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Cannabinoids and ceramide: Two lipids acting hand-by-hand
Abstract
Cannabinoids, the active components of Cannabis sativa (marijuana) and their endogenous counterparts, exert their effects by binding to specific G-protein-coupled receptors that modulate adenylyl cyclase and ion channels. Recent research has shown that the CB1 cannabinoid receptor is also coupled to the generation of the lipid second messenger ceramide via two different pathways: sphingomyelin hydrolysis and ceramide synthesis de novo. Sustained ceramide accumulation in tumor cells mediates cannabinoid-induced apoptosis, as evidenced by in vitro and in vivo studies. This effect seems to be due to the impact of ceramide on key cell signalling systems such as the extracellular signal-regulated kinase cascade and the Akt pathway. These findings provide a new conceptual view on how cannabinoids act, and raise interesting physiological and therapeutic questions.
... is is proposed to be mediated through the upregulation of endoplasmic reticulum stress-related genes (ATF-4, TRB3), accumulation of reactive oxygen species (ROS), cell cycle arrest in a p-53 independent manner, and activation of proapoptotic proteins (BAX, caspase 3/9) [38,39,[42][43][44]. Ceramide enhanced production is another mechanism of apoptosis induction by cannabinoids through the mitochondrial pathway [45]. In these cases, proapoptotic proteins, usually sequestered in the intermembrane space, are released into the cytosol, assembling the "apoptosome" (formed by the binding of cytochrome c, Apaf-1, and procaspase-9) [46]. ...
... Cannabinoids also regulate different proliferation, growth, and survival pathways [45]. Different carcinoma cell lines treated with ∆9-THC also exhibited inhibition of the RAS-MAPK pathway and the phosphatidylinositol 3-kinase pathway (PI3k-AKT) pathway through CB1-receptor activation or ceramide accumulation [34,45]. ...
... Cannabinoids also regulate different proliferation, growth, and survival pathways [45]. Different carcinoma cell lines treated with ∆9-THC also exhibited inhibition of the RAS-MAPK pathway and the phosphatidylinositol 3-kinase pathway (PI3k-AKT) pathway through CB1-receptor activation or ceramide accumulation [34,45]. ...
Objective. Colorectal cancer represents a heavy burden for health systems worldwide, being the third most common cancer worldwide. Despite the breakthroughs in medicine, current chemotherapeutic options continue to have important side effects and may not be effective in preventing disease progression. Cannabinoids might be substances with possible therapeutic potential for cancer because they can attenuate the side effects of chemotherapy and have antiproliferative and antimetastatic effects. We aim to determine, through a systematic review of experimental studies performed on animal CRC models, if cannabinoids can reduce the formation of preneoplastic lesions (aberrant crypt foci), number, and volume of neoplastic lesions. Materials and Methods. A systematic, qualitative review of the literature was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, Embase, and Scopus databases were searched. We use the following Medical Subject Headings (MESH) terms in PubMed: “colorectal neoplasms,” “colonic neoplasms,” “colorectal cancer,” “polyps,” “rimonabant,” “cannabidiol,” “cannabinoids,” “azoxymethane,” “xenograft,” and “mice.” Only studies that met the eligibility criteria were included. Results. Eight in vivo experimental studies were included in the analysis after the full-text evaluation. Seven studies were azoxymethane (AOM) colorectal cancer models, and four studies were xenograft models. Cannabidiol botanical substance (CBD BS) and rimonabant achieved high aberrant crypt foci (ACF) reduction (86% and 75.4%, respectively). Cannabigerol, O-1602, and URB-602 demonstrated a high capacity for tumor volume reduction. Induction of apoptosis, interaction with cell survival, growth pathways, and angiogenesis inhibition were the mechanisms extracted from the studies that explain cannabinoids’ actions on CRC. Conclusions. Cannabinoids have incredible potential as antineoplastic agents as experimental models demonstrate that they can reduce tumor volume and ACF formation. It is crucial to conduct more experimental studies to understand the pharmacology of cannabinoids in CRC better.
1. Background
Colorectal cancer (CRC) is the third most common cancer worldwide, only behind prostate and lung in males, and behind breast and lung in females [1]. It has high morbidity and mortality that represents a heavy burden for health systems worldwide. In the United States alone, with roughly 1.8 million new cases in 2018, healthcare costs exceed $14 billion annually [2]. In addition, it is the fourth cause of cancer-related deaths [3, 4]. CRC represents a significant public health concern because temporal projections estimate that its global burden will increase by 60% to more than 2.2 million new cases and 1.1 million cancer deaths by 2030 [5].
CRC is a type of cancer with a complex and heterogeneous pathophysiology. It is the result of the transformation of healthy colonic epithelial cells into cancer [6]. This process, called “adenoma-carcinoma sequence,” develops through an ordered series of events, in which the initial step is the transformation of normal colonic epithelium to aberrant crypt foci (ACF) [6]. ACF progress to CRC, in 10–15 years [7]. During this process, many risk factors play an essential role in pathogenesis, including unhealthy diet, smoking, alcohol use, physical inactivity, inflammatory bowel disease, and aging [2].
Breakthroughs in CRC therapy have decreased the mortality of patients with CRC. Current chemotherapeutic options continue to have important side effects due to cytotoxicity and may fail to prevent disease progression [8]. Thus, there is a great interest in new therapeutic approaches for CRC, including phytochemical agents.
Cannabinoids might be substances with possible therapeutic potential for cancer because of their chemotherapeutic effect and their ability to attenuate anorexia, pain, and emesis; these are common side effects of chemotherapy [9, 10]. This has been proved in several experimental models of CRC, brain cancer, breast cancer, lung cancer, prostate cancer, leukemia, and melanoma [11]. However, to the best of our knowledge, cannabinoids have not been tested in humans as medicines for CRC.
Animal models and cell lines of CRC have tested cannabinoids. This study aims to conduct a systematic review of the research about the effect of cannabinoids on in vivo azoxymethane (AOM) or xenograft CRC models. The outcomes used to assess the effects of cannabinoids, compared with no cannabinoid therapy, were a decrease in the number of preneoplastic lesions (aberrant crypt foci), number, and volume of neoplastic lesions.
2. Materials and Methods
The protocol for this study was registered in PROSPERO (International Prospective Register for Systematic Reviews) under CRD42019148356 [12]. This systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Supplementary file) [13].
2.1. Eligibility Criteria
Population. The population should be animal species (no restrictions), used for in vivo models of CRC, either chemically induced (Azoxymethane or DSS) or by xenograft injection. Dose and time of exposure to azoxymethane were not exclusion criteria for this review. We excluded all studies that included only in vitro assessment and studies that evaluated species for noncolorectal cancer models.
Intervention. Studies had to evaluate the beneficial effects of the following cannabinoids: CBD, CBG, O-1602, LYR-8, WIN 55, 212–2, AEA, HU-210, rimonabant, anandamide reuptake inhibitors (VDM11), FAAH inhibitors, and MAGL inhibitors.
Comparators. Studies had to include at least one comparator group of the same animal species used for the intervention group, with similar characteristics (weight, age, sex, exposure to the same environment, and feeding), without exposition to cannabinoid therapy.
Studies. Studies should be experimental in vivo studies of CRC in mice, with at least one control group. We excluded conference abstracts, narrative reviews, and systematic reviews.
Primary Outcome. There should be a reduction in tumor volume (mm³), number of aberrant crypt foci (ACF), and number of tumors comparing intervention and control group.
Secondary Outcome. There should be an expression of apoptosis markers (Bax, caspase-3, caspase-9, annexin V, PI), expression of proinflammatory markers (STAT3, NFκβ, TNF-α), and levels of endocannabinoids.
2.2. Search Strategy
We performed a methodologic and systematic strategic search in the following electronic bibliographic databases: PubMed, Scopus, and Embase (from their inception to December 18, 2019). The last search was run on December 18, 2019. Only full available articles written in English were suitable for assessment. We used the following Medical Subject Headings (MESH) terms in PubMed: “colorectal neoplasms”, “colonic neoplasms”, “colorectal cancer”, “polyps”, “rimonabant”, “cannabidiol”, “cannabinoids”, “azoxymethane”, “xenograft”, and “mice”.
2.3. Study Selection and Data Collection
The authors EOG and LLT conducted the search independently. Duplicate articles were moved to a different folder and registered in the flowchart. Before the selection process, a test was conducted to evaluate the agreement between evaluators. All titles and summaries of the articles were assessed by EOG and LLT independently based on a selection criterion. The full text of previously selected studies was then reviewed and analyzed. Any disagreement was discussed, and if not resolved, a third author (AVV) was consulted. All selected articles were summarized in a flowchart according to the PRISMA protocol. We used a standardized form with a pilot test to collect the following data: title, author, publication year, type of animal model, sample size, type of cannabinoid, the dose of cannabinoid, the dose of AOM, type of outcome measure, length of the experiment, reduction in ACF formation, reduction in the number of tumors, tumor volume reduction, pathway or function modified by cannabinoids, an increase of endocannabinoid levels, expression of apoptosis markers, and expression of proinflammatory markers.
2.4. Quality Assessment
The risk of bias was independently evaluated by two authors (EOG and LLT) following the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool [14]. The domains considered were random sequence generation, baseline characteristics, allocation concealment, random housing, blinding, random outcome assessment, outcome assessor blinding, incomplete outcome data, selective outcome report, and other sources of bias (contamination, the influence of funders, and analysis of errors) [14]. We reviewed each article, and we sought if any of these biases were present. Any discrepancy was discussed between 2 authors (EOG and LLT), and if not resolved, a third investigator intervened (AVV).
2.5. Data Analysis
Proportions were used as descriptive statistics for primary outcomes. Secondary outcomes were described qualitatively. A meta-analysis or measures of consistency were not performed due to characteristics of the studies and heterogeneity of articles.
3. Cannabinoids: Pharmacology and Generalities
Endocannabinoids are lipid mediators, including amides, esters, and ethers of polyunsaturated fatty acids, which were isolated from the porcine brain [15–17]. Anandamide’s structure resembles Δ9-THC structure, and it is synthesized from membrane phospholipids by the enzymes N-acyl phosphatidylethanolamine phospholipase D (NAPE-D) and lysophospholipase D (lyso-PLD) [18]. 2-Arachidonoyl-glycerol (2-AG) is an arachidonoyl ester, produced from diacylglycerols [18]. Endocannabinoids diffuse into the extracellular space and bind to CB1 and CB2 receptors, TRPV1, TRPM8, and GPR55 [18]. Anandamide and 2-AG are reuptake via an extraneuronal monoamine membrane transporter (EMT); then, they are degraded by the fatty acid amide hydrolase (FAAH) and the monoacylglycerol lipase (MAGL), respectively [16, 19]. Most known plant-derived cannabinoids include tetrahydrocannabinol (THC) and cannabidiol (CBD) [20]. These are tricyclic terpenoid compounds bearing a benzopyran moiety soluble in lipids and nonpolar organic solvents [20, 21].
Δ9-Tetrahydrocannabinol (THC) and anandamide have the highest affinity for the CB1 receptor, while CBD exhibits low affinity for these receptors [20, 21]. However, CBD has been proved to enhance endocannabinoid levels and indirectly activate CB receptors [8].
CB1 receptors constitute one of the most abundant receptors in the central nervous system. In the case of CB2 receptors, these are expressed in cells of the immune and hematopoietic system, spleen, and tonsils, modulating cytokine release and cellular immune migration [16]. Both receptors are metabotropic and belong to the G protein-coupled receptor (GPCR) family, and their activation produces inhibition of the adenylyl cyclase via G proteins (Gi/o) [16]. This decreases cAMP in the cell and activity of protein kinase A [16].
Cannabinoids may have alternative molecular targets other than classical CB1 and CB2 receptors [22]. Recently, orphan GPCRs like the GPR 55, GPR18, and GPR110 have been identified as new targets [22]. There is also increasing evidence that they can interact with ionotropic receptors such as the transient receptor potential cation channel subfamily V member 1 (TRPV1), and the transient receptor potential cation channel subfamily M member 8 (TRPM8) [23].
The transient receptor potential vanilloid receptor 1 (TRPV1) and the transient receptor potential cation channel subfamily M member 8 (TRPM8) are ionotropic channels that allow Na⁺ and Ca⁺⁺ entry to the cell [24]. Cannabidiol (CBD) and cannabigerol (CBG) close the TRPM8 channel, whereas CBD opens TRPV1 [24].
GPR55 is another GPCR, which is coupled to a Gα12/13 protein [25]. Several cancer lines like OVACAR3 (ovarian cancer cell line), PC-3, and DU145 (prostate cancer cell lines) exhibit expression of this orphan receptor [25]. Furthermore, Piñeiro et al. showed an autocrine activation of this receptor through his main endogenous agonist lysophosphatidylinositol (LPI) [25]. The receptor acts via activation of Gα12 and Gq family proteins, which activate Ras homolog gene family, member A (RhoA) kinase [26]. Overexpression of GPR55 produces increased levels of pERK in HEK-293, breast carcinoma, and glioma cells, while pAKT levels are increased in ovarian and prostate cancer cells [25, 27].
The high expression of GPR55 is also linked to high proliferation indices in human breast tumors and Glioblastoma [26]. The best-studied cannabinoid with actions on the GPR55 in colonic tissue is O-1602 [28]. It is highly speculative that the compound exerts its antineoplastic effects on CRC tissue through the GPR55 receptor, as the cannabinoid has shown agonist activity on this receptor [28]. More research is needed before we can conclude the actions of cannabinoids on this receptor.
Ceramide’s synthesis begins with the enzyme serine palmitoyltransferase (SPT) [29]. Gustaffson et al. have demonstrated that the cannabinoids Win55,212-2 and R (+)-methanandamide induce ceramide accumulation mainly through CB1 and CB2 activation, which acts on SPT [30, 31]. Both studies were performed in mantle cell lymphoma cells (L718, L1547, L1676, and Rec-1) [30, 31]. Furthermore, in neural tissues (rat glioma C6 line and H4 neuroglioma), R (+)-methanandamide and JWH-133 (CB2 agonist) also induce ceramide accumulation [32, 33]. Ceramide provokes a loss of mitochondrial membrane potential and caspase activation, subsequently [30, 31].
3.1. Reported Effects of Cannabinoids on CRC
Most cultured colonic cancer cells used for in vitro assessment express CB1, CB2, TRPM8, and GPR55 (G protein-coupled receptor) [34–39]. Additionally, adenomatous polyps and colorectal cancer tissue have increased the amounts of the endogenous cannabinoids AEA and 2-AG (3-fold versus 2-fold, respectively) [40]. This has been suggested to be a mechanism of self-protection against further tumor progression [40, 41]. Cannabinoids and phytocannabinoids have, therefore, effects on colonic cancer tissues since CRC tissues produce those (endogenous cannabinoids) and express some of their receptors (Figure 1).
... 9 In addition, increasing evidence suggest that ceramides are involved in cannabinoid-induced apoptosis. [10][11][12] The first clinical trials exploring the anti-tumor effect of cannabinoids are being conducted in some solid tumors (NCT02255292) including brain cancer (NCT01654497); other studies have recently been completed in glioma (NCT00314808) and glioblastoma (NCT01812616). ...
... Remarkably, the activity of this pathway is regulated by the SPT enzyme. 10 We found that exposure to cannabinoids upregulates SPT, increasing the levels of ceramides. In fact, blockage of ceramide synthesis confirmed the involvement of ceramide in cannabinoid-induced apoptosis. ...
... Akt is the most strongly activated signaling pathway by cannabinoids in MM cells, inducing a biphasic response similar to the cannabinoid-induced response reported in gliomas. 41 This inhibition of Akt is in line with previous observations reported using other cannabinoid derivatives in other tumors such as melanoma and breast cancer, and suggests that the apoptotic effect of cannabinoids also relies on Akt inhibition, [10][11][12]41 Concerning MAPKs pathways and given the low magnitude of regulation observed in this study, we suggest that it merely might be a consequence of crosstalk between pathways. ...
... Existing evidence indicated that cannabinoid-induced apoptosis was mediated via the activation of cannabinoid receptors, which in turn triggers the proapoptotic mitochondrial intrinsic pathway. In glioma cells and pancreatic cancer cells, activation of cannabinoid receptors resulted in two peaks of ceramide generation by the mechanisms of sphingomyelin hydrolysis and de novo synthesis, respectively [53,54]. Studies showed that the second peak of ceramide accumulation accounted for the apoptosis induced by cannabinoids [55]. ...
... Cannabinoid-induced inhibition of Akt was found to result from de novo synthesis of ceramide. In glioma cells, cannabinoids induce intracellular ceramide accumulation by mechanisms of sphingomyelin hydrolysis and ceramide synthesis de novo [54,89]. In turn, de novo synthesized ceramide leads to activation of ERK and inhibition of Akt. ...
Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the lack of estrogen receptors, progesterone receptors, and HER-2 receptors. Thus, TNBC tumors do not benefit from the current therapies targeting estrogen receptor or HER-2. Therefore, there is an urgent need to develop novel treatments for this subtype of breast cancer. Marijuana is a common name given to Cannabis plants, a group of plants in the Cannabis genus of the Cannabaceae family. Cannabis plants are among the oldest cultivated crops, traced back at least 12,000 years and are well known for their multipurpose usage, including medicinal purposes. The main active compounds extracted from Cannabis plants are 21-carbon-containing terpenophenolics, which are referred to as phytocannabinoids. Of these, the tetrahydrocannabinol (THC) group contains highly potent cannabinoids, including delta-9-tetrahydrocannabinol (9-THC) and delta-8-tetrahydrocannabinol(8-THC), which are the most abundant THCs and are primarily responsible for the psychological and physiological effects of marijuana. The use of Cannabis plants for medicinal purposes was first recorded in2337 BC in China, where Cannabis plants were used to treat pains, rheumatism, and gout. Recently, several cannabinoids have been approved for several treatments, one of which is the treatment of nausea and vomiting caused by chemotherapy in cancer patients. Furthermore, increasing evidence shows that cannabinoids not only attenuate side effects due to cancer treatment but might also potentially possess direct antitumor effects in several cancer types, including breast cancer. However, the antitumor activity of cannabinoids has been variable in different studies and even promoted tumor growth in some cases. In addition, mechanisms of cannabinoids actions in cancer remain unclear. This review summarizes evidence about the mixed actions and mechanisms of cannabinoids in cancer in general and TNBC in particular.
... Phytocannabinoids (PCs) refer to those cannabinoid compounds synthesized in plants. There are two other different types of cannabinoids made in the mammalians called endocannabinoids (ECs), as well as synthetic cannabinoids (SCs) [82][83][84][85]. The ECs, mainly anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), mediate their effects through two groups of receptors designated as cannabinoid-1 abbreviated as CB1 and cannabinoid-2 that is known as CB2 [64,86,87]. ...
... Phytocannabinoids (PCs) refer to those cannabinoid compounds synthesized in plants. There are two other different types of cannabinoids made in the mammalians called endocannabinoids (ECs), as well as synthetic cannabinoids (SCs) [82][83][84][85]. The ECs, mainly anandamide (AEA) and 2arachidonoyl glycerol (2-AG), mediate their effects through two groups of receptors designated as cannabinoid-1 abbreviated as CB1 and cannabinoid-2 that is known as CB2 [64,86,87]. ...
Cannabis is an annual plant with a long history of use as food, feed, fiber, oil, medicine, and narcotics. Despite realizing its true value, it has not yet found its true place. Cannabis has had a long history with many ups and downs, and now it is our turn to promote it. Cannabis contains approximately 600 identified and many yet unidentified potentially useful compounds. Cannabinoids, phenolic compounds, terpenoids, and alkaloids are some of the secondary metabolites present in cannabis. However, among a plethora of unique chemical compounds found in this plant, the most important ones are phytocannabinoids (PCs). Over hundreds of 21-22-carbon compounds exclusively produce in cannabis glandular hairs through either polyketide and or deoxyxylulose phosphate/methylerythritol phosphate (DOXP/MEP) pathways. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are those that first come to mind while talking about cannabis. Nevertheless, despite the low concentration, cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabinodiol (CBND), and cannabinidiol (CBDL) may have potentially some medical effects. PCs and endocannabinoids (ECs) mediate their effects mainly through CB1 and CB2 receptors. Despite all concerns regarding cannabis, nobody can ignore the use of cannabinoids as promising tonic, analgesic, antipyretic, antiemetic, anti-inflammatory, anti-epileptic, anticancer agents, which are effective for pain relief, depression, anxiety, sleep disorders, nausea and vomiting, multiple sclerosis, cardiovascular disorders, and appetite stimulation. The scientific community and public society have now increasingly accepted cannabis specifically hemp as much more than a recreational drug. There are growing demands for cannabinoids, mainly CBD, with many diverse therapeutic and nutritional properties in veterinary or human medicine. The main objective of this review article is to historically summarize findings concerning cannabinoids, mainly THC and CBD, towards putting these valuable compounds into food, feed and health baskets and current and future trends in the consumption of products derived from cannabis.
... In addition to these well-established events, the signalling pathways induced by cannabinoid receptors are more diverse than originally recognized. It is now well known that cannabinoids can modulate sphingolipid-metabolizing pathways by cAMP Ceramide accumulation ATP Control of cell function AC increasing the intracellular levels of ceramide [51], an ubiquitous lipid secondary messenger. In fact, CB1 receptor activation has been shown to induce sphingomyelin hydrolysis and acute ceramide production in both primary astrocytes [52] and glioma cell lines, through sphingomyelinases [53]. ...
... The functional coupling of CB1 with sphingomyelinases involves different adaptor proteins, one of which is the factor associated with neutral sphingomyelinase activation (FAN) [54]. The CB1 activation also induces an extended ceramide accumulation due to the stimulation of its de novo synthesis, by serine palmitoyltransferase [51]. ...
... For instance, a lower ECS tone in diet-induced insulin-resistant mice was associated with decreased hepatic ceramide synthesis (21). This finding corroborates the hypothesis that a higher ECS tone may lead to greater intracellular ceramide content (20,22,23), thus reducing mitochondrial respiratory activity and ultimately wholebody energy metabolism (20,24,25). The ECS may increase sphingolipid synthesis via gene expression regulation of the enzyme serine palmitoyltransferase (SPT), which catalyzes the rate-limiting step of ceramide and sphingomyelin synthesis (21,23,26). ...
... Additionally, sphingomyelins play a central role in pathways leading to ceramide accumulation, as mediated by the ECS via sphingomyelin hydrolysis (22) or by SPT-dependent de novo synthesis (21,22). Thus, an ECS-induced increase in ceramide synthesis and a subsequent ceramide-mediated decrease in mitochondrial respiratory activity may also involve sphingomyelins (20,(24)(25)(26). Our present results are consistent with a key role for sphingomyelins in EE control and with an influence of the ECS on skeletal muscle sphingomyelin content. ...
Context
Skeletal muscle endocannabinoids and sphingolipids (particularly, sphingomyelins) are inversely associated with sleeping energy expenditure (SLEEP) in humans. The endocannabinoid system may increase sphingolipid synthesis via cannabinoid receptor-1.
Objective
To investigate in human skeletal muscle whether endocannabinoids are responsible for the effect of sphingomyelins on SLEEP.
Design
Muscle endocannabinoid (anandamide, AEA; 2-arachidonoylglycerol, 2-AG), endocannabinoid congeners (oleoylethanolamide, OEA; palmitoylethanolamide, PEA) and sphingomyelin content were measured using liquid chromatography/mass spectrometry. SLEEP was assessed in a whole-room indirect calorimeter. Mediation analyses tested whether the inverse associations between sphingomyelins and SLEEP depended on endocannabinoids and endocannabinoid-related OEA and PEA.
Setting
Inpatient study.
Participants
Fifty-three overweight Native Americans.
Main Outcome Measure
SLEEP.
Results
AEA (r = 0.45, P = 0.001), 2-AG (r = 0.47, P = 0.0004), OEA (r = 0.27, P = 0.05) and PEA (r = 0.53, P < 0.0001) concentrations were associated with the total sphingomyelin content. AEA, OEA, and PEA correlated with specific sphingomyelins (SM18:1/23:0, SM18:1/23:1, and SM18:1/26:1) previously reported to be determinants of SLEEP in Native Americans (all r > 0.31, all P < 0.03). Up to 1/2 of the negative effect of these specific sphingomyelins on SLEEP was accounted by AEA (all P < 0.04), rendering the direct effect by sphingomyelin per se on SLEEP negligible (P > 0.05).
Conclusions
In skeletal muscle, AEA is responsible for the sphingomyelin effect on SLEEP, indicating that endocannabinoids and sphingomyelins may jointly reduce human whole-body energy metabolism.
... In addition to the relationship between phytocannabinoids and mitochondria, several studies in the literature have also focused their attention on the involvement of the ECS in mitochondrial function. The ECS has evolved in a living organism to ensure the survival of the animal, and because of this, it interacts and modulates other systems, such as the mitochondria (Gomez et al., 2011;Howlett et al., 2010;Moreira et al., 2012;Nunn et al., 2012;Puighermanal et al., 2009;Velasco et al., 2005). Several studies suggested that THC, AEA, and HU210 could inhibit mitochondrial function. ...
Introduction
The contribution of Cannabinoid type 1 receptor (CB1) in mitochondrial energy transduction mechanisms and mitochondrial activities awaits deeper investigations. Our study aims to assess the impact of CB1 absence on the mitochondrial compartment in the liver, focusing on both functional aspects and remodeling processes.
Methods
We used CB1−/− and CB1+/+ male mice. Cytochrome C Oxidase activity was determined polarographically. The expression and the activities of separated mitochondrial complexes and supercomplexes were performed by using Blue-Native Page, Western blotting and histochemical staining for in-gel activity. Key players of Mitochondrial Quality Control processes were measured using RT-qPCR and Western blotting. Liver fine sub-cellular ultrastructural features were analyzed by TEM analysis.
Results and discussion
In the absence of CB1, several changes in the liver occur, including increased oxidative capacity, reduced complex I activity, enhanced complex IV activity, general upregulation of respiratory supercomplexes, as well as higher levels of oxidative stress. The mitochondria and cellular metabolism may be affected by these changes, increasing the risk of ROS-related damage. CB1−/− mice show upregulation of mitochondrial fusion, fission and biogenesis processes which suggests a dynamic response to the absence of CB1. Furthermore, oxidative stress disturbs mitochondrial proteostasis, initiating the mitochondrial unfolded protein response (UPRmt). We noted heightened levels of pivotal enzymes responsible for maintaining mitochondrial integrity, along with heightened expression of molecular chaperones and transcription factors associated with cellular stress reactions. Additionally, our discoveries demonstrate a synchronized reaction to cellular stress, involving both UPRmt and UPRER pathways.
... The CB1 and CB2 receptors are grouped under the transmembrane-spanning G-protein coupled family of receptors [25]. Their activation results in downstream cellular physiological changes [25,27,[29][30][31][32][33], such as lowered cAMP and AC levels, initiation of mitogen-activated protein (MAP) kinases, and inhibition of calcium channels [27,32,[34][35][36]. ...
Background
A global increase in cannabis use has led to questions about its effects on fertility. The rise in consumption amongst women of reproductive age is a growing concern, as this group is vulnerable in terms of reproductive health. Ample evidence suggests that the psychoactive component of cannabis, Δ⁹-Tetrahydrocannabinol (THC), interacts with the endocannabinoid system (ECS), that helps regulate mammalian reproduction. This study aimed to research the epigenetic effects of THC in bovine granulosa cells (GCs) by (1) investigating global DNA methylation via measuring 5-mC and 5-hmC levels; (2) measuring key methylation regulators, including the methylating enzymes DNMT1, DNMT3a, DNMT3b and the demethylases TDG and TET1/2/3; and (3) assessing fertility-associated miRNAs key in developmental competency, including miR-21, -155, -33b, -324 and -346.
Methods
Bovine GCs were used as a translational model for reproductive toxicity in humans. To determine THC effects, GCs were isolated from Cumulus-Oocyte-Complexes (COCs) from bovine ovaries, cultured in vitro for 7 days, or until confluent, and cryopreserved at passage 1 (P1). For experimentation, cells were thawed, cultured until passage 2 (P2), serum restricted for 24-h and treated for 24-h in one of five groups: control, vehicle (1:1:18 ethanol: tween: saline) and three clinically relevant THC doses (0.032, 0.32 and 3.2 μM). Global methylation was assessed by measuring 5-mC and 5-hmC levels with flow cytometry. To assess mRNA and protein expression of methylation regulators and miRNA profiles, qPCR and Western Blotting were utilized. Shapiro-Wilk test was used to determine normality within datasets. One-way ANOVA was applied to determine statistical significance using GraphPad Prism 6.0.0.
Results
Results indicate a significant decrease (p = 0.0435) in 5-mC levels following low THC exposure, while no changes were observed in 5-hmC levels. A significant increase in DNMT1 following high THC exposure at the RNA level (p < 0.05) and a significant increase following low THC exposure at the protein level (p = 0.0048) were also observed. No significant differences were observed in DNMT3a/3b, TDG, TET1/2/3 mRNAs or in any of the miRNAs analyzed.
Conclusions
This research suggests that THC mainly affects DNA methylation, but not miRNA profiles, ultimately altering gene expression and likely impairing oocyte competence, maturation, and fertilization potential.
... Briefly, ceramides are sphingosine-based lipids that act as signaling molecules, and their proapoptotic activities are partially induced by the ability to inhibit the Akt pathway [87]. However, CB1 receptors and cannabinoids may modulate the metabolizing pathways of sphingolipids by inducing sphingomyelin breakdown and by increasing ceramide levels via enhanced de novo synthesis [88]. Hence, the interplay between the beneficial effects of cannabinoids and their ability to increase the level of pro-apoptotic molecules such as ceramides needs to be investigated in future studies. ...
In the last few decades, endocannabinoids, plant-derived cannabinoids and synthetic cannabinoids have received growing interest as treatment options in neurodegenerative conditions. In various experimental settings, they have displayed antioxidative, anti-inflammatory, antiapoptotic, immunomodulatory, and neuroprotective effects. However, due to numerous targets and downstream effectors of their action, the cellular and molecular mechanisms underlying these effects are rather complex and still under discussion. Cannabinoids are able to neutralize free radicals and modulate the production of reactive oxygen species and the activity of antioxidative systems acting on CB1 and CB2 cannabinoid receptors. The activation of CB1 receptors stimulates signaling pathways involved in antioxidative defense and survival (such as the phosphoinositide 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK), and Nrf2 pathways) and regulates glutamatergic signaling, the activation of N-methyl-D-aspartate (NMDA) receptors, calcium influx, and the induction of Ca2+-regulated signaling cascades, whereas the neuroprotective effects mediated by CB2 receptors are due to the suppression of microglial activation and the release of prooxidative and proinflammatory mediators. This review summarizes the main molecular mechanisms and new advances in understanding the antioxidative and neuroprotective effects of cannabinoids. Because of the plethora of possible pharmacological interventions related to oxidative stress and cannabinoid-mediated neuroprotection, future research should be directed towards a better understanding of the interplay between activated signal transduction pathways and molecular targets with the aim to improve treatment options and efficacy by targeting the endocannabinoid system.
... Activation of both the receptors vows the cellular signalling via G protein alpha subunits (Gi/o), causing inhibition of adenylyl cyclase. This also activates various other pathways like phosphoinositide 3-kinase (PI3K) pathways, mitogen-activated protein kinase (MAPK) pathways, protein kinase B (Akt), modulation of ion channels (through CB1 receptors), and ceramide signalling pathways, especially in tumorigenesis affects the proliferation [11][12][13][14][15]. Many other kinds of transmembrane proteins are also represented in the system in addition to CB1 and CB2. ...
Various preclinical and clinical studies exhibited the potential of cannabis against various diseases, including cancer and related pain. Subsequently, many efforts have been made to establish and develop cannabis-related products and make them available as prescription products. Moreover, FDA has already approved some cannabis-related products, and more advancement in this aspect is still going on. However, the approved product of cannabis is in oral dosage form, which exerts various limitations to achieve maximum therapeutic effects. A considerable translation is on a hike to improve bioavailability, and ultimately, the therapeutic efficacy of cannabis by the employment of nanotechnology. Besides the well-known psychotropic effects of cannabis upon the use at high doses, literature has also shown the importance of cannabis and its constituents in minimizing the lethality of cancer in the preclinical models. This review discusses the history of cannabis, its legal aspect, safety profile, the mechanism by which cannabis combats with cancer, and the advancement of clinical therapy by exploiting nanotechnology. A brief discussion related to the role of cannabinoid in various cancers has also been incorporated. Lastly, the information regarding completed and ongoing trials have also been elaborated.
... Plant-derived compounds such as cannabinoids may also indirectly influence sphingolipid metabolism. For example, G-coupled protein receptor cannabinoid1 (CB1) activation increased ceramide levels in primary astrocytes and glioma cells via factor associated with neutral sphingomyelinase activation which induces the breakdown of sphingomyelin into ceramide and phosphorylcholine [49]. Delta9-tetrahydrocannabinol and the synthetic cannabinoid agonist WIN-55,212-2 administration significantly regressed the malignant gliomas in Wistar rats and in mice with recombination-activating gene 2 deficiency. ...
Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.
... There are also evidences that CB1 activates the PI3K/Akt pathway [39] and modulates sphingolipid-metabolizing pathways by increasing the intracellular levels of ceramide [40]. The most pronounced cannabinoid-induced gene expression changes occur through the cAMP-responsive element-binding protein (CREB) and MAPK signalling. ...
Although cannabis use is increasing in general population, their prevalence among young adults is remarkably high. In recent years, their medical use gained a renewed interest. However, it can underline the reputation of cannabis being a harmless drug. Between cannabinoids, uniquely found on the cannabis plant, Δ9-tetrahydrocannabinol (THC) is the well-studied compound. It is responsible for the psychoactive effects via central cannabinoid receptors. Nevertheless, cannabinoids interact with other chemical signalling systems such as the hypothalamic-pituitary-gonadal axis. THC indirectly decreases gonadotropin-releasing hormone (GnRH) secretion by the hypothalamus. The consequences are diverse, and several key hormones are affected. THC disturbs important reproductive events like folliculogenesis, ovulation and sperm maturation and function. Although generally accepted that cannabinoid consumption impacts male and female fertility, prevailing evidence remains largely on pre-clinical studies. Here, we introduce cannabinoids and the endocannabinoid system, and we review the most prominent clinical evidence about cannabis consumption in reproductive potential and teratogenicity.
... On the other hand, 2-AG has unfavorable effects for example in the context of inflammation in obesity [73,74] and liver disease [75,76]. It is of note, that ceramides and endocannabinoid paths are inter-connected [67]. Hence, the lipid pattern under sapropterin rather than an individual lipid is likely to determine the outcome. ...
Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood–brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
... Thus, there is good evidence that the CB 1 receptor may activate mitogen-activated kinases (ERK1 and 2, JNK and p38 signaling involving bg subunits in the G protein or b-arrestin interactions), 87-89 the PI3K-Akt signaling, 90 and the generation of ceramide and other signals related to reticulum stress. 91 The CB 1 receptor signaling also involves the inhibition of voltage-dependent Ca 21 channels, a classic effect of GPCRs linked to G i/o proteins, which reduces the conductance of this cation, and also the increase in the conductance for K 1 through the GIRK-type channels facilitating output of this ion. 92 In both cases, the changes in ion conductance are dependent on metabotropic signaling and are always compatible with a reduction in neuronal activity (e.g. ...
Phytocannabinoids, the most singular chemical constituents present in the Cannabis sativa plant, were discovered and characterized up to the 1960s. By contrast, endocannabinoids and their receptors were discovered in the late 1980s and early 1990s, as a result of the efforts to identify the mechanism(s) of action of plant-derived cannabinoids. The discovery of this system, the so-called “endogenous cannabinoid system”, enabled by itself important advances in the knowledge of the biology of numerous cells, tissues and organs in the human body, but it has also provided a strong rationale to understand why the cannabis plant has been used from ancient times with therapeutic purposes for human pathologies. It also explained the recent advances in the development of cannabinoid-based medicines, including medicinal cannabis and also plant-derived and synthetic molecules formulated as Marinol®, Cesamet®, Sativex® and, more recently, Epidiolex®. The latter drugs have been approved in many countries for the treatment of vomiting and nausea, anorexia–cachexia syndrome, spasticity in multiple sclerosis, and seizures in infantile refractory epilepsies, respectively. This chapter aims to review the scientific knowledge generated around the research on plant-derived cannabinoids and the discovery of endocannabinoids, their receptors and molecular machinery for generating and inactivating these endogenous compounds, as a necessary step to develop novel medicines based on the activation, inhibition or modulation of the endocannabinoid system for the treatment of different central and peripheral pathologies.
... Different cannabinoid ligands may activate diverse pathways, leading to diverse responses, a concept termed 'biased signalling' (Ibsen et al., 2017). Furthermore, it has been demonstrated that cannabinoids, the natural or synthetic molecules able to bind the cannabinoid receptors, have the ability to modulate the sphingolipid-metabolizing pathways, by increasing ceramidase activity and leading to higher levels of ceramide, a second messenger that is able to control cell fate (Velasco et al., 2005) (Fig. 2). Besides the classic CB1 and CB2, other receptors can be activated by cannabinoids. ...
Background:
The endocannabinoid system (ECS) consists of the cannabinoid receptors CB1 and CB2, the main endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their metabolic enzymes N-acylphosphatidylethanolamine-specific phospholipase D, fatty acid amide hydrolase, diacylglycerol lipase and monoacylglycerol lipase. This system is involved in the modulation of essential physiological processes. Its role in the reproductive system has become significantly important in recent years, given its major role in events such as gametogenesis, decidualisation, implantation and placentation.
Objective and rationale:
In this paper, we review the literature and summarize the role of the ECS elements in reproduction and their potential as early markers for diagnosis of reproductive disorders or as pharmacological targets for treatment.
Search methods:
Original research and review papers published from 1964 to June 2019 were selected in terms of relevance, reliability and quality by searching PubMed, MEDLINE and Web of Science, using the following search terms: endocannabinoid system and endometriosis; endocannabinoid system and ectopic pregnancy; endocannabinoid system and miscarriage; endocannabinoid system and pre-eclampsia; endocannabinoid system and endometrial cancer; endocannabinoid system and reproduction; endocannabinoid, endometrium; placenta; N-acylethanolamines; anandamide; 2-arachidonoylglycerol; and cannabinoids.
Outcomes:
This review demonstrates relevant information concerning ECS alterations in endometriosis, ectopic pregnancy, miscarriage, pre-eclampsia and endometrial cancer. We highlight the importance of the endocannabinoids in endometrial and placental physiology and pathophysiology, from studies in vitro and in vivo and in clinical observations. The most studied of the endogenous cannabinoids is AEA. The levels of AEA were increased in plasma of patients with endometriosis and miscarriage, as well as in the fallopian tube of women with ectopic pregnancy and in endometrial biopsies of endometrial cancer. Changes in the pattern of expression of the cannabinoid receptor CB1 were also observed in endometrial biopsies of endometriosis, fallopian tube and decidua of patients with ectopic pregnancy and pre-eclamptic placenta. Moreover, alterations in CB2 expression have been reported in association with endometrial cancer. In general, studies on the cannabinoid signalling through CB2 and on the biological activities of the other major endocannabinoid, namely 2-AG, as well as its metabolic enzymes are scarce and avidly required.
Wider implications:
The pathophysiological mechanisms involved in the described endometrial and placental pathologies are still unclear and lack the means for an early diagnosis. Based on current evidence, though alterations in ECS are demonstrated at tissue level, it is difficult to associate plasmatic changes in AEA with specific endometrial and placental diseases. Thus, pairing alterations in AEA levels with 2-AG and/or other endocannabinoid-like molecules may provide more accurate and early diagnoses. In addition, patients may benefit from new therapies that target the ECS and endocannabinoid signalling.
... In particular, numerous studies have explored the anti-proliferative effects of these compounds in various tumours [8]. Following the interaction with their specific receptors, cannabinoids can trigger several different signalling pathways [9], including the accumulation of ceramide, the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK, as well as the increase in calcium concentration, reactive oxygen species (ROS) production and the modulation of pro-and anti-apoptotic members of the Bcl-2 family [10][11][12][13]. More recently, a relationship between the cannabinoid system and miRNA expression has been evidenced. ...
WIN55,212-2 (WIN) is a synthetic agonist of cannabinoid receptors that displays promising antitumour properties. The aim of this study is to demonstrate that WIN is able to block the migratory ability of osteosarcoma cells and characterize the mechanisms involved. Using wound healing assay and zymography, we showed that WIN affects cell migration and reduces the activity of the metalloproteases MMP2 and MMP9. This effect seemed to be independent of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein involved in tissue remodeling and extracellular matrix deposition. SPARC release was indeed prevented by WIN, and SPARC silencing by RNA interference did not influence the effect of the cannabinoid on cell migration. WIN also increased the release of extracellular vesicles and dramatically upregulated miR-29b1, a key miRNA that modulates cell proliferation and migration. Interestingly, reduced cell migration was observed in stably miR-29b1-transfected cells, similarly to WIN-treated cells. Finally, we show the absence of SPARC in the extracellular vesicles released by osteosarcoma cells and no changes in SPARC level in miR-29b1 overexpressing cells. Overall, these findings suggest that WIN markedly affects cell migration, dependently on miR-29b1 and independently of SPARC, and can thus be considered as a potential innovative therapeutic agent in the treatment of osteosarcoma.
... CB2-selective agonists regulate cell proliferation, differentiation, transformation, and death by stimulating major components of the MAPK pathway, including ERK1/2, p38, and c-Jun N-terminal kinases (40). CB2 also induces apoptosis, necrosis, and autophagy through modulations of the Akt-phosphoinositide 3-kinase pathways, as well as modulating arrestin activity and ceramide production (41)(42)(43)(44). Structurally, CB2 shares approximately 50% amino acid homology with CB1 in the transmembrane region; however, these two receptors are functionally distinct, as they exhibit differences in binding affinity for cannabinoid agonists and antagonists (45). ...
This paper reviews the endocannabinoid system and focuses on the role of endocannabinoids in bone metabolism and their potential use in the management of conditions associated with bone loss.
Context:
The endocannabinoid system uses tissue-specific lipid ligands and G protein-coupled transmembrane receptors to regulate neurological, metabolic, and immune responses. Recent studies demonstrate that the endocannabinoid system influences bone metabolism. With the increasing use of endocannabinoid mimetics, e.g. tetrahydrocannabinol (THC) and cannabidiol (CBD), endocannabinoids' involvement in bone growth and remodeling has become clinically relevant.
Evidence acquisition:
This literature review is based upon a search of Pubmed and Google Scholar databases, as of June 2019, for all English-language publications relating to cannabinoids and bone. We evaluated retrieved articles for relevance, experimental design, data acquisition, statistical analysis, and conclusions.
Evidence synthesis:
Preclinical studies establish a role for endocannabinoids in bone metabolism. These studies yield complex and often contradictory results attributed to differences in the specific experimental model examined. Studies using human cells or subjects are limited.
Conclusions:
In vitro and animal models document that endocannabinoids participate in bone biology. The relevance of these observations to humans is not clear. The increasing chronic use of medical and recreational cannabis underscores the need to better understand the role of endocannabinoids in human bone metabolism. Moreover, it is important to evaluate the role of endocannabinoids as a therapeutic target to prevent and treat disorders associated with bone loss.
... CRIP1a expression modulates ERK1/2 phosphorylation and the coupling selectivity among different G-protein α subunits [26], which are related to the functional selectivity of CB 1 ligands. CB 1 receptors also transduce signals through other G-protein independent pathways that employ ceramide as the second messenger [27]. In addition to regulating the activities of the protein kinases, CB 1 activation inhibits voltage-gated N-and P/Q-type calcium channels and activates A-type, and G-proteincoupled inwardly rectifying potassium channels (GIRK) [2]. Figure 2 illustrates the signaling network of the CB 1 receptor. ...
The cannabinoid type-1 (CB1) receptor, a G-protein-coupled receptor, is an attractive target for drug discovery due to its involvement in many physiological processes. Historically, drug discovery efforts targeting the CB1 receptor have focused on the development of orthosteric ligands that interact with the active site to which endogenous cannabinoids bind. Research performed over the last several decades has revealed substantial difficulties in translating CB1 orthosteric ligands into druggable candidates. The difficulty is mainly due to the adverse effects associated with orthosteric CB1 ligands. Recent discoveries of allosteric CB1 modulators provide tremendous opportunities to develop CB1 ligands with novel mechanisms of action; these ligands may potentially improve the pharmacological effects and enhance drug safety in treating the disorders by regulating the functions of the CB1 receptor. In this paper, we review and summarize the complex pharmacological profiles of each class of CB1 allosteric modulators, the development of new classes of CB1 allosteric modulators and the results from in vivo assessments of their therapeutic value.
... Ceramide may also display some regulatory activity on L-type Ca 2+ currents [115] but-to the best of our knowledge-not on the other AEA receptors, including CB1. From a clinical point of view, the exacerbated toxicity of AEA in the presence of ceramide [50] and the striking interplay between the two signaling systems [116] should be taken into consideration for developing new anti-tumoral strategies based on the use of endocannabinoids [50,117,118]. In this respect, understanding the molecular mechanisms underlying the biological activity of AEA and related endocannabinoids is of primary importance for the use of these molecules in the treatment of human diseases. ...
Anandamide is a lipid neurotransmitter derived from arachidonic acid, a polyunsaturated fatty acid. The chemical differences between anandamide and arachidonic acid result in a slightly enhanced solubility in water and absence of an ionisable group for the neurotransmitter compared with the fatty acid. In this review, we first analyze the conformational flexibility of anandamide in aqueous and membrane phases. We next study the interaction of the neurotransmitter with membrane lipids and discuss the molecular basis of the unexpected selectivity of anandamide for cholesterol and ceramide from among other membrane lipids. We show that cholesterol behaves as a binding partner for anandamide, and that following an initial interaction mediated by the establishment of a hydrogen bond, anandamide is attracted towards the membrane interior, where it forms a molecular complex with cholesterol after a functional conformation adaptation to the apolar membrane milieu. The complex is then directed to the anandamide cannabinoid receptor (CB1) which displays a high affinity binding pocket for anandamide. We propose that cholesterol may regulate the entry and exit of anandamide in and out of CB1 by interacting with low affinity cholesterol recognition sites (CARC and CRAC) located in transmembrane helices. The mirror topology of cholesterol binding sites in the seventh transmembrane domain is consistent with the delivery, extraction and flip-flop of anandamide through a coordinated cholesterol-dependent mechanism. The binding of anandamide to ceramide illustrates another key function of membrane lipids which may occur independently of protein receptors. Interestingly, ceramide forms a tight complex with anandamide which blocks the degradation pathway of both lipids and could be exploited for anti-cancer therapies.
... Short-term 163 ceramide generation involves sphingomyelin hydrolysis via sphingomyelinase 164 (SMase) activation through the adaptor protein FAN, acting on metabolic regula-165 tion (Sanchez et al. 2001b). Long-term ceramide generation may occur via serine 166 palmitoyltransferase (SPT) induction and enhanced de novo ceramide synthesis 167 (Velasco et al. 2005;Herrera et al. 2006). 168 Moreover, besides these signalling pathways mediated by cannabinoid receptors 169 and directly involved in cell survival, proliferation and apoptosis, there are also 170 evidences that some cannabinoids may stimulate transient receptor potential 171 vanilloid subtype 1 (TRPV1) (Zygmunt et al. 1999) and peroxisome proliferator- Numerous studies demonstrate that cannabinoids may affect cell proliferation and 177 death by a multitude of signalling pathways. ...
Endocannabinoids are bioactive lipids that modulate various physiological processes through G-protein-coupled receptors (CB1 and CB2) and other putative targets. By sharing the activation of the same receptors, some phytocannabinoids and a multitude of synthetic cannabinoids mimic the effects of endocannabinoids. In recent years, a growing interest has been dedicated to the study of cannabinoids properties for their analgesic, antioxidant, anti-inflammatory and neuroprotective effects. In addition to these well-recognized effects, various studies suggest that cannabinoids may affect cell survival, cell proliferation or cell death. These observations indicate that cannabinoids may play an important role in the regulation of cellular homeostasis and, thus, may contribute to tissue remodelling and cancer treatment. For a long time, the study of cannabinoid receptor signalling has been focused on the classical adenylyl cyclase/cyclic AMP/protein kinase A (PKA) pathway. However, this pathway does not totally explain the wide array of biological responses to cannabinoids. In addition, the diversity of receptors and signalling pathways that endocannabinoids modulate offers an interesting opportunity for the development of specific molecules to disturb selectively the endogenous system. Moreover, emerging evidences suggest that cannabinoids ability to limit cell proliferation and to induce tumour-selective cell death may offer a novel strategy in cancer treatment. This review describes the main properties of cannabinoids in cell death and attempts to clarify the different pathways triggered by these compounds that may help to understand the complexity of respective molecular mechanisms and explore the potential clinical benefit of cannabinoids use in cancer therapies.
... Cannabinoids can promote cell death in tumours by inducing autophagy and modulating membrane sphingolipids 3,4 . JWH015 induced conversion from the soluble form of LC3 (LC3-I) to autophagosome-associated form (LC3-II) and mediated inhibition of mTORc1, evaluated as reduced phosphorylation of p70S6, was detected already 6 hrs after agonist stimulation and counteracted by the autophagosome fusion inhibitor bafilomycin. ...
... There are more links between S1P, TNF-alpha and the rest of lipid mediators found significant in the present study, because one feature was putatively identified as heneicosatetraenoyl ethanolamine and the CB1 cannabinoid receptor is coupled to ceramide production mediated by sphingomyelinase (acute response) and de novo synthesis by serine-palmitoyl transferase (long-term response) [20]. ...
Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The current study has examined differences in metabolite profiles (fingerprints) of plasma from mice (strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months. One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was established, whereas the other group did not receive the treatment. Age and sex-matched mice not exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints (untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The signals were processed and a large number of possible metabolites were found (23944). Multivariate data analysis provided models that highlighted the differences between control and smoke exposed mice in both conditions. Accurate masses of features (possible compounds) representing significant differences were searched using online public databases. Lipid mediators, related to intracellular signaling in inflammation, were found among the metabolites putatively identified as markers of the different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids point at the relevance of such metabolites in the regulation of the processes related to tissue regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could potentially guide the characterization of relevant metabolites leading the regeneration of lungs in emphysema disease.
... In contrast, CB2Rs are associated with a more circumscribed repertoire of downstream effector pathways and do not modulate PI3K/PKB signaling, inward rectifying K + channels, or Ca 2+ channels [54]. Both CB1R and CB2R increase ceramide levels either by increasing sphingomyelin hydrolysis or de novo ceramide synthesis and can modulate gene transcription [55]. ...
The phenomenon of functional selectivity, whereby a ligand preferentially directs the information output of a G-protein coupled receptor (GPCR) along (a) particular effector pathway(s) and away from others, has redefined traditional GPCR signaling paradigms to provide a new approach to structure-based drug design. The two principal cannabinoid receptors (CBRs) 1 and 2 belong to the class-A GPCR subfamily and are considered tenable therapeutic targets for several indications. Yet conventional orthosteric ligands (agonists, antagonists/inverse agonists) for these receptors have had very limited clinical utility due to their propensity to incite on-target adverse events. Chemically distinct classes of cannabinergic ligands exhibit signaling bias at CBRs toward individual subsets of signal transduction pathways. In this review, we discuss the known signaling pathways regulated by CBRs and examine the current evidence for functional selectivity at CBRs in response to endogenous and exogenous cannabinergic ligands as biased agonists. We further discuss the receptor and ligand structural features allowing for selective activation of CBR-dependent functional responses. The design and development of biased ligands may offer a pathway to therapeutic success for novel CBR-targeted drugs.
... 18 While the antitumor effects of cannabinoids have been demonstrated in a variety of tumor models, the intracellular pathway(s) by which cannabinoid antitumor activity occurs remain unclear. Several cannabinoid mechanisms have been reported, some conflicting, that identify cannabinoid activity through Gα i , 19 Gα q , 20 mTORC1, 21 AKT/PI3K inhibition, 17 AKT/PI3K stimulation, 22 MAPK/ERK modulation, 23 p38/ MAPK modulation, 24 ceramide accumulation, 25 induction of reactive oxygen species, 26 modification of matrix metalloproteinases, 10 Id-1 inhibition, 9 and the involvement of extraneous receptors including TRPV1 27 and GPR55. 28 To further complicate matters, it has also been suggested that the action of cannabinoids on tumor cells alone is not sufficient to produce in vivo antitumor effects and also requires immune system interaction to achieve full efficacy. ...
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.
... Induction de l'apoptose L'apoptose induite par les cannabinoïdes peut être dépendante ou non de la voie de signalisation des caspases. De plus, les effets pro-apoptotiques induits par les cannabinoïdes peuvent également résulter d'une accumulation de céramide dans les cellules tumorales [237].Il a été montré que, dans le cancer colorectal, l'activation de CB1 induit l'apoptose des cellules tumorales via l'inhibition de la voie de signalisation RAS-MAPK et PI3K-AKT, la diminution des facteurs anti-apoptotiques et l'accumulation de céramide[238]. L'activation des récepteurs CB1 et CB2 entraine la synthèse de novo de céramide dans plusieurs types de tumeurs dont les gliomes, les cellules pancréatiques malignes et les leucémies. ...
Endocannabinoïds, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), belong to the andocannabinoïd system are involved in regulating of many biological systems. They have a role in modulating the immune system and anti-tumor defense. In this study, we show that AEA and 2-AG may modulate the activation of natural killer cells CD56+CD3- (NK) cells and NK92. At low doses , they potentiate the production of IFN gamma induced by suboptimal doses of IL-12, IL-15 and IL-2. In addition, AEA and 2-AG potentiate the cytotoxicity of NK cells. Experiments to quantify transcripts encoding FasL, TRAIL, perforin and granzyme and inhibition experiments indicate that this increase in cytotoxicity was correlated with increased expression of FasL, while production of granzyme, perforin and expression TRAIL remains unchanged. Endocannabinoïd effects on NK cells are inhibited in the presence of antagonist of the type 2 endocannabinoïd receptor (CB2). These in vitro studies have been confirmed in vivo in a murine tumor model. Murine NK cells stimulated ex-vivo by endocannabinoïds and reinjected directly in the tumor, induce a significant slowing of tumor growth. These results show for the first time that endocannabinoïds AEA and 2-AG used at low doses are involved in the modulation of regulatory activities (objectified by the production of IFN gamma) and cytotoxic activities of NK cells.
... Cannabinoids, the active ingredients of marijuana, induced an acute ceramide synthesis via sphingomyelin hydrolysis, which mediated the regulation of metabolic functions; and a sustained generation of ceramide via the de novo pathway. This production leads to apoptosis (Velasco et al., 2005). In addition, ceramide levels increased specifically from the de novo synthesis after treating cells with N-(4-hydroxyphenyl)retinamide, an event that lead to apoptosis (Darwiche et al., 2005). ...
... CB1 agonist promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation [108]. Furthermore, CB1 agonists induce biosynthesis of ceramide by sphingomyelinase [109]. Thus, the CB1 appears to contribute to polarization and maintenance of M1 macrophages and microglia. ...
Psychiatric disorders such as schizophrenia and major depressive disorder were thought to be caused by neurotransmitter abnormalities. Patients with these disorders often experience relapse and remission; however the underlying molecular mechanisms of relapse and remission still remain unclear. Recent advanced immunological analyses have revealed that M1/M2 polarization of macrophages plays an important role in controlling the balance between promotion and suppression in inflammation. Microglial cells share certain characteristics with macrophages and contribute to immune-surveillance in the central nervous system (CNS). In this review, we summarize immunoregulatory functions of microglia and discuss a possible role of microglial M1/M2 polarization in relapse and remission of psychiatric disorders and diseases. M1 polarized microglia can produce pro-inflammatory cytokines, reactive oxygen species, and nitric oxide, suggesting that these molecules contribute to dysfunction of neural network in the CNS. Alternatively, M2 polarized microglia express cytokines and receptors that are implicated in inhibiting inflammation and restoring homeostasis. Based on these aspects, we propose a possibility that M1 and M2 microglia are related to relapse and remission, respectively in psychiatric disorders and diseases. Consequently, a target molecule skewing M2 polarization of microglia may provide beneficial therapies for these disorders and diseases in the CNS.
... Involvement of toxic intracellular lipids in ECS-mediated disruption of mitochondrial function. One way that the ECS may act to impair mitochondrial integrity and function is through stimulating the generation of toxic lipid intermediates such as ceramide (26,147). For example, both R-(ϩ)-methanandamide, a stable analog of AEA, and WIN55,212-2, a mixed CB1R/CB2R synthetic agonist, have been shown to promote the CB1R/CB2R-dependent accumulation of ceramide in lymphoma cells, concomitant with mitochondrial depolarization (47). ...
The endocannabinoid system (ECS) regulates numerous cellular and physiological processes through the activation of receptors targeted by endogenously produced ligands called endocannabinoids. Importantly, this signalling system is known to play an important role in modulating energy balance and glucose homeostasis. For example, current evidence indicates that the ECS becomes overactive during obesity whereby its central and peripheral stimulation drives metabolic processes that mimic the metabolic syndrome. Herein, we examine the role of the ECS in modulating the function of mitochondria which play a pivotal role in maintaining cellular and systemic energy homeostasis, in large part due to their ability to tightly coordinate glucose and lipid utilisation. Because of this, mitochondrial dysfunction is often associated with peripheral insulin resistance and glucose intolerance, as well as the manifestation of excess lipid accumulation in the obese state. This review aims to highlight the different ways through which the ECS may impact upon mitochondrial abundance and/or oxidative capacity, and where possible, relate these findings to obesity-induced perturbations in metabolic function. Furthermore, we explore the potential implications of these findings in terms of the pathogenesis of metabolic disorders and how these may be used to strategically develop therapies targeting the ECS.
... Cannabinoids can trigger several different signalling pathways. Following the interaction with the specific CB1 and CB2 receptors, cannabinoids have been shown to induce accumulation of ceramide [6][7] or activation of c-Jun N-terminal kinase (JNK) and p38 MAPK [8], as well as, increase in calcium Ivyspring International Publisher concentration, ROS production, inhibition of PKA or NF-kB and modulation of pro-and anti-apoptotic members of the Bcl-2 family [9][10]. ...
The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.
... As in other tumour cells, an increase in Cer is important for the activity of many cytotoxic treatments in glioma cells [94][95][96]. In particular, the accumulation of de novo synthesised Cer is crucial for cannabinoid-triggered ER stress and apoptosis in these cells [73,97], and the accumulation of Cer in the ER owing to impaired Cer traffic is associated with the antiproliferative effect of nitric oxide [98], which suggests that Cer levels in the ER can be crucial for glioma cell fate. The control of Cer levels in the ER can involve specific enzymes that utilise Cer, such as SMS and GCS [99,100], as well as the vesicular-and protein-mediated transport of Cer, from the ER to the Golgi apparatus in the sphingolipid biosynthetic pathway [19,98,[101][102][103]. ...
Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.
... The CB1 is also coupled to ionic channels, inhibiting Nand P/Q-type voltage-gated calcium channels, activating Atype voltage-gated calcium channels, and inwardly rectifying potassium channels [5][6][7]. Furthermore, cannabinoids can modulate sphingolipid-metabolizing pathways by increasing intracellular levels of ceramide, an ubiquitous lipid second messenger [8]. ...
Although the detrimental effects of cannabis consumption during gestation are known for years, the vast majority of studies established a link between cannabis consumption and foetal development. The complex maternal-foetal interrelationships within the placental bed are essential for normal pregnancy, and decidua definitively contributes to the success of this process. Nevertheless, the molecular signalling network that coordinates strategies for successful decidualization and placentation are not well understood. The discovery of the endocannabinoid system highlighted new signalling mediators in various physiological processes, including reproduction. It is known that endocannabinoids present regulatory functions during blastocyst development, oviductal transport, and implantation. In addition, all the endocannabinoid machinery was found to be expressed in decidual and placental tissues. Additionally, endocannabinoid's plasmatic levels were found to fluctuate during normal gestation and to induce decidual cell death and disturb normal placental development. Moreover, aberrant endocannabinoid signalling during the period of placental development has been associated with pregnancy disorders. It indicates the existence of a possible regulatory role for these molecules during decidualization and placentation processes, which are known to be particularly vulnerable. In this review, the influence of the endocannabinoid system in these critical processes is explored and discussed.
... AEA and C6-ceramide induce activation of p38 MAPK cascade but not p44/42 or JNK We have previously shown that AEA induces apoptosis of primary decidual cells [19]. Since there is evidence that MAPK activation acts at an early step prior to mitochondrial dysfunction and caspase activation [24], it was analysed the involvement of MAPK activation in AEA and C6-ceramidemediated cell death to elucidate the signalling events. ...
Anandamide (AEA) belongs to an endogenous family of lipid messengers, called endocannabinoids (ECs), which exert pharmacological effects by binding to selective membrane receptors, the CB1 and CB2 receptors. Increasing evidence suggests that AEA is involved in the regulation of a variety of cell signalling pathways both in experimental models and humans. We have previously demonstrated that ECs machinery operates in decidual cells and found that AEA, the principal EC, induced apoptosis in decidual cells through CB1. Here, we investigated in rat primary decidual cells the signal transduction pathways activated upon AEA binding to CB1. We found that AEA induces a significant increase in the level of intracellular ceramide. These effects were reversed by inhibiting CB1 receptor activation with AM251. The ceramide analogue, C6-ceramide, induced a decrease in decidual cell viability and of p38 MAPK phosphorylation. Additionally, the pharmacologic inhibition of de novo ceramide biosynthesis with L-cycloserine and fumonisin B reduced the AEA-effects on cell viability and p38 MAPK phosphorylation. Furthermore, AEA and C6-ceramide induced a drop in ΔΨm, an increase in ROS production and caspase-3/-7 activation, effects partially reverted by inhibitors of ceramide synthesis and of p38 MAPK. Taken together, we showed that AEA induces a reduction in decidual cell viability by a mechanism involving CB1 activation, which results in ceramide synthesis de novo and p38 phosphorylation, followed by mitochondrial stress and ROS production, leading to apoptosis.
... This suggests that there are other mechanisms that influence cathepsin D levels in the CB1 knockout mice. It is also known that CB1 receptor activation can induce ceramide synthesis (Velasco et al., 2005), and ceramide can directly activate cathepsin D (Heinrich et al., 1999). Therefore, absence of CB1 receptor activity can indirectly influence cathepsin D activation by affecting ceramide synthesis. ...
Recent research connecting choline-containing lipids to basal forebrain cholinergic neurons (BFCN) degeneration in neuropathological states highlights a challenge for balancing lipid integrity with optimal acetylcholine (ACh) levels. Warranting an adequate choline source to maintain ACh levels in this pathway is crucial for preserving memory. The endocannabinoid (eCB) system plays a role in modulating learning and memory processes controlled by cholinergic neurotransmission. Consequently, we propose that activation of this system is neuroprotective against cholinergic degeneration. In the present study, we investigated the neuroprotective effect of a subchronic treatment with the CB1 cannabinoid agonist, WIN55,212-2, using both ex vivo and in vivo 192IgG-Saporin models of specific cholinergic damage. Degeneration of baso-cortical cholinergic pathways induced memory deficits and a downregulation of saturated and mono-unsaturated lysophosphatidylcholines (LPC) cortical levels. WIN55,212-2 not only restored memory deficits but also increased cortical ACh levels and modified cortical choline-containing lipids such as sphingomyelins (SM) and LPCs, which are essential for correct memory functioning, in lesioned animals. Given these results, we propose that WIN55,212-2 generates an alternative choline source through the breakdown of SMs, which is enough to increase cortical ACh levels and LPCs. These findings suggest that modification of choline-containing lipids by the activation of CB1 receptors is a promising therapy for dementia associated with cholinergic dysfunction, such as in Alzheimer`s disease (AD).
Introduction: The endogenous cannabinoid (endocannabinoid) system is an emerging target for the treatment of chronic inflammatory disease with the potential to advance treatment for many respiratory illnesses. The varied effects of endocannabinoids across tissue types makes it imperative that we explore their physiologic impact within unique tissue targets. The aim of this scoping review is to explore the impact of endocannabinoid activity on eicosanoid production as a measure of human airway inflammation. Methods: A scoping literature review was conducted according to PRISMA-ScR (Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews) guidelines. Search strategies using MeSH terms related to cannabinoids, eicosanoids, cyclooxygenase (COX), and the respiratory system were used to query Medline, Embase, Cochrane, CINAHL, Web of Science, and Biosis Previews in December 2021. Only studies that investigated the relationship between endocannabinoids and the eicosanoid system in mammalian respiratory tissue after 1992 were included. Results: Sixteen studies were incorporated in the final qualitative review. Endocannabinoid activation increases COX-2 expression, potentially through ceramide-dependent or p38 and p42/44 Mitogen-Activated Protein Kinase pathways and is associated with a concentration-dependent increase in prostaglandin (PG)E2. Inhibitors of endocannabinoid hydrolysis found either an increase or no change in levels of PGE2 and PGD2 and decreased levels of leukotriene (LT)B4, PGI2, and thromboxane A2 (TXA2). Endocannabinoids increase bronchial epithelial cell permeability and have vasorelaxant effects in human pulmonary arteries and cause contraction of bronchi and decreased gas trapping in guinea pigs. Inhibitors of endocannabinoid hydrolysis were found to have anti-inflammatory effects on pulmonary tissue and are primarily mediated by COX-2 and activation of eicosanoid receptors. Direct agonism of endocannabinoid receptors appears to play a minor role. Conclusion: The endocannabinoid system has diverse effects on the mammalian airway. While endocannabinoid-derived PGs can have anti-inflammatory effects, endocannabinoids also produce proinflammatory conditions, such as increased epithelial permeability and bronchial contraction. These conflicting findings suggest that endocannabinoids produce a variety of effects depending on their local metabolism and receptor agonism. Elucidation of the complex interplay between the endocannabinoid and eicosanoid pathways is key to leveraging the endocannabinoid system as a potential therapeutic target for human airway disease.
Colorectal cancer (CRC) is one of the common types of cancer affecting humans. The treatment of CRC involves surgery and chemotherapy. CRC treatment using the conventional chemotherapeutics has a negative burden on the patient's health as a result of high toxicity, occurrence of side effects, and drug resistance. Therefore, there is a pressing need to discover more effective and efficient approaches and drugs for treating CRC. This chapter will shed more light on the conventional treatment of colorectal cancer. This chapter discusses the natural products that have anti-CRC effects such as the polyphenols (curcumin, resveratrol), irinotecan, Ganoderma lucidum, cannabinoids, flavonoids, and terpenes. Furthermore, this chapter also highlights the importance of combination chemotherapy (conventional therapy and natural products) in treating CRC. It is believed that this area of research could be a promising approach to minimize side effects and drug resistance linked to the conventional chemotherapy.
Cannabis plant has been used from ancient times with therapeutic purposes for treating human pathologies, but the identification of the cellular and molecular mechanisms underlying the therapeutic properties of the phytocannabinoids, the active compounds in this plant, occurred in the last years of the past century. In the late 1980s and early 1990s, seminal studies demonstrated the existence of cannabinoid receptors and other elements of the so-called endocannabinoid system. These G protein-coupled receptors (GPCRs) are a key element in the functions assigned to endocannabinoids and appear to serve as promising pharmacological targets. They include CB1, CB2, and GPR55, but also non-GPCRs can be activated by endocannabinoids, like ionotropic receptor TRPV1 and even nuclear receptors of the PPAR family. Their activation, inhibition, or simply modulation have been associated with numerous physiological effects at both central and peripheral levels, which may have therapeutic value in different human pathologies, then providing a solid experimental explanation for both the ancient medicinal uses of Cannabis plant and the recent advances in the development of cannabinoid-based specific therapies. This chapter will review the scientific knowledge generated in the last years around the research on the different endocannabinoid-binding receptors and their signaling mechanisms. Our intention is that this knowledge may help readers to understand the relevance of these receptors in health and disease conditions, as well as it may serve as the theoretical basis for the different experimental protocols to investigate these receptors and their signaling mechanisms that will be described in the following chapters.Key wordsCannabinoidsEndocannabinoid systemCB1 receptorCB2 receptorGPR55TRPV1 receptorPPARsCannabinoid-based therapies
The global prevalence of metabolic diseases such as type 2 diabetes mellitus, steatohepatitis, myocardial infarction, and stroke has increased dramatically over the past two decades. These obesity-fueled disorders result, in part, from the aberrant accumulation of harmful lipid metabolites in tissues not suited for lipid storage (e.g., the liver, vasculature, heart, and pancreatic beta-cells). Among the numerous lipid subtypes that accumulate, sphingolipids such as ceramides are particularly impactful, as they elicit the selective insulin resistance, dyslipidemia, and ultimately cell death that underlie nearly all metabolic disorders. This review summarizes recent findings on the regulatory pathways controlling ceramide production, the molecular mechanisms linking the lipids to these discrete pathogenic events, and exciting attempts to develop therapeutics to reduce ceramide levels to combat metabolic disease.
Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Brassinosteroids (BRs) are a group of naturally occurring endogenous steroidal plant growth regulators (PGRs) which are hydroxylated derivatives of a 5α- cholestane ring structure. Both spectroscopy and X-ray based structural elucidation methods have revealed the structure of the first discovered BR, i.e. Brassinolide (BL) as (22R, 23R, 24S) - 2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α- cholestan-6-one. Generally, the natural BRs are hydroxylated at the C-2, C-3, C-22 and C-23 positions. The most biologically active BRs differ from other BRs solely on the basis of either a 6-oxo functional group or a lactone structure at one of its rings. Presently, more than 60 types of BRs have been identified and isolated from a diverse range of plants. Recently, BRs have emerged as important PGRs that are a pre-requisite for growth and various developmental processes along with other PGRs namely auxins, gibberellins, cytokinins, ethylene and abscisic acid. Further, these BRs also provide protection to the plants under both biotic and abiotic stresses. Therefore, the present chapter highlights the structure, occurrence, mode of action, biosynthetic pathway, plant stress ameliorating properties of BRs and their interactions with other PGRs.
Cannabis is the most popular illicit drug in the Western world, and has a long history of medical use. Its active ingredients, known as cannabinoids, can elicit various biological activities through activation of certain G-protein coupled receptors, denoted by the name of cannabinoid receptors. By far, two cannabinoid receptors have been identified, namely the cannabinoid receptor 1 (CB1) and 2 (CB2). The CB1 receptors are primarily found in the central nervous system (CNS) while the CB2 receptors largely present in the periphery. Besides the plant-derived cannabinoids (phytocannabinoids), a group of endogenous lipid-derived molecules can similarly activate the cannabinoid receptors; therefore they are referred to as endocannabinoids. Activation and blockade of cannabinoid receptors can lead to a variety of responses relevant to pharmacotherapy. This chapter provides a synopsis of phytocannabinoids, and an overview of cannabinoid receptors with respects to: what they are; where they are; and what they do.
The endocannabinoid system (ECS) and reactive oxygen species (ROS) constitute two key cellular signalling systems that participate in the modulation of diverse cellular functions. Importantly, growing evidence suggests that cross-talk between these two prominent signalling systems acts to modulate functionality of the ECS as well as redox homeostasis in different cell types. Herein, we review and discuss evidence pertaining to ECS-induced regulation of ROS generating and scavenging mechanisms, as well as highlighting emerging work that supports redox modulation of ECS function. Functionally, the studies outlined reveal that interactions between the ECS and ROS signalling systems can be both stimulatory and inhibitory in nature, depending on cell stimulus, the source of ROS species and cell context. Importantly, such cross-talk may act to maintain cell function, whereas abnormalities in either system may propagate and undermine the stability of both systems, thereby contributing to various pathologies associated with their dysregulation.
Cannabinoids, lipid mediators presents in Cannabis sativa plant, have been used for medicinal purposes for centuries. In the first half of the twentieth century they were removed from medicine due to their psychoactive adverse effects and the emergence of safer drugs. Nevertheless at the end of this century the discovery of endogenous cannabinoid system and its participation in multiple basic physiological functions have attracted much attention to the researchers as possible tools in the treatment of several pathological conditions such as neurodegenerative and metabolic disorders, autoimmune diseases, glaucoma and cancer.
The proapoptotic and antineoplastic properties of cannabinoids with emphasis on effects of N-acylethanolamines were analyzed. Cannabinoids enhanced apoptotic and necrotic processes in many types of tumour cells and tissues. Involvement of different types of receptors and signaling pathways in mediating the proapoptotic effects of cannabinoids are discussed. The evidences in favour of both proapoptotic, pronecrotic and protective, antiapoptotic effects of cannabinoids and, especially N-acylethanolamines, are evaluated. The hypothesis is suggested that N-acylethanolamines, formed in some tissues under strong stress conditions, can be not a consequence of tissue damage but cause such damage. The conclusion is made on promising of cannabinoids as potential anticancer agents.
Key Words: Cannabinoids, N-acylethanolamines, apoptosis, necrosis, tumours, ischemia.
RESUMEN Los cannabinoides, mediadores lipídicos presentes en la planta Cannabis sativa, han sido utilizados en medicina desde la antigüedad; retirándose del arsenal terapéutico en la primera mitad del siglo XX debido a sus efectos adversos psicoactivos y a la aparición de alternativas más seguras. No obstante el descubrimiento del sistema cannabinoide endógeno y su participación reguladora en múltiples funciones fisiológicas básicas del organismo suscitaron de nuevo a finales del siglo pasado el interés de los investigadores como posibles herramientas terapéuticas en diversas patologías como trastornos neurodegenerativos y metabólicos, enfermedades autoinmunes, glaucoma e, incluso, cáncer. ABSTRACT Cannabinoids: a promissing tool for the development of novel therapies Cannabinoids, lipid mediators presents in Cannabis sativa plant, have been used for medicinal purposes for centuries. In the first half of the twentieth century they were removed from medicine due to their psychoactive adverse effects and the emergence of safer drugs. Nevertheless at the end of this century the discovery of endogenous cannabinoid system and its participation in multiple basic physiological functions have attracted much attention to the researchers as possible tools in the treatment of several pathological conditions such as neurodegenerative and metabolic disorders, autoimmune diseases, glaucoma and cancer.
Global metabolic profiling using quantitative nuclear magnetic resonance spectroscopy (MRS) and mass spectrometry (MS) is useful for biomarker discovery. The objective of this study was to discover biomarkers of acute lung injury induced by mechanical ventilation (ventilator-induced lung injury [VILI]), by using MRS and MS.
Male Sprague-Dawley rats were subjected to two ventilatory strategies for 2.5 h: tidal volume 9 ml/kg, positive end-expiratory pressure 5 cm H2O (control, n = 14); and tidal volume 25 ml/kg and positive end-expiratory pressure 0 cm H2O (VILI, n = 10). Lung tissue, bronchoalveolar lavage fluid, and serum spectra were obtained by high-resolution magic angle spinning and H-MRS. Serum spectra were acquired by high-performance liquid chromatography coupled to quadupole-time of flight MS. Principal component and partial least squares analyses were performed.
Metabolic profiling discriminated characteristics between control and VILI animals. As compared with the controls, animals with VILI showed by MRS higher concentrations of lactate and lower concentration of glucose and glycine in lung tissue, accompanied by increased levels of glucose, lactate, acetate, 3-hydroxybutyrate, and creatine in bronchoalveolar lavage fluid. In serum, increased levels of phosphatidylcholine, oleamide, sphinganine, hexadecenal and lysine, and decreased levels of lyso-phosphatidylcholine and sphingosine were identified by MS.
This pilot study suggests that VILI is characterized by a particular metabolic profile that can be identified by MRS and MS. The metabolic profile, though preliminary and pending confirmation in larger data sets, suggests alterations in energy and membrane lipids.
The growth-related gene product β (GROβ) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and
GROβ receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express
the GROβ receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase
C, GROβ activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase
1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GROβ-induced JNK1 activation is an event probably
independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity.
This is the first report on the activation of the SMase pathway by chemokines.
Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands
for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry
and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a
radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent
inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids.
These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.
Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.
The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.
Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide
increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted
from the inhibition of adenylyl cyclase and cyclic adenosine 3,5-monophosphate-dependent protein kinase. One of the proteins
phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in
neurons. Focal adhesion kinaseis a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton.
Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.
This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type
2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin
or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular
glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, β-chloro-l-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and β-chloro-l-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.
The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.
Cannabinoid CB1 receptor mRNA was detected using reverse transcription-polymerase chain reaction (RT-PCR) in endothelial cells from human aorta and hepatic artery and in the ECV304 cell line derived from human umbilical vein endothelial cells. CB1 receptor-binding sites were detected by the high-affinity antagonist radioligand [(125)I]AM-251. In ECV304 cells, both the highly potent synthetic cannabinoid agonist HU-210 and the endogenous ligand anandamide induce activation of mitogen-activated protein (MAP) kinase, and the effect of HU-210 was completely blocked, whereas the effect of anandamide was partially inhibited by SR141716A, a selective CB1 receptor antagonist. Transfection of ECV304 cells with CB1 receptor antisense, but not sense, oligonucleotides caused the same pattern of inhibition as SR141716A. This provides more definitive evidence for the involvement of CB1 receptors in MAP kinase activation and suggests that anandamide may also activate MAP kinase via an additional, CB1 receptor-independent, SR141716A-resistant mechanism. The MAP kinase activation by anandamide in ECV304 cells requires genistein-sensitive tyrosine kinases and protein kinase C (PKC), and anandamide also activates p38 kinase and c-Jun kinase. These findings indicate that CB1 receptors located in human vascular endothelium are functionally coupled to the MAP kinase cascade. Activation of protein kinase cascades by anandamide may be involved in the modulation of endothelial cell growth and proliferation.
Delta9-Tetrahydrocannabinol, the main active component of marijuana, induces apoptosis of transformed neural cells in culture. Here, we show that intratumoral administration of Delta9-tetrahydrocannabinol and the synthetic cannabinoid agonist WIN-55,212-2 induced a considerable regression of malignant gliomas in Wistar rats and in mice deficient in recombination activating gene 2. Cannabinoid treatment did not produce any substantial neurotoxic effect in the conditions used. Experiments with two subclones of C6 glioma cells in culture showed that cannabinoids signal apoptosis by a pathway involving cannabinoid receptors, sustained ceramide accumulation and Raf1/extracellular signal-regulated kinase activation. These results may provide the basis for a new therapeutic approach for the treatment of malignant gliomas.
Cannabinoids exert most of their effects in the central nervous system through the CB(1) cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin and wortmannin blocked the CB(1) receptor-evoked activation of PKB, pointing to the sequential involvement of a G(i)/G(o) protein and phosphoinositide 3'-kinase. The functionality of the cannabinoid-induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB(1) receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB(2) receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB(1) receptor.
Cannabinoids exert most of their effects through the CB(1) receptor. This G protein-coupled receptor signals inhibition of adenylyl cyclase, modulation of ion channels, and stimulation of mitogen- and stress-activated protein kinases. In this article, we report that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces sphingomyelin hydrolysis in primary astrocytes but not in other cells expressing the CB(1) receptor, such as primary neurons, U373 MG astrocytoma cells, and Chinese hamster ovary cells transfected with the CB(1) receptor cDNA. THC-evoked sphingomyelin breakdown in astrocytes was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoid HU-210 and was prevented by the selective CB(1) antagonist SR141716. By contrast, the effect of THC was not blocked by pertussis toxin, pointing to a lack of involvement of G(i/o) proteins. A role for the adaptor protein FAN in CB(1) receptor-coupled sphingomyelin breakdown is supported by two observations: 1) coimmunoprecipitation experiments show that the binding of FAN to the CB(1) receptor is enhanced by THC and prevented by SR141716; 2) cells expressing a dominant-negative form of FAN are refractory to THC-induced sphingomyelin breakdown. This is the first report showing that a G-protein-coupled receptor induces sphingomyelin hydrolysis through FAN and that the CB(1) cannabinoid receptor may signal independently of G(i/o) proteins.
The principal psychoactive ingredient in marijuana, Delta(9)-tetrahydrocannabinol, has been shown to inhibit adenylyl cyclase activity in vitro and can lead to impairment of memory in vivo. cAMP-induced changes in synaptic plasticity are thought to underlie memory formation. We examined the effects of cannabinoid receptor agonists on forskolin-induced formation of new synapses between rat hippocampal neurons in culture. Functional synaptic boutons were identified with FM1-43-based digital imaging. Cannabimimetic drugs prevented the recruitment of new synapses by inhibiting the formation of cAMP. The inhibition produced by Win55212-2, a synthetic cannabinoid receptor agonist, was stereoselective and was reversed by a selective CB1 receptor antagonist. Both Delta(9)-tetrahydrocannabinol and the endogenous ligand, anandamide, inhibited the formation of new synapses. Win55212-2 blocked the formation of new synapses induced by forskolin, but not those evoked by a membrane permeant cAMP analog. Thus, activation of cannabinoid receptors can modulate synaptic plasticity independent of direct effects on neurotransmitter release. Preventing the formation of new synapses may contribute to the impairment of memory produced by cannabinoids.
TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.
The development of new therapeutic strategies is essential for the management of gliomas, one of the most malignant forms of cancer. We have shown previously that the growth of the rat glioma C6 cell line is inhibited by psychoactive cannabinoids (I. Galve-Roperh et al., Nat. Med., 6: 313-319, 2000). These compounds act on the brain and some other organs through the widely expressed CB(1) receptor. By contrast, the other cannabinoid receptor subtype, the CB(2) receptor, shows a much more restricted distribution and is absent from normal brain. Here we show that local administration of the selective CB(2) agonist JWH-133 at 50 microg/day to Rag-2(-/-) mice induced a considerable regression of malignant tumors generated by inoculation of C6 glioma cells. The selective involvement of the CB(2) receptor in this action was evidenced by: (a) the prevention by the CB(2) antagonist SR144528 but not the CB(1) antagonist SR141716; (b) the down-regulation of the CB(2) receptor but not the CB(1) receptor in the tumors; and (c) the absence of typical CB(1)-mediated psychotropic side effects. Cannabinoid receptor expression was subsequently examined in biopsies from human astrocytomas. A full 70% (26 of 37) of the human astrocytomas analyzed expressed significant levels of cannabinoid receptors. Of interest, the extent of CB(2) receptor expression was directly related with tumor malignancy. In addition, the growth of grade IV human astrocytoma cells in Rag-2(-/-) mice was completely blocked by JWH-133 administration at 50 microg/day. Experiments carried out with C6 glioma cells in culture evidenced the internalization of the CB(2) but not the CB(1) receptor upon JWH-133 challenge and showed that selective activation of the CB(2) receptor signaled apoptosis via enhanced ceramide synthesis de novo. These results support a therapeutic approach for the treatment of malignant gliomas devoid of psychotropic side effects.
Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.
Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.
New uses warranted for cannabinoids? Recent data indicate that synthetic or endogenous substances activating the receptor for marijuana's psychotropic component might be used as templates for the development of new anti-cancer drugs.
Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central
nervous system by binding to the CB1cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However,
those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic
lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show
the following: (i) cannabinoids rescue primary astrocytes from C2-ceramide-induced apoptosis in a dose- and time-dependent manner; (ii) triggering of this anti-apoptotic signal depends on
the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might
be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects
of focal C2-ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol
3-kinase/protein kinase B pathway. These findings constitute the first evidence for an “astroprotective” role of cannabinoids.
Endocannabinoids are neuromodulators that act as retrograde synaptic messengers inhibiting the release of different neurotransmitters in cerebral areas such as hippocampus, cortex, and striatum. However, little is known about other roles of the endocannabinoid system in brain. In the present work we provide substantial evidence that the endocannabinoid anandamide (AEA) regulates neuronal differentiation both in culture and in vivo. Thus AEA, through the CB(1) receptor, inhibited cortical neuron progenitor differentiation to mature neuronal phenotype. In addition, human neural stem cell differentiation and nerve growth factor-induced PC12 cell differentiation were also inhibited by cannabinoid challenge. AEA decreased PC12 neuronal-like generation via CB(1)-mediated inhibition of sustained extracellular signal-regulated kinase (ERK) activation, which is responsible for nerve growth factor action. AEA thus inhibited TrkA-induced Rap1/B-Raf/ERK activation. Finally, immunohistochemical analyses by confocal microscopy revealed that adult neurogenesis in dentate gyrus was significantly decreased by the AEA analogue methanandamide and increased by the CB(1) antagonist SR141716. These data indicate that endocannabinoids inhibit neuronal progenitor cell differentiation through attenuation of the ERK pathway and suggest that they constitute a new physiological system involved in the regulation of neurogenesis.
Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the nonselective cannabinoid agonists HU210 and (+)-Win-55212-2 enhanced cell survival. To investigate intracellular signaling involved in cannabinoid protection, we focused on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. HU210, (+)-Win-55212-2, and ACEA elicited a time-dependent phosphorylation of Akt. Pertussis toxin abolished Akt activation, indicating the involvement of G(i)/G(o)-protein-coupled receptors. The CB1 receptor antagonist SR141716A partially inhibited Akt phosphorylation in response to HU210 and (+)-Win-55212-2 and abolished the effects of ACEA. Trophic support deprivation downregulated Akt activity, and cannabinoids recovered phospho-Akt levels. Inhibition of PI3K abrogated the survival action and the recovery of Akt activity in response to cannabinoids. SR141716A prevented only the protection conferred by ACEA. Nevertheless, SR141716A and the selective CB2 receptor antagonist SR144528 in combination inhibited the prosurvival action of HU210, which is in accordance with the finding of CB2 receptor expression by oligodendroglial cells. These data identify oligodendrocytes as potential targets of cannabinoid action in the CNS.
Nonmelanoma skin cancer is one of the most common malignancies in humans. Different therapeutic strategies for the treatment of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the wide tissue distribution of the two subtypes of cannabinoid receptors (CB(1) and CB(2)), we studied the potential utility of these compounds in anti-skin tumor therapy. Here we show that the CB(1) and the CB(2) receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic death of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CB(1)/CB(2) agonist WIN-55,212-2 or the selective CB(2) agonist JWH-133 induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by altered blood vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth factor, and angiopoietin 2). Abrogation of EGF-R function was also observed in cannabinoid-treated tumors. These results support a new therapeutic approach for the treatment of skin tumors.
Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression in rodents (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell migration and survival as well as the decrease of the expression of proangiogenic factors (vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of cannabinoid-based antitumoral therapies.
A key role for DAG lipase activity in the control of axonal growth and guidance in vitro and in vivo has been established. For example, DAG lipase activity is required for FGF-stimulated calcium influx into neuronal growth cones, and this response is both necessary and sufficient for an axonal growth response. The mechanism that couples the hydrolysis of DAG to the calcium response is not known. The initial hydrolysis of DAG at the sn-1 position (by DAG lipase) will generate 2-arachidonylglycerol, and this molecule is well established as an endogenous cannabinoid receptor agonist in the brain. In the present paper, we show that in rat cerebellar granule neurons, CB1 cannabinoid receptor antagonists inhibit axonal growth responses stimulated by N-cadherin and FGF2. Furthermore, three CB1 receptor agonists mimic the N-cadherin/FGF2 response at a step downstream from FGF receptor activation, but upstream from calcium influx into cells. In contrast, we could find no evidence for the CB1 receptor coupling the TrkB neurotrophin receptor to an axonal growth response in the same neurons. The observation that the CB1 receptor can couple the activated FGF receptor to an axonal growth response raises novel therapeutic opportunities.