Expression of Lysophosphatidylinositol Signaling-relevant Molecules in Colorectal Cancer.

BACKGROUND/AIM: Lysophosphatidylinositol (LPI) is a subspecies of the lysophospholipid mediators produced when phospholipase hydrolyzes membrane phosphatidylinositol. Previously, we used mass spectrometry-based lipidomics to demonstrate that LPI is selectively elevated in colorectal cancer (CRC) tissues. Here, we hypothesized that the expression levels of the LPI biosynthetic enzyme and LPI receptor - DDHD domain containing 1 (DDHD1) and G protein-coupled receptor 55 (GPR55), respectively - may be correlated with malignant potential, and we evaluated their roles in the context of CRC. MATERIALS AND METHODS: Colorectal specimens from 92 CRC patients underwent DDHD1 and GPR55 immunolabeling. Correlation between protein expression levels and clinicopathological variables was examined. RESULTS: Depth of tumor invasion was positively correlated with DDHD1 expression. Regardless of the degree of invasion depth, GPR55 was highly expressed in CRC tissues. Neither DDHD1 nor GPR55 expression levels were associated with disease-free survival. CONCLUSION: DDHD1 expression is associated with depth of tumor invasion in CRC tissues and may be involved in tumor progression.

Suppression of STAT3 Signaling by Δ9-Tetrahydrocannabinol (THC) Induces Trophoblast Dysfunction.

AIMS: Marijuana is a widely used illicit drug and its consumption during pregnancy has been associated with adverse reproductive outcomes. The purpose of this study was to determine the effects of chronic intake of Δ9-tetrahydrocannabinol (THC), the major component of marijuana, on trophoblast function, placental development, and birth outcomes. METHODS: The pathological characteristics and distribution of cannabinoid receptors in placenta were observed by immunohistochemical (IHC) staining. Cell migration in response to THC was measured by transwell assays. The levels of cannabinoid receptors and Signal Transducer and Activator of Transcription 3 (STAT3) were detected by western blot. RESULTS: We found the placenta expressed two main cannabinoid receptors, suggesting that THC induced biological responses in placental cells. Supporting this hypothesis, we observed dramatic alterations of placental morphology in marijuana users. Using THC and inhibitors of cannabinoid receptors, we demonstrated that THC impaired trophoblast cell migration and invasion partly via cannabinoid receptors. Additionally, pregnant mice injected with THC showed adverse reproductive events including reduced number of fetuses, lower maternal and placental weights. Mechanistically, STAT3 signaling pathway was involved in the THC-induced suppression of trophoblast cell motility and pregnancy outcomes. CONCLUSION: Our study indicates that the STAT3 signaling pathway plays a critical role in THC-induced trophoblast dysfunction.

Cannabinoid receptor expression in the bladder is altered in detrusor overactivity.

INTRODUCTION: Immunohistochemical (IHC) evidence shows that cannabinoid receptors (CB) are expressed in human bladders and cannabinoid agonists are known to inhibit detrusor contractility. However, the mechanism for this inhibition remains unknown. In addition, the role of CB in detrusor overactivity (DO) is under-investigated. The aim of this study was to compare CB expression in normal and DO human bladders and to further characterise these receptors. METHODS: Polymer chain reaction (PCR) was used to detect differences in CB transcripts in bladder samples. Differences in CB protein expression was assessed by IHC. Immunofluorescence (IF) was used to evaluate co-localisation of CB with nerve fibres. Receptor density and binding affinity were measured using the cannabinoid radioligand [(3)H]-CP-55,940. RESULTS: There were higher levels of CB1 transcripts in the urothelium of patients with DO and lower levels in the detrusor, compared with normal bladders. Radioligand binding revealed CB density of 421 ± 104 fmol/mg protein in normal human bladders. IHC confirmed these findings at the protein level. IF staining demonstrated co-localisation of CB1 with choline acetyltransferase-(ChAT)-positive nerves in the detrusor and co-localisation with PGP9.5 in both urothelium and detrusor. CB2 was co-localised with both ChAT and PGP9.5 in the urothelium and the detrusor. CONCLUSIONS: Cannabinoid receptor expression is reduced in the detrusor of patients with DO, which may play a role in the pathophysiology of the disease. Co-localisation of CB receptors with cholinergic nerves may suggest that CB1, being localised on pre- and postsynaptic terminals, could influence neurotransmitter release. Our findings suggest the potential role of cannabinoid agonists in overactive bladder pharmacotherapy.

Involvement of cannabinoid receptors in infrasonic noise-induced neuronal impairment.

Excessive exposure to infrasound, a kind of low-frequency but high-intensity sound noise generated by heavy transportations and machineries, can cause vibroacoustic disease which is a progressive and systemic disease, and finally results in the dysfunction of central nervous system. Our previous studies have demonstrated that glial cell-mediated inflammation may contribute to infrasound-induced neuronal impairment, but the underlying mechanisms are not fully understood. Here, we show that cannabinoid (CB) receptors may be involved in infrasound-induced neuronal injury. After exposure to infrasound at 16 Hz and 130 dB for 1-14 days, the expression of CB receptors in rat hippocampi was gradually but significantly decreased. Their expression levels reached the minimum after 7- to 14-day exposure during which the maximum number of apoptotic cells was observed in the CA1. 2-Arachidonoylglycerol (2-AG), an endogenous agonist for CB receptors, reduced the number of infrasound-triggered apoptotic cells, which, however, could be further increased by CB receptor antagonist AM251. In animal behavior performance test, 2-AG ameliorated the infrasound-impaired learning and memory abilities of rats, whereas AM251 aggravated the infrasound-impaired learning and memory abilities of rats. Furthermore, the levels of proinflammatory cytokines tumor necrosis factor alpha and interleukin-1ß in the CA1 were upregulated after infrasound exposure, which were attenuated by 2-AG but further increased by AM251. Thus, our results provide the first evidence that CB receptors may be involved in infrasound-induced neuronal impairment possibly by affecting the release of proinflammatory cytokines.

Endocannabinoid crosstalk between placenta and maternal fat in a baboon model (Papio spp.) of obesity.

INTRODUCTION: Maternal obesity (MO) remains a serious obstetric problem with acute and chronic morbidities for both mothers and offspring. The mechanisms underlying these adverse consequences of MO remain unknown. Endocannabinoids (ECB) are neuromodulatory lipids released from adipocytes and other tissues. Metabolic crosstalk between placenta and adipocytes may mediate sequelae of MO. The goal of this study was to elucidate placental and systemic ECB in MO. MATERIAL AND METHODS: Placentas, sera, and subcutaneous fat were collected at Cesarean sections performed near term (0.9 G) in four non-obese (nOB) and four obese (OB) baboons (Papio spp.). Concentrations of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled to tandem mass spectrometry. AEA and 2-AG pathways were characterized in placentas by Q-RT-PCR, Western blot and immunohistochemistry. RESULTS: Placental 2-AG levels were lower and maternal fat AEA levels were higher in OB (1254.1 ± 401.3 nmol/kg and 17.3 ± 4 nmol/kg) vs. nOB (3124.2 ± 557.3 nmol/kg and 3.1 ± 0.6 nmol/kg) animals. Concentrations of 2-AG correlated positively between maternal fat and placenta (r = 0.82, p = 0.013), but correlated negatively with maternal leptin concentrations (r = -0.72, p = 0.04 and r = -0.83, p = 0.01, respectively). CONCLUSION: This is the first study to demonstrate differential ECB pathway regulation in maternal fat and placenta in MO. Differential regulation and function exist for AEA and 2-AG as the major ECB pathways in placenta.

A novel monoclonal antibody against cannabinoid receptor 1.

The cannabinoid receptor 1 (CBR1) is being widely investigated because of its specific structure and functions compared with other cannabinoid receptors. In this study, we immunized BALB/c mice with synthesized human CBR1 polypeptide and obtained a novel monoclonal antibody (MAb) against human CBR1. Analysis through enzyme-linked immunosorbent assay (ELISA), spot-ELISA, Western blot, and immunohistochemistry revealed that the MAb was specifically against recombinant human CBR1 protein, and its subtype and affinity constant (Kaff) were IgG2b/k and 7.85 × 10(8) M/L, respectively. Using this MAb we found that CBR1 is expressed on HL-7702 cells and lipid tissue, raising the possibility that the CBR1 may take a role in glucose and lipid metabolism. Thus, this antibody might facilitate studies for pathophysiology of diseases associated with glucose and lipid metabolism abnormality.

Localization of mGluR5, GABAB, GABAA, and cannabinoid receptors on the vago-vagal reflex pathway responsible for transient lower esophageal sphincter relaxation in humans: an immunohistochemical study.

BACKGROUND: Transient lower esophageal sphincter relaxations (TLESRs) are the predominant mechanisms underlying gastro-esophageal reflux. TLESRs are mediated by a vago-vagal reflex, which can be blocked by interaction with metabotropic Glutamate Receptor 5 (mGluR5), γ-aminobutyric acid type B (GABA(B)), γ-aminobutyric acid type A (GABA(A)), and cannabinoid (CB) receptors. However, the distribution of these receptors in the neural pathway underlying the triggering of TLESRs has not been evaluated in humans. METHODS: Using immunohistochemistry, we investigated the distribution of mGluR5, GABA(A), GABA(B), CB1, and CB2 receptors in the human nodose ganglion, the brain stem, and the myenteric plexus of the esophagus. KEY RESULTS: MGluR5, GABA(B), CB1, and CB2 receptors are abundantly expressed in neurons of the myenteric plexus of the LES, nodose ganglion cell bodies and nerve fibers, the dorsal motor nucleus, and nucleus of the solitary tract in the brain stem. GABA(A) receptors are expressed in the same regions except in the nodose ganglion and myenteric plexus of the LES. CONCLUSIONS & INFERENCES: Human mGluR5, GABA(A,B), and CB(1,2) receptors are abundantly expressed along the vago-vagal neural pathway and involved in the triggering of TLESRs. These findings are not only in line with the central side effects observed during treatment with reflux inhibitors such as GABA(B) receptor agonists and mGluR5 antagonists, but also suggest that peripherally acting compounds may be effective.

Differential role of anandamide and 2-arachidonoylglycerol in memory and anxiety-like responses.

BACKGROUND: Cannabinoid agonists are potential therapeutic agents because of their antinociceptive and anxiolytic-like effects, although an important caveat to their use is the possible adverse responses related to memory impairment. An alternative approach to circumvent this limitation consists of enhancing the concentration of the endocannabinoids anandamide and 2-arachidonoylglycerol. METHODS: Using low doses of the specific inhibitors of the endocannabinoid metabolizing enzymes fatty acid amide hydrolase, URB597, and monoacylglycerol lipase, JZL184, we analyzed their acute and chronic effects on memory consolidation, anxiolytic-like effects, and nociception in mice (n = 6-12 per experimental group). RESULTS: We show that anandamide is a central component in the modulation of memory consolidation, whereas 2-arachidonoylglycerol is not involved in this process. Interestingly, both URB597 and JZL184 induce anxiolytic-like effects through different cannabinoid receptors. In addition, the results show that the antinociceptive and anxiolytic-like responses of both inhibitors, as well as their acute effects on memory consolidation, are maintained after chronic treatment. CONCLUSIONS: These results dissociate the role of anandamide and 2-arachidonoylglycerol in memory consolidation and anxiety and reveal the interest of cannabinoid receptor 2 as a novel target for the treatment of anxiety-related disorders.

Cannabinoid receptor expression and phosphorylation are differentially regulated between male and female cerebellum and brain stem after repeated stress: implication for PTSD and drug abuse.

Recent study demonstrated a close relationship between cerebellum atrophy and symptom severity of pediatric maltreatment-related posttraumatic stress disorder (PTSD). It has also been known that females are more vulnerable than males in developing anxiety disorders after exposure to traumatic stress. The mechanisms are unknown. Because cannabinoid receptors (CB1 and CB2) are neuroprotective and highly expressed in the cerebellum, we investigated cerebellar CB expression in stressed rats. Young male and female Sprague-Dawley rats were given 40 unpredictable electric tail-shocks for 2h daily on 3 consecutive days. CB1 and CB2 mRNA and protein levels in rat cerebellum and brain stem were determined using quantitative real-time PCR and Western blot, respectively. Two-way ANOVA revealed significant gender and stress effects on cerebellar CB1 mRNA expression, with females and non-stressed rats exhibiting higher CB1 mRNA levels than the males (3 fold, p<0.01) and stressed rats (30%, p<0.01), respectively. CB1 and CB2 mRNA levels in brain stem were also greater in female rats than males (p<0.01, p<0.05, respectively). Repeated stress increased the level of phosphorylated CB1 receptors, the inactivated CB1, in rat cerebellum (p<0.01), particularly in female rats as revealed by the significant gender × stress interaction. Thus, repeated severe stress caused greater CB1 mRNA suppression and CB1 receptor phosphorylation in female cerebellum that could lead to increased susceptibility to stress-related anxiety disorders including PTSD.

Cannabinoids attenuate cancer pain and proliferation in a mouse model.

We investigated the effects of cannabinoid receptor agonists on (1) oral cancer cell viability in vitro and (2) oral cancer pain and tumor growth in a mouse cancer model. We utilized immunohistochemistry and Western blot to show that human oral cancer cells express CBr1 and CBr2. When treated with WIN55,212-2 (non-selective), ACEA (CBr1-selective) or AM1241 (CBr2-selective) agonists in vitro, oral cancer cell proliferation was significantly attenuated in a dose-dependent manner. In vivo, systemic administration (0.013M) of WIN55,212-2, ACEA, or AM1241 significantly attenuated cancer-induced mechanical allodynia. Tumor growth was also significantly attenuated with systemic AM1241 administration. Our findings suggest a direct role for cannabinoid mechanisms in oral cancer pain and proliferation. The systemic administration of cannabinoid receptor agonists may have important therapeutic implications wherein cannabinoid receptor agonists may reduce morbidity and mortality of oral cancer.

Immunoregulation of a CB2 receptor agonist in a murine model of neuroAIDS.

Chronic HIV-1 infection commonly affects behavioral, cognitive, and motor functions in the infected human host and is commonly referred to as HIV-1-associated neurocognitive disorders (HAND). This occurs, in measure, as a consequence of ingress of leukocytes into brain perivascular regions. Such cells facilitate viral infection and disease by eliciting blood-brain barrier and neuronal network dysfunctions. Previous works demonstrated that the endocannabinoid system modulates neuroimmunity and as such neuronal and glial functions. Herein, we investigated CB2R receptor expression in murine HIV-1 encephalitis (HIVE) and the abilities of a highly selective CB2R agonist, Gp1a, to modulate disease. HIV-1-infected human monocyte-derived macrophages were injected into the caudate and putamen of immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL/HIVE). Brains of hu-PBL/HIVE mice showed microglial activation and increased expression of CB2R, but not CB1R or GPR55. Gp1a substantively reduced infiltration of human cells into the mouse brain and reduced HLA DQ activation. Gp1a down modulated CCR5 expression on human cells in the spleen with an increase in Fas ligand expression. Our results support the notion that CB2 receptor agonists may be a viable therapeutic candidate for HAND.

Cannabinoids and the immune system: an overview.

Cannabinoids can influence the immune network. Data on the impact of exogenous cannabinoid ligands on immune function serve not only to understand how the endocannabinoid system modulates immune phenomena associated with infection or inflammation, but also to identify therapeutic targets for immune diseases. Cannabinoids can modulate immune reactions in the periphery but also in the brain, influence T cell subset balance and cytokine expression and play a role in the balance between neuroinflammation and neurodegeneration. Immune cells can synthesize endocannabinoids and also be influenced by cannabinoid analogues. Cannabinoid receptors show different expression on immune cells depending on activation status and stimuli. The complexity of relation between cannabinoid ligands of various classes and cannabinoid receptors brought the need to refine the simple conceptual frame of agonist-antagonists and offered potential implications for understanding interactions in pathological conditions. The immune influence of cannabinoid ligands is not fully elucidated. However, aspects of their immunomodulatory effects provide the basis for a context-dependent targeted therapeutic approach, thus leading to the possibility for the use of cannabinoids in the treatment of inflammatory disease.

Anandamide inhibits cholangiocyte hyperplastic proliferation via activation of thioredoxin 1/redox factor 1 and AP-1 activation.

The endocannabinoid system regulates various aspects of hepatic fibrosis; however, nothing is known about its role in regulating cholangiocyte proliferation and function. We evaluated the effects of anandamide (AEA) on cholangiocyte proliferation and explored the effects of AEA on the thioredoxin 1 (TRX1)/redox factor 1 (Ref1)/activator protein-1 (AP-1) pathway. Mice underwent bile duct ligation (BDL) and were infused with AEA for 3 days postsurgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis were studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. mRNA expression of the cannabinoid receptors Cb1 and VR1 was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos, and c-Jun expression, increased nuclear localization of TRX1, and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. We conclude that AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.

Preclinical assessment of novel therapeutics on the cough reflex: cannabinoid agonists as potential antitussives.

Cough, a reflex defense mechanism, is a common symptom of many airway inflammatory diseases. At present there are no satisfactory treatments for cough that have an acceptable side effect profile. Recent data have described the inhibitory effect of selective cannabinoid CB(2) receptor agonists on sensory nerve activity in vitro and the cough reflex in a guinea pig model. CB(2) receptor expression is limited in the central nervous system (CNS) and hence the development of selective agonists may provide a new therapeutic strategy for treatment of cough devoid of the CNS-mediated side effects that are normally associated with nonselective cannabinoid agonists.

Cannabinoid receptors expression in bone marrow trephine biopsy of chronic lymphocytic leukaemia patients treated with purine analogues.

BACKGROUND: Cannabinoid receptors CB1 and CB2 are part the endocannabinoid system that plays an important role in the process of proliferation and apoptosis of different neoplastic cells. B-cell chronic lymphocytic leukaemia is one of the diseases in which these processes are altered. AIM: The aim of our study was the assessment of cannabinoid receptor expression on the B-lymphocytes in bone marrow trephine biopsy from leukaemic patients at diagnosis and after purine analogue treatment. METHODS: The biopsy was taken routinely and standard immunohistochemical staining procedure for paraffin embedded sections was applied. The cannabinoid receptors were detected using specific primary polyclonal antibody anti-CB1 and anti-CB2. Additionally, an existence of cannabinoid receptors was confirmed by flow cytometry. RESULTS: The results showed that the expression of CB1 receptor on the surface of neoplastic cells was lower than that of CB2 (17.0+/-3.1% and 92.1+/-1.7% respectively, p<0.001). Nine of the patients responded to applied treatment with a reduction in leukaemic infiltration (77.2+/-6.9% to 30.2+/-6.5%, p=0.007) and CB1 receptor expression (24.4+/-4.8% to 8.6+/-2.9%, p=0.01), but there was no change in CB2 expression (91.7+/-2.7% vs 90.9+/-2.8%, p=0.69). Four patients without remission expressed even greater number of the receptors. In all of the cases both cannabinoid receptor types antibodies gave positive reaction. Furthermore, the existence of cannabinoid receptors on neoplastic lymphocytes was confirmed by flow cytometry. CONCLUSION: The study provides original evidence for the existence of cannabinoid receptors on B-lymphocytes in chronic lymphocytic leukaemia patients. The receptors are thought to be a new structure that can modify the course of the disease and may be considered as a new target in leukaemia treatment.

The expression of receptors for endocannabinoids in human and rodent skeletal muscle.

The endocannabinoid system is a lipid derived signalling system that has been shown to regulate appetite and energy metabolism. The most abundant endogenous endocannabinoid, anandamide, has been shown to activate the cannabinoid receptor type 1 (CB1) and type 2 (CB2) as well as the 'non-cannabinoid' transient receptor potential channel-vanilloid sub-family member 1 (TRPV1), before being rapidly metabolised by fatty acid amide hydrolase (FAAH). We have previously demonstrated the expression of CB1 and studied the effects of CB1 activation and inhibition in human skeletal muscle myotubes, however, not all results could be explained by CB1 mediated effects. This suggests that other receptors which are activated by endocannabinoids may be present in skeletal muscle. In this study we describe the presence of not only CB1, but also CB2, TRPV1 and the degrading enzyme FAAH in human and rodent skeletal muscle using reverse transcription polymerase chain reaction (RT-PCR).

[Preneural transmitters as regulators of embryogenesis. Current state of the problem].

Our knowledge about the preneural neurotransmitter systems and their functions were based on the old pharmacological and biochemical data that have recently been confirmed and substantially supplemented. Specific components of the preneural serotoninergic and endocannabinoid systems were identified in developing echinoderm embryos using immunocytochemistry, Western immunoelectroblotting, and HPLC-mass spectroscopy. These data were corroborated by the results of pharmacological experiments: it was found that some ligands of serotonin receptors, as well as the agonist of cannabinoid receptors anandamide induced the appearance of abnormal embryonic phenotypes, whose expression depended on the ligand-teratogen concentration. Their appearance was prevented, correspondingly, by serotonin and its lipophilic (or hydrophilic) analogs and antagonists of cannabinoid (CB1/CB2)-receptors.

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treatment of abdominal pain and irritable bowel syndrome.

Cannabinoid receptors in human astroglial tumors.

In animal models, cannabinoids are reported to inhibit the growth of tumors, including gliomas. These effects have been claimed to be mediated via cannabinoid receptors 1 and 2 (CB1, CB2). To elucidate a possible relevance for treatment of human gliomas, we investigated receptor subtype expression in surgical material of solid human astrocytomas, gliomas and cultivated glioma cells by quantitative reverse transcriptase polymerase chain reaction, western blot and immunohistochemistry and assayed their functionality. In normal brain, cultivated glioma cells and solid tumors, CB1 mRNA was expressed to a much greater extent than CB2, which in some samples was even undetectable. Expression of both receptor subtypes was unrelated to malignancy, varied between patients, and was not significantly increased in relation to normal brain tissues. In normal brain, CB1 protein was localized on astroglial and other cell types; in gliomas, it was found on astroglial/glioma cells. CB2 protein was detected on microglial cells/macrophages but rarely on astroglial cells. Functionally, CB1 receptor agonists reduced elevated cyclic AMP levels and slightly reduced proliferation of glioma cells in vitro, but did not induce apoptosis. We conclude that cannabinoid therapy of human gliomas targets not only receptors on tumor, but also on other cell types. Therefore, complex and potential side-effects should be considered carefully.