Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

Varenicline decreases alcohol consumption in heavy-drinking smokers

Rationale Emerging evidence suggests that the α4β2 form of the nicotinic acetylcholine receptor (nAChR) modulates the rewarding effects of alcohol. The nAChR α4β2 subunit partial agonist varenicline (Chantix™), which is approved by the Food and Drug Administration for smoking cessation, also decreases ethanol consumption in rodents (Steensland et al., Proc Natl Acad Sci U S A 104:12518–12523, 2007) and in human laboratory and open-label studies (Fucito et al., Psychopharmacology (Berl) 215:655–663, 2011; McKee et al., Biol Psychiatry 66:185–190 2009). Objectives We present a randomized, double-blind, 16-week study in heavy-drinking smokers (n = 64 randomized to treatment) who were seeking treatment for their smoking. The study was designed to determine the effects of varenicline on alcohol craving and consumption. Outcome measures included number of alcoholic drinks per week, cigarettes per week, amount of alcohol craving per week, cumulative cigarettes and alcoholic drinks consumed during the treatment period, number of abstinent days, and weekly percentage of positive ethyl glucuronide and cotinine screens. Results Varenicline significantly decreases alcohol consumption (χ 2 = 35.32, p < 0.0001) in smokers. Although varenicline has previously been associated with suicidality and depression, side effects were low in this study and declined over time in the varenicline treatment group. Conclusions Varenicline can produce a sustained decrease in alcohol consumption in individuals who also smoke. Further studies are warranted to assess varenicline efficacy in treatment-seeking alcohol abusers who do not smoke and to ascertain the relationship between varenicline effects on smoking and drinking.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Real-time measurement of F-Actin remodelling during exocytosis using lifeact-EGFP transgenic animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.

Treatment of cannabis use among people with psychotic disorders : critical review of randomised controlled trials

There is growing and converging evidence that cannabis may be a major risk factor in people with psychotic disorders and prodromal psychotic symptoms. The lack of available pharmacological treatments for cannabis use indicates that psychological interventions should be a high priority, especially among people with psychotic disorders. However, there have been few randomised controlled trials (RCTs) of psychological interventions among this group. In the present study we critically overview RCTs of psychological and pharmacologic interventions among people with psychotic disorders, giving particular attention to those studies which report cannabis use outcomes. We then review data regarding treatment preferences among this group. RCTs of interventions within "real world" mental health systems among adults with severe mental disorders suggest that cannabis use is amenable to treatment in real world settings among people with psychotic disorders. RCTs of manual guided interventions among cannabis users indicate that while brief interventions are associated with reductions in cannabis use, longer interventions may be more effective. Additionally, RCTs reviewed suggest treatment with antipsychotic medication is not associated with a worsening of cannabis cravings or use and may be beneficial. The development of cannabinoid agonist medication may be an effective strategy for cannabis dependence and suitable for people with psychotic disorders. The development of cannabis use interventions for people with psychotic disorders should also consider patients' treatment preferences. Initial results indicate face-to-face interventions focussed on cannabis use may be preferred. Further research investigating the treatment preferences of people with psychotic disorders using cannabis is needed.

Molecular and structural requirements of the ß1L-adrenoceptor

Noradrenaline which occurs naturally in the body binds to beta-adrenoceptors on the heart, causing the heart to beat faster and with greater force in response to increased demand. This enables the heart to provide oxygenated blood to vital organs. Prolonged overstimulation by noradrenaline can be harmful to the heart and lead to the progression of heart disease. In these circumstances beta-adrenoceptors are blocked with drugs called beta-blockers. Beta-blockers block the effects of noradrenaline by binding to the same site on the beta-adrenoceptor. Some beta-blockers such as CGP12177 can also cause increases in heart rate. Therefore it was proposed that CGP12177 could bind in a different place to noradrenaline. The aim of this study was to determine where CGP12177 binds to on the beta-adrenoceptor. The results have revealed a separate binding site named beta-1-low. These results may lead to the development of improved -blockers for the management of heart conditions.

The effects and interactions of GABAergic and dopaminergic agents in the prevention of form deprivation myopia by brief periods of normal vision

Intravitreal injections of GABA antagonists, dopamine agonists and brief periods of normal vision have been shown separately to inhibit form-deprivation myopia (FDM). Our study had three aims: (i) establish whether GABAergic agents modify the myopia protective effect of normal vision, (ii) investigate the receptor sub-type specificity of any observed effect, and (iii) consider an interaction with the dopamine (DA) system. Prior to the period of normal vision GABAergic agents were applied either (i) individually, (ii) in combination with other GABAergic agents (an agonist with an antagonist), or (iii) in combination with DA agonists and antagonists. Water injections were given to groups not receiving drug treatments so that all experimental eyes received intravitreal injections. As shown previously, constant form-deprivation resulted in high myopia and when diffusers were removed for 2 h per day the period of normal vision greatly reduced the FDM that developed. GABA agonists inhibited the protective effect of normal vision whereas antagonists had the opposite effect. GABAA/C agonists and D2 DA antagonists when used in combination were additive in suppressing the protective effect of normal vision. A D2 DA agonist restored some of the protective effect of normal vision that was inhibited by a GABA agonist (muscimol). The protective effect of normal vision against form-deprivation is modifiable by both the GABAergic and DAergic pathways.

Exenatide extended-release; clinical trials, patient preference, and economic considerations

Type 2 diabetes remains an escalating world-wide problem, despite a range of treatments. The revelation that insulin secretion is under the control of a gut hormone, glucagon-like peptide 1 (GLP-1) led to a new paradigm in the management of type 2 diabetes, medicines that directly stimulate, or that prolong the actions of the endogenous GLP-1, at its receptors. Exenatide is an agonist at the GLP-1 receptors, and was initially developed as a subcutaneous twice daily medication, ExBID. The clinical trials with ExBID established a role for exenatide in the treatment of type 2 diabetes. Subsequently, once weekly exenatide (ExQW) was shown to have advantages over ExBID, and there is now more emphasis on the development of ExQW. ExQW alone reduces glycosylated haemoglobin (HbA1c) and body weight, and is well tolerated. ExQW has been compared to sitagliptin, pioglitazone and metformin, and shown to have a greater ability to reduce HbA1c than these other medicines. The only preparation of insulin, which ExQW has been compared to, is insulin glargine, and the ExQW has some favourable properties in this comparison, notably causing weight loss, compared to the gain with insulin glargine. ExQW has been compared to another GLP-1 receptor agonist, liraglutide, and ExQW is non-inferior to liraglutide in reducing HbA1c. The small amount of evidence available, shows that subjects with type 2 diabetes, prefer ExQW to ExBID, and that adherence was high to these in the clinical trial setting. Healthcare and economic modelling suggests that ExQW will reduce diabetic complications and be cost-effective, compared to other medications, with long term use. Little is known about whether subjects with type 2 diabetes prefer ExQW to other medicines, and whether adherence is good to ExQW in practice, and these important topics require further study.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Neutrophil oxidative burst is primed by supernatant from stored red cells : implications for Transfusion-Related Acute Lung Injury (TRALI)

Aim/Background: Transfusion-related acute lung injury (TRALI) is a potentially fatal adverse transfusion reaction. It is hypothesised to occur via a two-insult mechanism: the recipient’s underlying co-morbidity in addition to the transfusion of blood products activate neutrophils in the lung resulting in damaged endothelium and capillary leakage. Neutrophil activation may occur by antibody or non-antibody related mechanisms, with the length of storage of cellular blood products implicated in the latter. This study investigated non-antibody mediated priming and/or activation of neutrophil oxidative burst. Methods: A cytochrome C reduction assay was used to assess priming and activation of neutrophil oxidative burst by pooled supernatant (SN) from day 1 (D1; n=75) and day 42 (D42; n=113) packed red blood cells (PRBC). Pooled PRBC-SN were assessed in parallel with PAF (priming), fMLP (activating), PAF + fMLP (priming + activating) and buffer only (negative) controls. Cytochrome C reduction was measured over 30min at 37oC (inclusive of 10min priming). Neutrophil activation by PRBC-SN was assessed cf. buffer only and neutrophil priming by PRBC-SN was assessed by co-incubation with fMLP cf. fMLP alone. One-way ANOVA; Newman-Keuls post-test; p<0.05; n=10 independent assays. Results: Neither D1- nor D42- PRBC-SN alone activated neutrophil oxidative burst. In addition, D1-PRBC-SN did not prime fMLP-activated neutrophil oxidative burst. D42-PRBC-SN did, however, prime neutrophils for subsequent activation of oxidative burst by fMLP, the magnitude of response being similar to PAF (a known neutrophil priming agonist). Conclusion: These findings are consistent with the two-insult mechanism of TRALI. Factors released into the SN during PRBC storage contributed to neutrophil priming synergistically with other neutrophil stimulating agonists. This implicates PRBC storage duration as a key factor contributing to non-immune neutrophil activation in the development of TRALI in patients with pre-disposing inflammatory conditions.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Heparan sulfate proteoglycans and human breast cancer epithelial cell tumorigenicity

Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1–4) and glypicans (GPC1–6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression.

Factors regulating basement membrane invasion by tumor cells

Basement membranes serve as significant barriers to the passage of tumor cells but ones which metastatic cells can pass. This involves the production of a cascade of proteases leading to the activation of a specific collagenase that degrades the unique collagen network in basement membrane. Breast cancer cells, when estrogen dependent, show a requirement for estrogen for invasive activity. However, when these cells progress to an estrogen independent state and increased malignancy, they express an invasive phenotype constitutively. Studies with various anti-estrogens suggest that these responses are mediated via the estrogen receptor. Anti-estrogens lacking agonist activity suppress invasiveness as well as growth of the breast cancer cells.

Role of the N-terminal region in G protein-coupled receptor functions : negative modulation revealed by 5-HT2B receptor polymorphisms

The putative role of the N-terminal region of rhodopsin-like 7 transmembrane biogenic amine receptors in agonist-induced signaling has not yet been clarified despite recent advances in 7 transmembrane receptor structural biology. Given the existence of N-terminal nonsynonymous polymorphisms (R6G;E42G) within the HTR2B gene in a drug-abusing population, we assessed whether these polymorphisms affect 5-hydroxytryptamine 2B (5-HT2B) receptor in vitro pharmacologic and coupling properties in transfected COS-7 cells. Modification of the 5-HT2B receptor N terminus by the R6G;E42G polymorphisms increases such agonist signaling pathways as inositol phosphate accumulation as assessed by either classic or operational models. The N-terminal R6G;E42G mutations of the 5-HT2B receptor also increase cell proliferation and slow its desensitization kinetics compared with the wild-type receptor, further supporting a role for the N terminus in transduction efficacy. Furthermore, by coexpressing a tethered wild-type 5-HT2B receptor N terminus with a 5-HT2B receptor bearing a N-terminal deletion, we were able to restore original coupling. This reversion to normal activity of a truncated 5-HT2B receptor by coexpression of the membrane-tethered wild-type 5-HT2B receptor N terminus was not observed using a membrane-tethered 5-HT2B receptor R6G;E42G N terminus. These data suggest that the N terminus exerts a negative control over basal as well as agonist-stimulated receptor activity that is lost in the R6G;E42G mutant. Our findings reveal a new and unanticipated role of the 5-HT2B receptor N terminus as a negative modulator, affecting both constitutive and agonist-stimulated activity. Moreover, our data caution against excluding the N terminus and extracellular loops in structural studies of this 7 transmembrane receptor family