RESUMO
Background JWH015 is a cannabinoid (CB) receptor type 2 agonist that produces immunomodulatory effects. Since skin cells play a key role in inflammatory conditions and tissue repair, we investigated the ability of JWH015 to promote an anti-inflammatory and pro-wound healing phenotype in human primary skin cells. Methods Human primary keratinocytes and fibroblasts were stimulated with lipopolysaccharide. The mRNA expression of cannabinoid receptors was determined using RT-PCR. The effects of JWH015 (0.05, 0.1, 0.5, and 1 µM) in pro- and anti-inflammatory factors were tested in lipopolysaccharide-stimulated cells. A scratch assay, using a co-culture of keratinocytes and fibroblasts, was used to test the effects of JWH015 in wound healing. In addition, the topical and transdermal penetration of JWH015 was studied in Franz diffusion cells using porcine skin and LC-MS. Results The expression of CB1 and CB2 receptors (mRNA) and the production of pro- and anti-inflammatory factors enhanced in keratinocytes and fibroblasts following lipopolysaccharide stimulation. JWH015 reduced the concentration of major pro-inflammatory factors (IL-6 and MCP-1) and increased the concentration of a major anti-inflammatory factor (TGF-ß) in lipopolysaccharide-stimulated cells. JWH015 induced a faster scratch gap closure. These JWH015'seffects were mainly modulated through both CB1 and CB2 receptors. Topically administered JWH015 was mostly retained in the skin and displayed a sustained and low level of transdermal permeation. Conclusions Our findings suggest that targeting keratinocytes and fibroblasts with cannabinoid drugs could represent a therapeutic strategy to resolve peripheral inflammation and promote tissue repair.
Assuntos
Agonistas de Receptores de Canabinoides/administração & dosagem , Citocinas/metabolismo , Fibroblastos/efeitos dos fármacos , Indóis/farmacologia , Queratinócitos/efeitos dos fármacos , Administração Cutânea , Antagonistas de Receptores de Canabinoides/farmacologia , Células Cultivadas , Citocinas/genética , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , RNA Mensageiro/metabolismo , Fatores de Tempo , Cicatrização/efeitos dos fármacosRESUMO
The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague-Dawley rats, weighing 160-180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors-mechanical allodynia and ambulatory pain-were assessed on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.
Assuntos
Neoplasias Ósseas/metabolismo , Dor do Câncer/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Indóis/administração & dosagem , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Animais , Neoplasias Ósseas/tratamento farmacológico , Dor do Câncer/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Injeções Intraperitoneais , Injeções Espinhais , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
JWH-015, a cannabinoid receptor 2 (CB2) agonist has tumor regressive property in various cancer types. However, the underlying mechanism by which it acts in lung cancer is still unknown. Tumor associated macrophage (TAM) intensity has positive correlation with tumor progression. Also, macrophages recruited at the tumor site promote tumor growth by enhancing epithelial to mesenchymal (EMT) progression. In this study, we analyzed the role of JWH-015 on EMT and macrophage infiltration by regulation of EGFR signaling. JWH-015 inhibited EMT in NSCLC cells A549 and also reversed the mesenchymal nature of CALU-1 cells by downregulation of EGFR signaling targets like ERK and STAT3. Also, in vitro co-culture experiments of A549 with M2 polarized macrophages provided evidence that JWH-015 decreased migratory and invasive abilities which was proved by reduced expression of FAK, VCAM1, and MMP2. Furthermore, it decreased macrophage induced EMT in A549 by attenuating the mesenchymal character by downregulating EGFR and its targets. These results were confirmed in an in vivo subcutaneous syngenic mouse model where JWH-015 blocks tumor growth and also inhibits macrophage recruitment and EMT at the tumor site which was regulated by EGFR pathway. Finally, JWH-015 reduced lung tumor lesions in an in vivo tumorigenicity mouse model. These data confer the impact of this cannabinoid on anti-proliferative and anti-tumorigenic effects, thus enhancing our understanding of its therapeutic efficacy in NSCLC. Our findings open new avenues for cannabinoid receptor CB2 agonist-JWH-015 as a novel and potential therapeutic target based on EGFR downregulation mechanisms in NSCLC. © 2016 Wiley Periodicals, Inc.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Indóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Células A549 , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Indóis/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Based on its wide expression in immune cells, type-2 cannabinoid (CB2) receptors were traditionally thought to act as "peripheral receptors" with an almost exclusively immunomodulatory function. However, their recent identification in mammalian brain areas, as well as in distinct neuronal cells, has opened the way to a re-consideration of CB2 signaling in the context of brain pathophysiology, synaptic plasticity and neuroprotection. To date, accumulated evidence from several independent preclinical studies has offered new perspectives on the possible involvement of CB2 signaling in brain and spinal cord traumatic injury, as well as in the most relevant neurodegenerative disorders like Alzheimer's disease, Parkinson's disease and Huntington's chorea. Here, we will review available information on CB2 in these disease conditions, along with data that support also its therapeutic potential to treat them.
Assuntos
Encéfalo/metabolismo , Degeneração Neural , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Medula Espinal/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Agonistas de Receptores de Canabinoides/uso terapêutico , Endocanabinoides/metabolismo , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Receptor CB2 de Canabinoide/agonistas , Transdução de Sinais , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
The misuse of prescription opiates is on the rise with combination therapies (e.g. acetaminophen or NSAIDs) resulting in severe liver and kidney damage. In recent years, cannabinoid receptors have been identified as potential modulators of pain and rewarding behaviors associated with cocaine, nicotine and ethanol in preclinical models. Yet, few studies have identified whether mu opioid agonists and CB2 agonists act synergistically to inhibit chronic pain while reducing unwanted side effects including reward liability. We determined if analgesic synergy exists between the mu-opioid agonist morphine and the selective CB2 agonist, JWH015, in rodent models of acute and chronic inflammatory, post-operative, and neuropathic pain using isobolographic analysis. We also investigated if the MOR-CB2 agonist combination decreased morphine-induced conditioned place preference (CPP) and slowing of gastrointestinal transit. Co-administration of morphine with JWH015 synergistically inhibited preclinical inflammatory, post-operative and neuropathic-pain in a dose- and time-dependent manner; no synergy was observed for nociceptive pain. Opioid-induced side effects of impaired gastrointestinal transit and CPP were significantly reduced in the presence of JWH015. Here we show that MOR + CB2 agonism results in a significant synergistic inhibition of preclinical pain while significantly reducing opioid-induced unwanted side effects. The opioid sparing effect of CB2 receptor agonism strongly supports the advancement of a MOR-CB2 agonist combinatorial pain therapy for clinical trials.
Assuntos
Analgésicos não Narcóticos/farmacologia , Analgésicos Opioides/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Dor Crônica/tratamento farmacológico , Indóis/farmacologia , Morfina/farmacologia , Analgésicos Opioides/efeitos adversos , Animais , Dor Crônica/metabolismo , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/tratamento farmacológico , Constipação Intestinal/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Masculino , Camundongos Endogâmicos ICR , Morfina/efeitos adversos , Ratos Sprague-Dawley , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , RecompensaRESUMO
INTRODUCTION: Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands. METHODS: The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model. RESULTS: JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling. CONCLUSION: The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.
RESUMO
BACKGROUND: Although in vivo studies have implicated endocannabinoids in metabolic dysfunction, little is known about direct, chronic activation of the endocannabinoid system (ECS) in human islets. Therefore, this study investigated the effects of prolonged exposure to cannabinoid agonists on human islet gene expression and function. METHODS: Human islets were maintained for 2 and 5 days in the absence or presence of CB1r (ACEA) or CB2r (JWH015) agonists. Gene expression was quantified by RT-PCR, hormone levels by radioimmunoassay and apoptosis by caspase activities. RESULTS: Human islets express an ECS, with mRNAs encoding the biosynthetic and degrading enzymes NAPE-PLD, FAAH and MAGL being considerably more abundant than DAGLα, an enzyme involved in 2-AG synthesis, or CB1 and CB2 receptor mRNAs. Prolonged activation of CB1r and CB2r altered expression of mRNAs encoding ECS components, but did not have major effects on islet hormone secretion. JWH015 enhanced insulin and glucagon content at 2 days, but had no effect after 5 days. Treatment with ACEA or JWH015 for up to 5 days did not have marked effects on islet viability, as assessed by morphology and caspase activities. CONCLUSIONS: Maintenance of human islets for up to 5 days in the presence of CB1 and CB2 receptor agonists causes modifications in ECS element gene expression, but does not have any major impact on islet function or viability. GENERAL SIGNIFICANCE: These data suggest that the metabolic dysfunction associated with over-activation of the ECS in obesity and diabetes in humans is unlikely to be secondary to impaired islet function.
RESUMO
During human immunodeficiency virus (HIV) infection, alcohol has been known to induce inflammation while cannabinoids have been shown to have an anti-inflammatory role. For instance cannabinoids have been shown to reduce susceptibility to HIV-1 infection and attenuate HIV replication in macrophages. Recently, we demonstrated that alcohol induces cannabinoid receptors and regulates cytokine production by monocyte-derived dendritic cells (MDDC). However, the ability of alcohol and cannabinoids to alter MDDC function during HIV infection has not been clearly elucidated yet. In order to study the potential impact of alcohol and cannabinoids on differentiated MDDC infected with HIV, monocytes were cultured for 7 days with GM-CSF and IL-4, differentiated MDDC were infected with HIV-1Ba-L and treated with EtOH (0.1 and 0.2%), THC (5 and 10 µM), or JWH-015 (5 and 10 µM) for 4-7 days. HIV infection of MDDC was confirmed by p24 and Long Terminal Repeats (LTR) estimation. MDDC endocytosis assay and cytokine array profiles were measured to investigate the effects of HIV and substances of abuse on MDDC function. Our results show the HIV + EtOH treated MDDC had the highest levels of p24 production and expression when compared with the HIV positive controls and the cannabinoid treated cells. Although both cannabinoids, THC and JWH-015 had lower levels of p24 production and expression, the HIV + JWH-015 treated MDDC had the lowest levels of p24 when compared to the HIV + THC treated cells. In addition, MDDC endocytic function and cytokine production were also differentially altered after alcohol and cannabinoid treatments. Our results show a differential effect of alcohol and cannabinoids, which may provide insights into the divergent inflammatory role of alcohol and cannabinoids to modulate MDDC function in the context of HIV infection.
RESUMO
Interstitial cystitis is a debilitating bladder inflammation disorder. To date, the understanding of the causes of interstitial cystitis remains largely fragmentary and there is no effective treatment available. Recent experimental results have shown a functional role of the endocannabinoid system in urinary bladder. In this study, we evaluated the anti-inflammatory effect of selective cannabinoid CB1 and CB2 receptor agonists in a mouse model of interstitial cystitis. Bladder inflammation was induced in mice by lipopolysaccharide (LPS) and whole bladders were removed 24h later. LPS induced a significant increase of the contractile amplitude in spontaneous activity and a hypersensitivity to exogenous acetylcholine-induced contraction of whole-isolated bladder. Next, we evaluated the anti-inflammatory activity of cannabinoidergic compounds by pretreating mice with CB1 or CB2 selective agonist compounds, respectively ACEA and JWH015. Interestingly, JWH015, but not ACEA, antagonized LPS-induced bladder inflammation. Additionally, anti-inflammatory activity was studied by evaluation, leukocytes mucosa infiltration, myeloperoxidase activity, and mRNA expression of pro-inflammatory interleukin (IL-1α and IL-1ß), tumor necrosis factor-alpha (TNF-α) and cannabinoid CB1 and CB2 receptors. JWH015 significantly decreased leukocytes infiltration in both submucosa and mucosa, as well as the myeloperoxydase activity, in LPS treated mice. JWH015 reduced mRNA expression of IL-1α, IL-1ß, and TNF-α. LPS treatment increased expression of bladder CB2 but not CB1 mRNA. Taken together, these findings strongly suggest that modulation of the cannabinoid CB2 receptors might be a promising therapeutic strategy for the treatment of bladder diseases and conditions characterized by inflammation, such as interstitial cystitis.