The endocrine disruptor DEHP and the ECS: analysis of a possible crosstalk.

Studies of the last decade associated the environmental contamination by di-(2-ethylhexyl)-phthalate (DEHP) with obesity and endocrine malfunction. DEHP was found to interact with several receptors - among them are receptors of the endocannabinoid system (ECS) with high expression levels in adipose tissue. Furthermore, the correlation for BMI and body fat to the serum endocannabinoid level raises the question if the obesogenic and endocrine-disrupting DEHP effects are mediated via the ECS. We therefore characterized the ECS in a human cell model of adipogenesis using the SGBS preadipocytes to subsequently investigate if DEHP exposure affects the intrinsic ECS. The receptors of the ECS and the endocannabinoid-metabolizing enzymes were upregulated during normal adipogenesis, accompanied by an increasing secretion of the adipokines adiponectin and leptin. DEHP affected the secretion of both adipokines but not the ECS, suggesting DEHP to alter the endocrine function of adipocytes without the involvement of the intrinsic ECS.

Protective Effect of N-Arachidonoyl Glycine-GPR18 Signaling after Excitotoxical Lesion in Murine Organotypic Hippocampal Slice Cultures.

N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)2 receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.

On the influence of cannabinoids on cell morphology and motility of glioblastoma cells.

The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways.

Synthetic Cannabinoids Influence the Invasion of Glioblastoma Cell Lines in a Cell- and Receptor-Dependent Manner.

The current treatment of glioblastoma is not sufficient, since they are heterogeneous and often resistant to chemotherapy. Earlier studies demonstrated effects of specific cannabinoid receptor (CB) agonists on the invasiveness of glioblastoma cell lines, but the exact mechanism remained unclear. Three human glioblastoma cell lines were treated with synthetic CB ligands. The effect of cannabinoids on microRNAs (miRs), Akt, and on the expression of proliferation and apoptosis markers were analyzed. Furthermore, in a model of organotypic hippocampal slice cultures cannabinoid mediated changes in the invasiveness were assessed. MicroRNAs and the activation of Akt which are related to cell migration, apoptosis, and proliferation were evaluated and found not to be associated with changes in the invasiveness after treatment with CB ligands. Also proliferation and/or apoptosis were not altered after treatment. The effects of cannabinoids on invasiveness could be blocked by the application of receptor antagonists and are likely mediated via CB1/CB2. In conclusion, our results suggest that cannabinoids can influence glioblastoma cell invasion in a receptor and cell type specific manner that is independent of proliferation and apoptosis. Thus, cannabinoids can potentially be used in the future as an addition to current therapy.

Automatic detection of DNA double strand breaks after irradiation using an γH2AX assay.

Radiation therapy belongs to the most common approaches for cancer therapy leading amongst others to DNA damage like double strand breaks (DSB). DSB can be used as a marker for the effect of radiation on cells. For visualization and assessing the extent of DNA damage the γH2AX foci assay is frequently used. The analysis of the γH2AX foci assay remains complicated as the number of γH2AX foci has to be counted. The quantification is mostly done manually, being time consuming and leading to person-dependent variations. Therefore, we present a method to automatically analyze the number of foci inside nuclei, facilitating and quickening the analysis of DSBs with high reliability in fluorescent images. First nuclei were detected in fluorescent images. Afterwards, the nuclei were analyzed independently from each other with a local thresholding algorithm. This approach allowed accounting for different levels of noise and detection of the foci inside the respective nucleus, using Hough transformation searching for circles. The presented algorithm was able to correctly classify most foci in cases of "high" and "average" image quality (sensitivity>0.8) with a low rate of false positive detections (positive predictive value (PPV)>0.98). In cases of "low" image quality the approach had a decreased sensitivity (0.7-0.9), depending on the manual control counter. The PPV remained high (PPV>0.91). Compared to other automatic approaches the presented algorithm had a higher sensitivity and PPV. The used automatic foci detection algorithm was capable of detecting foci with high sensitivity and PPV. Thus it can be used for automatic analysis of images of varying quality.

N-Arachidonoyl Dopamine: A Novel Endocannabinoid and Endovanilloid with Widespread Physiological and Pharmacological Activities.

N-arachidonoyl dopamine (NADA) is a member of the family of endocannabinoids to which several other N-acyldopamines belong as well. Their activity is mediated through various targets that include cannabinoid receptors or transient receptor potential vanilloid (TRPV)1. Synthesis and degradation of NADA are not yet fully understood. Nonetheless, there is evidence that NADA plays an important role in nociception and inflammation in the central and peripheral nervous system. The TRPV1 receptor, for which NADA is a potent agonist, was shown to be an endogenous transducer of noxious heat. Moreover, it has been demonstrated that NADA exerts protective and antioxidative properties in microglial cell cultures, cortical neurons, and organotypical hippocampal slice cultures. NADA is present in very low concentrations in the brain and is seemingly not involved in activation of the classical pathways. We believe that treatment with exogenous NADA during and after injury might be beneficial. This review summarizes the recent findings on biochemical properties of NADA and other N-acyldopamines and their role in physiological and pathological processes. These findings provide strong evidence that NADA is an effective agent to manage neuroinflammatory diseases or pain and can be useful in designing novel therapeutic strategies.

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

The Impact of Non-Lethal Single-Dose Radiation on Tumor Invasion and Cytoskeletal Properties.

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a "generalized stiffness" and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, "generalized stiffness" was shown to be a profound marker of the invasiveness of a tumor cell population in our model.

The influence of biomechanical properties and cannabinoids on tumor invasion.

BACKGROUND: Cannabinoids are known to have an anti-tumorous effect, but the underlying mechanisms are only sparsely understood. Mechanical characteristics of tumor cells represent a promising marker to distinguish between tumor cells and the healthy tissue. We tested the hypothesis whether cannabinoids influence the tumor cell specific mechanical and migratory properties and if these factors are a prognostic marker for the invasiveness of tumor cells. METHODS: 3 different glioblastoma cell lines were treated with cannabinoids and changes of mechanical and migratory properties of single cells were measured using atomic force microscopy and time lapse imaging. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. RESULTS: We found that cannabinoids are capable of influencing migratory and mechanical properties in a cell line specific manner. A network analysis revealed a correlation between a "generalized stiffness" and the invasiveness for all tumor cell lines after 3 and 4 d of invasion time: r3d = -0.88 [-0.52;-0.97]; r4d = -0.90 [-0.59;-0.98]. CONCLUSIONS: Here we could show that a "generalized stiffness" is a profound marker for the invasiveness of a tumor cell population in our model and thus might be of high clinical relevance for drug testing. Additionally cannabinoids were shown to be of potential use for therapeutic approaches of glioblastoma.

Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells.

UNLABELLED: The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2ß1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. STATEMENT OF SIGNIFICANCE: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.

The endocannabinoid system in the human granulosa cell line KGN.

Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis.

Expression and functional relevance of cannabinoid receptor 1 in Hodgkin lymphoma.

BACKGROUND: Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date. DESIGN AND METHODS: We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated. RESULTS: A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells. CONCLUSIONS: The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Impact of MACC1 on human malignant glioma progression and patients' unfavorable prognosis.

BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) has been established as an independent prognostic indicator of metastasis formation and metastasis-free survival for patients with colon cancer and other solid tumors. However, no data are available concerning MACC1 expression in human astrocytic tumors. Glioblastoma multiforme (GBM) is the most prevalent primary brain tumor of adulthood, and due to its invasive and rapid growth, patients have unfavorable prognoses. Although these tumors rarely metastasize, their invasive and migratory behavior is similar to those of metastatic cells of tumors of different origin. Thus, we hypothesized that MACC1 may be involved in progression of human gliomas. METHODS: We performed real-time measurements of proliferation and migration in MACC1-transfected GBM cell lines (U138, U251) and evaluated tumor formation in organotypic hippocampal slice cultures of mice. Semiquantitative and quantitative real-time reverse transcription PCR analyses were performed for MACC1 and for its transcriptional target c-Met in human astrocytoma of World Health Organization grade II (low-grade astrocytoma) and GBM biopsies. Data were validated by MACC1 immunohistochemistry in independent matched samples of low-grade astrocytoma and GBM. RESULTS: MACC1 increases the proliferative, migratory, and tumor-formation abilities of GBM cells. The c-Met inhibitor crizotinib reduced MACC1-induced migration and tumor formation in organotypic hippocampal slice cultures of mice. Analyzing patients' biopsies, MACC1 expression increased concomitantly with increasing World Health Organization grade. Moreover, MACC1 expression levels allowed discrimination of dormant and recurrent low-grade astrocytomas and of primary and secondary GBM. Strong MACC1 expression correlated with reduced patient survival. CONCLUSIONS: MACC1 may represent a promising biomarker for prognostication and a new target for treatment of human gliomas.

The G protein-coupled receptor 55 ligand l-α-lysophosphatidylinositol exerts microglia-dependent neuroprotection after excitotoxic lesion.

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein-coupled cannabinoid receptor 1 (CB1 ) and microglial G protein-coupled cannabinoid receptor 2 (CB2 ). However, pharmacological experiments with CB1 /CB2 -deficient mice unraveled the existence of further, so-called non-CB1 /non-CB2 G protein-coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l-α-Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI-mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca(2+) and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI-mediated modulation of microglia function.

The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB(1)).

Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N-methyl-d-aspartate (NMDA, 50 µM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM-50 µM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a slight reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 µM NADA (p < 0.05). To identify the responsive receptor type of NADA mediated neuroprotection, we applied the cannabinoid (CB) receptor 1 (CB(1)) inverse agonist/antagonist AM251, CB(2) inverse agonist/antagonist AM630, abnormal-cannabidiol (abn-CBD)-sensitive receptor antagonist O-1918, transient receptor potential channel V1 (TRPV1) antagonist 6-iodonordihydrocapsaicin and A1 (TRPA1) antagonist HC-030031. Neuroprotective properties of low (1 nM) but not high (10 µM) NADA concentrations were solely blocked by AM251 and were absent in CB(1)(-/-) mice. AM630, O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all NADA concentrations applied. Our findings demonstrate that NADA protects dentate gyrus granule cells by acting via CB(1). NADA reduced the number of microglial cells at distinct concentrations. TRPV1 and TRPA1 were not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA mediated activation of neuronal CB(1) may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.

The cannabinoid WIN 55,212-2-mediated protection of dentate gyrus granule cells is driven by CB1 receptors and modulated by TRPA1 and Cav 2.2 channels.

Cannabinoids regulate numerous physiological and pathological events like inflammation or neurodegeneration via CB(1) and CB(2) receptors. The mechanisms behind cannabinoid effects show a high variability and may also involve transient receptor potential channels (TRP) and N-type voltage-gated Ca(2+) channels (Ca(v) 2.2). In the present study we investigated the neuroprotective effects of the synthetic cannabinoid WIN 55,212-2 (WIN) on dentate gyrus (DG) granule cells and elucidated the involvement of TRP and Ca(v) 2.2 that are shown to participate in inflammatory processes. Organotypic hippocampal slice cultures were excitotoxically lesioned using NMDA and subsequently incubated with different WIN concentrations (0.001-10 µM). WIN showed neuroprotective properties in an inverse concentration-dependent manner, most effectively at 0.01 µM. The CB(1) receptor antagonist AM251 blocked neuroprotection mediated by WIN whereas the CB(2) receptor antagonist AM630 showed no effects. Application of the TRPA1 blocker HC-030031 enhanced the neuroprotective efficacy of high (10 µM) WIN concentrations and the number of degenerating neurons became equal to that seen after application of the most effective WIN dose (0.01 µM). In contrast, the application of TRPA1 agonist icilin or allyl isothiocyanate (AITC) led to a stronger neurodegeneration. The use of TRPV1 blocker 6-iodo-nordihydrocapsaicin did not affect WIN-mediated neuroprotection. The selective Ca(v) 2.2 blocker ω-conotoxin (GVIA) completely blocked neuroprotection shown by 10 µM WIN. GVIA and HC-030031 exerted no effects at WIN concentrations lower than 10 µM. Our data show that WIN protects dentate gyrus granule cells in a concentration dependent manner by acting upon CB(1) receptors. At high (10 µM) concentrations WIN additionally activates TRPA1 and Ca(v) 2.2 within the hippocampal formation that both interfere with CB(1) receptor-mediated neuroprotection. This leads to the conclusion that physiological and pharmacological effects of cannabinoids strongly depend on their concentration and the neuroprotective efficacy of cannabinoids may be determined by interaction of activated CB(1) receptor, TRPA1, and Ca(v) 2.2.