzyme for biosynthesis of serotonin and is upregulated in PAH arterial endothelial cells, supporting TPH1 inhibition to treat PAH. Targeting the serotonin pathway via inhibition of peripheral serotonin and local production in diseased tissues, rather than individual receptor‐mediated or receptor‐independent mechanisms, may result in the ability to halt or reverse pulmonary vascular remodeling. Rodatristat ethyl, a prodrug for rodatristat, a potent, peripheral inhibitor of TPH1, has demonstrated efficacy in monocrotaline and SUGEN hypoxia nonclinical models of PAH and robust dose‐dependent reductions of 5‐hydroxyindoleacetic acid, the major metabolite of serotonin in plasma and urine of healthy human subjects. ELEVATE 2 (NCT04712669) is a Phase 2b, double‐blind, multicenter trial where patients with PAH are randomized to placebo, 300 or 600 mg twice daily of rodatristat ethyl. The trial incorporates endpoints to generate essential clinical efficacy, safety, pharmacokinetic, and pharmacodynamic data needed to evaluate the ability of rodatristat ethyl to ameliorate PAH by halting or reversing pulmonary vascular remodeling through its unique mechanism of TPH1 inhibition. Herein we describe the experimental design highlighting the trial's unique features.

PACAP-38 Induces Transcriptomic Changes in Rat Trigeminal Ganglion Cells Related to Neuroinflammation and Altered Mitochondrial Function Presumably via PAC1/VPAC2 Receptor-Independent Mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a broadly expressed neuropeptide which has diverse effects in both the peripheral and central nervous systems. While its neuroprotective effects have been shown in a variety of disease models, both animal and human data support the role of PACAP in migraine generation. Both PACAP and its truncated derivative PACAP(6-38) increased calcium influx in rat trigeminal ganglia (TG) primary sensory neurons in most experimental settings. PACAP(6-38), however, has been described as an antagonist for PACAP type I (known as PAC1), and Vasoactive Intestinal Polypeptide Receptor 2 (also known as VPAC2) receptors. Here, we aimed to compare the signaling pathways induced by the two peptides using transcriptomic analysis. Rat trigeminal ganglion cell cultures were incubated with 1 µM PACAP-38 or PACAP(6-38). Six hours later RNA was isolated, next-generation RNA sequencing was performed and transcriptomic changes were analyzed to identify differentially expressed genes. Functional analysis was performed for gene annotation using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. We found 200 common differentially expressed (DE) genes for these two neuropeptides. Both PACAP-38 and PACAP(6-38) treatments caused significant downregulation of NADH: ubiquinone oxidoreductase subunit B6 and upregulation of transient receptor potential cation channel, subfamily M, member 8. The common signaling pathways induced by both peptides indicate that they act on the same target, suggesting that PACAP activates trigeminal primary sensory neurons via a mechanism independent of the identified and cloned PAC1/VPAC2 receptor, either via another target structure or a different splice variant of PAC1/VPAC2 receptors. Identification of the target could help to understand key mechanisms of migraine.

Dynamic alteration of poroelastic attributes as determinant membrane nanorheology for endocytosis of organ specific targeted gold nanoparticles

BackgroundEfficacy of targeted drug delivery using nanoparticles relies on several factors including the uptake mechanisms such as phagocytosis, macropinocytosis, micropinocytosis and receptor mediated endocytosis. These mechanisms have been studied with respect to the alteration in signaling mechanisms, cellular morphology, and linear nanomechanical properties (NMPs). Commonly employed classical contact mechanics models to address cellular NMPs fail to address mesh like structure consisting of bilayer lipids and proteins of cell membrane. To overcome this technical challenge, we employed poroelastic model which accounts for the biphasic nature of cells including their porous behavior exhibiting both solid like (fluid storage) and liquid like (fluid dissipate) behavior.ResultsIn this study, we employed atomic force microscopy to monitor the influence of surface engineering of gold nanoparticles (GNPs) to the alteration of nonlinear NMPs such as drained Poisson’s ratio, effective shear stress, diffusion constant and pore dimensions of cell membranes during their uptake. Herein, we used pancreatic cancer (PDAC) cell lines including Panc1, AsPC-1 and endothelial cell (HUVECs) to understand the receptor-dependent and -independent endocytosis of two different GNPs derived using plectin-1 targeting peptide (PTP-GNP) and corresponding scrambled peptide (sPEP-GNP). Compared to untreated cells, in case of receptor dependent endocytosis of PTP-GNPs diffusion coefficient altered ~ 1264-fold and ~ 1530-fold and pore size altered ~ 320-fold and ~ 260-fold in Panc1 and AsPC-1 cells, respectively. Whereas for receptor independent mechanisms, we observed modest alteration in diffusion coefficient and pore size, in these cells compared to untreated cells. Effective shear stress corresponding to 7.38 ± 0.15 kPa and 20.49 ± 0.39 kPa in PTP-GNP treatment in Panc1 and AsPC-1, respectively was significantly more than that for sPEP-GNP. These results demonstrate that with temporal recruitment of plectin-1 during receptor mediated endocytosis affects the poroelastic attributes of the membrane.ConclusionThis study confirms that nonlinear NMPs of cell membrane are directly associated with the uptake mechanism of nanoparticles and can provide promising insights of the nature of endocytosis mechanism involved for organ specific drug delivery using nanoparticles. Hence, nanomechanical analysis of cell membrane using this noninvasive, label-free and live-cell analytical tool can therefore be instrumental to evaluate therapeutic benefit of nanoformulations.Graphical

Monitoring Modified Lipoprotein Uptake and Macropinocytosis Associated with Macrophage Foam Cell Formation.

Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosclerosis. Macrophages uptake native and modified low density lipoprotein (LDL) through either receptor-dependent or receptor-independent mechanisms to transform into lipid laden foam cells. Foam cells are involved in the formation of fatty streak that is seen during the early stages of atherosclerosis development and therefore represents a promising therapeutic target. Normal or modified lipoproteins labeled with fluorescent dyes such as 1,1'-dioctadecyl-3-3-3',3'-tetramethylindocarbocyanine perchlorate (Dil) are often used to monitor their internalization during foam cell formation. In addition, the fluorescent dye Lucifer Yellow (LY) is widely used as a marker for macropinocytosis activity. In this chapter, we describe established methods for monitoring modified lipoprotein uptake and macropinocytosis during macrophage foam cell formation.

Preparations from Campomanesia reitziana reduce the gastrointestinal motility and castor oil-induced diarrhea in a non-opioid and non-dopaminergic pathway in mice and display antimicrobial activity in vitro.

This study investigated the antidiarrheal potential of the aqueous extract (AECR) and hydroalcoholic extract ofCampomanesia reitzianaleaves (HECR), its ethyl acetate (EAF) and dichloromethane fractions (DCMF), and myricitrin isolated from EAF. The total phenols and flavonoids were measured, followed by chromatography and myricitrin isolation. The 2,2-diphenyl-1-picryl-hydrazyl scavenger activity, the cytotoxicity, and the effects on LPS-induced nitrite production in intestinal epithelial cells (IEC-6) were quantified. The effect of HECR, EAF, DCMF, and AECR on intestinal motility (IT), gastric emptying (GE), and castor oil-induced diarrhea in mice was determined, as well as its antimicrobial activity. The administration of AECR 10% (10ml/kg, p.o), but not HECR (300mg/kg), reduced the GE and IT by 52 and 51%. The EAF and DCMF at 300mg/kg also reduced IT but did not change GE. Moreover, AECR and EAF, but not DCMF, inhibited the castor oil-induced diarrhea and naloxone or metoclopramide pretreatment did not change these effects. Myricitrin did not change IT and the evacuation index of mice. Finally, the dry residue of AECR inhibited bacterial growth and EAF showed bacteriostatic activity againstS.aureus,E.coli,andS.typhimuriumand antifungal forC.albicans. However, none of the preparations alter the viability ofGiardia spp. trophozoites. The AECR and EAF can be effective to treat diarrhea acting through opioid- or dopaminergic type 2 receptor-independent mechanisms and by its antimicrobial actions.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface β2AR (β2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces β2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases β-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Activity of Mitragyna speciosa ("Kratom") Alkaloids at Serotonin Receptors.

Kratom alkaloids have mostly been evaluated for their opioid activity but less at other targets that could contribute to their physiological effects. Here, we investigated the in vitro and in vivo activity of kratom alkaloids at serotonin receptors (5-HTRs). Paynantheine and speciogynine exhibited high affinity for 5-HT1ARs and 5-HT2BRs, unlike the principal kratom alkaloid mitragynine. Both alkaloids produced antinociceptive properties in rats via an opioid receptor-independent mechanism, and neither activated 5-HT2BRs in vitro. Paynantheine, speciogynine, and mitragynine induced lower lip retraction and antinociception in rats, effects blocked by a selective 5-HT1AR antagonist. In vitro functional assays revealed that the in vivo 5-HT1AR agonistic effects may be due to the metabolites 9-O-desmethylspeciogynine and 9-O-desmethylpaynantheine and not the parent compounds. Both metabolites did not activate 5-HT2BR, suggesting low inherent risk of causing valvulopathy. The 5-HT1AR agonism by kratom alkaloids may contribute to the mood-enhancing effects associated with kratom use.

Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

Optimizing Active Tumor Targeting Biocompatible Polymers for Efficient Systemic Delivery of Adenovirus.

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA–FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.

Chronic cerebral lipocalin 2 exposure elicits hippocampal neuronal dysfunction and cognitive impairment

Lipocalin 2 (LCN2) is a pleiotropic molecule that is induced in the central nervous system (CNS) in several acute and chronic pathologies. The acute induction of LCN2 evolved as a beneficial process, aimed at combating bacterial infection through the sequestration of iron from pathogens, while the role of LCN2 during chronic, non-infectious disease remains unclear, and recent studies suggest that LCN2 is neurotoxic. However, whether LCN2 is sufficient to induce behavioral and cognitive alterations remains unclear. In this paper, we sought to address the role of cerebral LCN2 on cognition in both acute and chronic settings. We demonstrate that LCN2 is robustly induced in the CNS during both acute and chronic inflammatory conditions, including LPS-based sepsis and cancer cachexia. In vivo, LPS challenge results in a global induction of LCN2 in the central nervous system, while cancer cachexia results in a distribution specific to the vasculature. Similar to these in vivo observations, in vitro modeling demonstrated that both glia and cerebral endothelium produce and secrete LCN2 when challenged with LPS, while only cerebral endothelium secrete LCN2 when challenged with cancer-conditioned medium. Chronic, but not short-term, cerebral LCN2 exposure resulted in reduced hippocampal neuron staining intensity, an increase in newborn neurons, microglial activation, and increased CNS immune cell infiltration, while gene set analyses suggested these effects were mediated through melanocortin-4 receptor independent mechanisms. RNA sequencing analyses of primary hippocampal neurons revealed a distinct transcriptome associated with prolonged LCN2 exposure, and ontology analysis was suggestive of altered neurite growth and abnormal spatial learning. Indeed, LCN2-treated hippocampal neurons display blunted neurite processes, and mice exposed to prolonged cerebral LCN2 levels experienced a reduction in spatial reference memory as indicated by Y-maze assessment. These findings implicate LCN2 as a pathologic mediator of cognitive decline in the setting of chronic disease.

Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-Low Induction of Therapy Resistance.

Recent evidence has highlighted the role of N 6-methyladenosine (m6A) in the regulation of mRNA expression, stability, and translation, supporting a potential role for posttranscriptional regulation mediated by m6A in cancer. Here, we explore prostate cancer as an exemplar and demonstrate that low levels of N 6-adenosine-methyltransferase (METTL3) is associated with advanced metastatic disease. To investigate this relationship, we generated the first prostate m6A maps, and further examined how METTL3 regulates expression at the level of transcription, translation, and protein. Significantly, transcripts encoding extracellular matrix proteins are consistently upregulated with METTL3 knockdown. We also examined the relationship between METTL3 and androgen signaling and discovered the upregulation of a hepatocyte nuclear factor-driven gene signature that is associated with therapy resistance in prostate cancer. Significantly, METTL3 knockdown rendered the cells resistant to androgen receptor antagonists via an androgen receptor-independent mechanism driven by the upregulation of nuclear receptor NR5A2/LRH-1. IMPLICATIONS: These findings implicate changes in m6A as a mechanism for therapy resistance in metastatic prostate cancer.

Adenosine Inhibits Cell Proliferation Differently in Human Astrocytes and in Glioblastoma Cell Lines

Glioblastoma (GBM) is the most common brain primary tumour. Hypoxic regions in GBM are associated to tumour growth. Adenosine accumulates in hypoxic regions and can affect cell proliferation and survival. However, how proliferating GBM cells respond/adapt to increased adenosine levels compared to human astrocytes (HA) is not clarified and was addressed in the present work. GBM cell lines and HA were treated for 3 days with test drugs. Thirty Adenosine (30 µM) caused a 43% ± 5% (P < 0.05) reduction of cell proliferation/viability in HA, through an adenosine receptor-independent mechanism, but had no effect in GBM cell lines U87MG, U373MG and SNB19. Contrastingly, inhibition of adenosine phosphorylation (using the adenosine kinase (ADK) inhibitor 5-iodotubercidin (ITU) (25 µM)), produced a strong and similar decrease on cell proliferation in both HA and GBM cells. The effect of adenosine on HA proliferation/viability was potentiated by 100 µM-homocysteine. Combined application of 30 µM-adenosine and 100 µM-homocysteine reduced the cell proliferation/viability in all three GBM cell lines, but this reduction was much lower than that observed in HA. Adenosine alone did not induce cell death, assessed by lactate dehydrogenase (LDH) release, both in HA and GBM cells, but potentiated the cytotoxic effect of homocysteine in HA and in U87MG and U373MG cells. Results show a strong attenuation of adenosine anti-proliferative effect in GBM cells compared to HA, probably resulting from increased adenosine elimination by ADK, suggesting a proliferative-prone adaptation of tumour cells to increased adenosine levels.

The emerging role of FTY720 as a sphingosine 1-phosphate analog for the treatment of ischemic stroke: The cellular and molecular mechanisms.

Finding novel and effective drugs for the treatment of ischemic stroke is warranted because there is not a definitive treatment for this prevalent disease. Due to the relevance between the sphingosine 1‐phosphate (S1P) receptor and several neurological diseases including ischemic stroke, it seems that fingolimod (FTY720), as an agonist of S1P receptor, can be a useful therapeutic strategy in these patients. FTY720 is the first oral drug approved by the US food and drug administration for the treatment of multiple sclerosis. Three important mechanisms for neuroprotective effects of FTY720 have been described. First, the functional antagonistic mechanism that is associated with lymphopenia and reduced lymphocytic inflammation. This effect results from the down‐regulation and degradation of lymphocytes' S1P receptors, which inhibits lymph node lymphocytes from entering the bloodstream. Second, a functional agonistic activity that is mediated through direct effects via targeting S1P receptors on the membrane of various cells including neurons, microglia, oligodendrocytes, astrocytes, and endothelial cells of blood vessels in the central nervous system (CNS), and the third, receptor‐independent mechanisms that are displayed by binding to specific cellular proteins that modulate intracellular signaling pathways or affect epigenetic transcriptions. Therefore, we review these mechanisms in more detail and describe the animal model and in clinical trial studies that support these three mechanisms for the neuroprotective action of FTY720 in ischemic stroke.

An opioid receptor-independent mechanism underlies motility dysfunction and visceral hyperalgesia in opioid-induced bowel dysfunction.

Constipation and abdominal pain are commonly encountered in opioid-induced bowel dysfunction (OBD). The underlying mechanisms are incompletely understood, and treatments are not satisfactory. As patients with OBD often have fecal retention, we aimed to determine whether fecal retention plays a pathogenic role in the development of constipation and abdominal pain in OBD, and if so to investigate the mechanisms. A rodent model of OBD was established by daily morphine treatment at 10 mg/kg for 7 days. Bowel movements, colonic muscle contractility, visceromotor response to colorectal distention, and cell excitability of colon-projecting dorsal root ganglion neurons were determined in rats fed with normal pellet food, or with clear liquid diet. Morphine treatment (Mor) reduced fecal outputs starting on day 1, and caused fecal retention afterward. Compared with controls, Mor rats demonstrated suppressed muscle contractility, increased neuronal excitability, and visceral hypersensitivity. Expression of cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) was upregulated in the smooth muscle of the distended colon in Mor rats. However, prevention of fecal retention by feeding rats with clear liquid diet blocked upregulation of COX-2 and NGF, restored muscle contractility, and attenuated visceral hypersensitivity in Mor rats. Moreover, inhibition of COX-2 improved smooth muscle function and fecal outputs, whereas anti-NGF antibody administration attenuated visceral hypersensitivity in Mor rats. Morphine-induced fecal retention is an independent pathogenic factor for motility dysfunction and visceral hypersensitivity in rats with OBD. Liquid diet may have therapeutic potential for OBD by preventing fecal retention-induced mechanotranscription of COX-2 and NGF.NEW & NOTEWORTHY Our preclinical study shows that fecal retention is a pathogenic factor in opioid-induced bowel dysfunction, as prevention of fecal retention with liquid diet improved motility and attenuated visceral hyperalgesia in morphine-treated animals by blocking expression of cyclooxygenase-2 and nerve growth factor in the colon.

Bile acid sequestrants: a review of mechanism and design.

Bile acid sequestrants (BAS) are used extensively in the treatment of hypercholesterolaemia. This brief review aimed to describe the design and evaluation of three types of BAS: amphiphilic copolymers, cyclodextrin/poly-cyclodextrin and molecular imprinted polymers. The mechanisms underlying the action of BAS are also discussed. BAS could lower plasma cholesterol, improve glycemic control in patients with type 2 diabetes and regulate balance energy metabolism via receptors or receptor-independent mediated mechanisms. Different types of BAS have different levels of ability to bind to bile acids, different stability and different in-vivo activity. A growing amount of evidence suggests that bile acids play important roles not only in lipid metabolism but also in glucose metabolism. The higher selectivity, specificity, stability and in-vivo activity of BAS show considerable potential for lipid-lowering therapy.

Developmental Role of Adenosine Kinase in the Cerebellum.

Adenosine acts as a neuromodulator and metabolic regulator of the brain through receptor dependent and independent mechanisms. In the brain, adenosine is tightly controlled through its metabolic enzyme adenosine kinase (ADK), which exists in a cytoplasmic (ADK-S) and nuclear (ADK-L) isoform. We recently discovered that ADK-L contributes to adult hippocampal neurogenesis regulation. Although the cerebellum (CB) is a highly plastic brain area with a delayed developmental trajectory, little is known about the role of ADK. Here, we investigated the developmental profile of ADK expression in C57BL/6 mice CB and assessed its role in developmental and proliferative processes. We found high levels of ADK-L during cerebellar development, which was maintained into adulthood. This pattern contrasts with that of the cerebrum, in which ADK-L expression is gradually downregulated postnatally and largely restricted to astrocytes in adulthood. Supporting a functional role in cell proliferation, we found that the ADK inhibitor 5-iodotubericine (5-ITU) reduced DNA synthesis of granular neuron precursors in a concentration-dependent manner in vitro. In the developing CB, immunohistochemical studies indicated ADK-L is expressed in immature Purkinje cells and granular neuron precursors, whereas in adulthood, ADK is absent from Purkinje cells, but widely expressed in mature granule neurons and their molecular layer (ML) processes. Furthermore, ADK-L is expressed in developing and mature Bergmann glia in the Purkinje cell layer, and in astrocytes in major cerebellar cortical layers. Together, our data demonstrate an association between neuronal ADK expression and developmental processes of the CB, which supports a functional role of ADK-L in the plasticity of the CB.

Oncogenic role of PinX1 in prostate cancer cells through androgen receptor dependent and independent mechanisms

Coregulators play an important role in prostate cancer (PCa), modulating androgen receptor (AR) action and representing a possible cause of androgen deprivation therapy failure. Pin2-interacting protein X1 (PinX1) is a nucleolar protein described as a steroid hormone receptor coregulator in breast cancer cell lines. In this work, we studied the effect of PinX1 on AR action in PCa. Our results demonstrate that PinX1 acts as an AR coactivator, increasing its transcriptional activity and target gene expression, as well as proliferation, migration and colony formation in PCa cell lines. These effects are observed in the presence and absence of AR agonist and antagonists, suggesting a possible androgen independent pathway for PinX1. We present the first oncogenic roles described for PinX1, acting as a coactivator of the AR.

Mechanisms and markers of resistance to androgen signaling inhibitors in patients with metastatic castration-resistant prostate cancer

Next-generation androgen signaling inhibitors such as abiraterone and enzalutamide are widely used for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, baseline and acquired resistance to these treatments is commonly observed. In the last few years, significant effort has been devoted to uncover the molecular mechanisms and predictive markers of resistance. These analyses identified various DNA (single nucleotide variations, amplifications) and RNA variants (e.g., the splice variant AR-V7) of androgen receptor in association with resistance to abiraterone and enzalutamide therapies. Additionally, androgen receptor independent resistance mechanisms were also described. Some of these alterations can be detected in tumor tissues and/or in liquid biopsies of prostate cancer patients and therefore may serve as predictive biomarkers. According to the diversity of potential resistance mechanisms, it appears that a combination of markers representing various resistance mechanisms may provide better performance as single markers. In the present review, we summarize the most important androgen receptor dependent and independent resistance mechanisms and pay attention to methodological details. Recent data has highlighted that some of the resistance mechanisms to next-generation antiandrogen agents are associated with a better response to other therapies, we give an overview on currently ongoing clinical studies evaluating this promising aspect.

Heat Shock Protein 70 Is Associated With Cardioversion Outcome and Recurrence of Symptomatic Recent Onset Atrial Fibrillation in Hypertensive Patients.

Accumulating evidence indicates that heat shock proteins (HSPs) may represent a suitable biomarker to predict atrial fibrillation (AF). We investigated the relation of circulating serum HSP70 (sHSP70) with inflammatory cytokines and recurrence of symptomatic recent onset AF (ROAF). We enrolled 90 patients with ROAF (the duration from onset of symptoms ≤24 hours) and 30 controls. Patients received amiodarone for cardioversion and rhythm control. The association of serum HSP70, serum interleukin-2 (sIL-2), and serum interleukin-4 (sIL-4) with the presence of cardioversion and AF recurrence within a year was investigated. Toll-like receptor 4 (TLR4) signaling dependence for IL-2 and IL-4 induction in response to stimulation with HSP70 was tested in rat aortic vascular smooth muscle cell cultures. Patients had higher sHSP70 and sIL-2 and lower sIL-4 compared with controls. Serum HSP70 was independently associated with ROAF (P = 0.005) and correlated with sIL-2 (r = 0.494, P < 0.001) and sIL-4 (r = -0.550, P < 0.001). By 48 hours, 71 of the 90 patients were cardioverted, with noncardioverted patients having higher sHSP70 and sIL-2 and lower sIL-4, which were the only independent factors associated with cardioversion. AF recurred in 38 of the 71 cardioverted patients in 1 year. A cutoff value of sHSP70 ≥0.65 ng/mL and sIL-2 ≥0.21 pg/mL was the only independent factor associated with AF recurrence (hazard ratio: 3.311, 95% confidence interval: 1.503-7.293, P = 0.003 and hazard ratio: 3.144, 95% confidence interval: 1.341-7.374, P = 0.008, respectively). The exposure of smooth muscle cell to HSP70 in vitro increased the expression of IL-2 (5×) and IL-4 (1.5×) through TLR4-dependent and receptor-independent mechanisms. In conclusion, sHSP70 and sIL-2 might constitute a prognostic tool for determining the cardioversion and recurrence likelihood in ROAF.

Oleamide Induces Cell Death in Glioblastoma RG2 Cells by a Cannabinoid Receptor-Independent Mechanism.

The endocannabinoid system has been associated with antiproliferative effects in several types of tumors through cannabinoid receptor-mediated cell death mechanisms. Oleamide (ODA) is a CB1/CB2 agonist associated with cell growth and migration by adhesion and/or ionic signals associated with Gap junctions. Antiproliferative mechanisms related to ODA remain unknown. In this work, we evaluated the effects of ODA on cell viability and morphological changes in a rat RG2 glioblastoma cell line and compared these effects with primary astrocyte cultures from 8-day postnatal rats. RG2 and primary astrocyte cultures were treated with ODA at increasing concentrations (25, 50, 100, and 200μM) for different periods of time (12, 24, and 48h). Changes in RG2 cell viability and morphology induced by ODA were assessed by viability/mitochondrial activity test and phase contrast microscopy, respectively. The ratios of necrotic and apoptotic cell death, and cell cycle alterations, were evaluated by flow cytometry. The roles of CB1 and CB2 receptors on ODA-induced changes were explored with specific receptor antagonists. ODA (100μM) induced somatic damage, detachment of somatic bodies, cytoplasmicpolarization, and somatic shrinkage in RG2 cells at 24 and 48h. In contrast, primary astrocytes treated at the same ODA concentrations exhibited cell aggregation but not cell damage. ODA (100μM) increased apoptotic cell death and cell arrest in the G1 phase at 24h in the RG2 line. The effects induced by ODA on cell viability of RG2 cells were independent of CB1 and CB2 receptors or changes in intracellular calcium transient. Results of this novel study suggest that ODA exerts specific antiproliferative effects on RG2 glioblastoma cells through unconventional apoptotic mechanisms not involving canonical signals.