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Background: The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA). Methodology/Principal Findings: The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 mu M, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 mu M. Conclusions/Significance: The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer

High Tumour Cannabinoid CB1 Receptor Immunoreactivity Negatively Impacts Disease-Specific Survival in Stage II Microsatellite Stable Colorectal Cancer

BACKGROUND: There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB(1) receptor immunoreactive intensity (CB(1)IR intensity) is associated with disease severity and outcome. METHODOLOGY/PRINCIPAL FINDINGS: CB(1)IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB(1)IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB(1)IR <2 and ≥2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB(1)IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB(1)IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB(1)IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB(1)IR upon disease specific survival. The 5 year probabilities of event-free survival were: 85±5 and 66±8%; tumour interior, 86±4% and 63±8% for the CB(1)IR<2 and CB(1)IR≥2 groups, respectively. CONCLUSIONS/SIGNIFICANCE: The level of CB(1) receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB(1)IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients

Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

Background: Exposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide. Methods: Retinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein beta III-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue (TM) cell viability assay and the TMRE mitochondrial membrane potential assay. Results: Differentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 mu M in the P19 neurons and 1 mu M in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (Delta Psi m) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells. Conclusions: P19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals

Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction

Expression of serotonin transporter and monoamine oxidase A in P19 cells and P19 neurons.

<p>(A) Reverse transcription PCR analysis of the mRNA expressions of SERT in P19 cells and P19 neurons (at day 8 in the serum-free media) with a mouse brain lysate (MB) as a positive control. Total RNA was isolated and reverse transcribed into cDNA. The PCR products were analysed by agarose gel electrophoresis and fragment size estimated using a 100 bp marker. The arrow shows the expected amplicon size for SERT (127 bp) with the primer pair used. (B) qPCR analysis of mRNA expression levels of SERT in P19 cells and neuronally differentiated P19 cells (at days 8 and 10 in the serum-free media) with RPL19 as housekeeping gene. Results are expressed as a percentage of P19 cells. Values are means ± SEM of n = 5 independent experiments. (C) Expression of SERT in P19 cells and P19 neurons (at day 8 in the serum-free media) as measured with ELISA. Data are means ± SEM of n = 4 independent cell preparations (P19 neurons) and 5 (P19 cells). Statistical analysis (unpaired t test) showed a significant difference between P19 cells and neurons (***p< 0.001). (D) Effects of the selective SERT inhibitor citalopram and MDMA on 5-HT uptake in P19 neurons (at day 8 in the serum-free media). The cells (or wells without cells) were preincubated for 10 min with 1 μM citalopram, 1 mM MDMA or 0.002% DMSO as vehicle control followed by 30 min incubation with 100 nM [³H]-5-HT at 37°C. Data are means ± SEM of n = 8 independent experiments. Statistical analysis was performed using one-way repeated measures ANOVA with post hoc Bonferroni’s multiple comparison test (***p<0.001 for citalopram- and MDMA- vs. vehicle-treated control cells, no statistically significant difference was observed between citalopram-treated and MDMA-treated cells). (E) Western blot analysis with rabbit anti-monoamine oxidase A monoclonal antibody (ab126751) (Abcam). Comparison of immunoreactivity between P19 cells, P19 neurons (at day 10 in the serum-free media) and the positive control human liver hepatocellular carcinoma cell line (HepG2). Cell lysates: 10 μg per lane. The arrow shows the expected size of MAO-A (60 kDA).</p

Effects of MDMA, clorgyline, deprenyl, fluoxetine and the combinations on MTT reduction in P19 neurons.

<p>Effects of MDMA, clorgyline, deprenyl, fluoxetine and the combinations on MTT reduction in P19 neurons.</p

Effects of clorgyline, deprenyl, ketanserin, α-tocopherol (VitE) and N-acetylcysteine (NAC) on MDMA-induced toxicity in P19 neurons.

<p>P19 neurons were cultured for eight days in the serum-free media and incubated for 48 h with the test compounds in absence or presence of 1 mM MDMA. Cell viability was measured with (A) MTT reduction assay and (B) LDH activity assay. Data are means ± SEM of n = 3–4 independent experiments. For MTT reduction, data are expressed as percentage of non-treated control cells. For LDH release, the results are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was performed using repeated measures one-way ANOVA with post hoc Bonferroni's multiple comparisons test (#p< 0.0001 for comparison between untreated control cells and the treatments, and *p< 0.05 when treatments in presence of MDMA are compared to 1 mM MDMA).</p

Time-dependent effects of MDMA on mitochondrial membrane potential.

<p>P19 neurons, cultured for seven to nine days in serum-free medium, were exposed to 1 mM MDMA for 10 min up to 48 h. Mitochondrial membrane potential was measured by using the TMRE assay. The mitochondrial oxidative phosphorylation uncoupler FCCP (5 μM) applied for 10 min was used as a positive control. Data are means ± SEM of n = 4–6 independent experiments. Statistical analysis was performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test (****p< 0.0001) compared to the untreated control.</p

Effects of MDMA treatment prior to high temperature upon the MTT reduction produced by P19 neurons.

<p>P19 neurons were treated with MDMA on day 7 in the serum-free media at 37°C, 5% CO₂ for 48 h without (white bars) or with (grey bars) an additional step whereby medium was changed to remove MDMA and the cells were incubated for a further 24 h at 42°C, in a humified atmosphere with 5% CO₂. Cell viability was assessed with MTT reduction assay. Data (means ± SEM of n = 4 independent experiments) are presented as percentage of non-treated control cells. Statistical analysis was performed using two-way repeated-measures ANOVA matching both treatment and temperature. The interaction term treatment x temperature was not significant. The significant difference within temperature groups were followed up with post hoc Dunnett’s multiple comparison test compared to non-treated control cells (*p< 0.05, ****p< 0.0001).</p

Concentration-, time- and temperature- dependent effects of MDMA on the viability of P19 neurons.

<p>MTT reduction and LDH release were measured in P19-derived neurons exposed to MDMA at day 7 in the serum-free media for 24 h, 48 h and 72 h at the temperatures 37°C, 40°C and 42°C, in a humified atmosphere with 5% CO₂. Data are means ± SEM of n = 4–5 independent experiments. For MTT reduction, data are expressed as percentage of non-treated control cells. For LDH release, the results are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test (*p< 0.05, †p< 0.01, ‡p< 0.001) compared to corresponding controls.</p