Maternal Cannabis Use during Lactation and Potential Effects on Human Milk Composition and Production: A Narrative Review.

Cannabis use has increased sharply in the last 20 y among adults, including reproductive-aged women. Its recent widespread legalization is associated with a decrease in risk perception of cannabis use during breastfeeding. However, the effect of cannabis use (if any) on milk production and milk composition is not known. This narrative review summarizes current knowledge related to maternal cannabis use during breastfeeding and provides an overview of possible pathways whereby cannabis might affect milk composition and production. Several studies have demonstrated that cannabinoids and their metabolites are detectable in human milk produced by mothers who use cannabis. Due to their physicochemical properties, cannabinoids are stored in adipose tissue, can easily reach the mammary gland, and can be secreted in milk. Moreover, cannabinoid receptors are present in adipocytes and mammary epithelial cells. The activation of these receptors directly modulates fatty acid metabolism, potentially causing changes in milk fatty acid profiles. Additionally, the endocannabinoid system is intimately connected to the endocrine system. As such, it is probable that interactions of exogenous cannabinoids with the endocannabinoid system might modify release of critical hormones (e.g., prolactin and dopamine) that regulate milk production and secretion. Nonetheless, few studies have investigated effects of cannabis use (including on milk production and composition) in lactating women. Additional research utilizing robust methodologies are needed to elucidate whether and how cannabis use affects human milk production and composition.

Cannabinoids in the treatment of glioblastoma.

Glioblastoma (GBM) is the most prevalent primary malignant tumor of the nervous system. While the treatment of other neoplasms is increasingly more efficacious the median survival rate of GBM patients remains low and equals about 14 months. Due to this fact, there are intensive efforts to find drugs that would help combat GBM. Nowadays cannabinoids are becoming more and more important in the field of cancer and not only because of their properties of antiemetic drugs during chemotherapy. These compounds may have a direct cytotoxic effect on cancer cells. Studies indicate GBM has disturbances in the endocannabinoid system-changes in cannabinoid metabolism as well as in the cannabinoid receptor expression. The GBM cells show expression of cannabinoid receptors 1 and 2 (CB1R and CB2R), which mediate various actions of cannabinoids. Through these receptors, cannabinoids inhibit the proliferation and invasion of GBM cells, along with changing their morphology. Cannabinoids also induce an intrinsic pathway of apoptosis in the tumor. Hence the use of cannabinoids in the treatment of GBM may be beneficial to the patients. So far, studies focusing on using cannabinoids in GBM therapy are mainly preclinical and involve cell lines and mice. The results are promising and show cannabinoids inhibit GBM growth. Several clinical studies are also being carried out. The preliminary results show good tolerance of cannabinoids and prolonged survival after administration of these drugs. In this review, we describe the impact of cannabinoids on GBM and glioma cells in vitro and in animal studies. We also provide overview of clinical trials on using cannabinoids in the treatment of GBM.

Hormetic effects of a cannabinoid system component, 2-arachidonoyl glycerol, on cell viability and expression profile of growth factors in cultured mouse Sertoli cells: Friend or foe of male fertility?

The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells' survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors' production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors' gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 µM). Opposite effects were observed by a higher concentration of 2-AG (3 µM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

Dual effect of anandamide on spinal nociceptive transmission in control and inflammatory conditions.

Anandamide (AEA) is an important modulator of nociception in the spinal dorsal horn, acting presynaptically through Cannabinoid (CB1) and Transient receptor potential vanilloid (TRPV1) receptors. The role of AEA (1 µM, 10 µM, and 30 µM) application on the modulation of nociceptive synaptic transmission under control and inflammatory conditions was studied by recording miniature excitatory postsynaptic currents (mEPSCs) from neurons in spinal cord slices. Inhibition of the CB1 receptors by PF514273, TRPV1 by SB366791, and the fatty acid amide hydrolase (FAAH) by URB597 was used. Under naïve conditions, the AEA application did not affect the mEPSCs frequency (1.43±0.12 Hz) when all the recorded neurons were considered. The mEPSC frequency increased (180.0±39.2%) only when AEA (30 µM) was applied with PF514273 and URB597. Analysis showed that one sub-population of neurons had synaptic input inhibited (39.1% of neurons), the second excited (43.5%), whereas 8.7% showed a mixed effect and 8.7% did not respond to the AEA. With inflammation, the AEA effect was highly inhibitory (72.7%), while the excitation was negligible (9.1%), and 18.2% were not modulated. After inflammation, more neurons (45.0%) responded even to low AEA by mEPSC frequency increase with PF514273/URB597 present. AEA-induced dual (excitatory/inhibitory) effects at the 1st nociceptive synapse should be considered when developing analgesics targeting the endocannabinoid system. These findings contrast the clear inhibitory effects of the AEA precursor 20:4-NAPE application described previously and suggest that modulation of endogenous AEA production may be more favorable for analgesic treatments.

The Interaction of the Endocannabinoid Anandamide and Paracannabinoid Lysophosphatidylinositol during Cell Death Induction in Human Breast Cancer Cells.

Endocannabinoid anandamide (AEA) and paracannabinoid lysophosphatidylinositol (LPI) play a significant role in cancer cell proliferation regulation. While anandamide inhibits the proliferation of cancer cells, LPI is known as a cancer stimulant. Despite the known endocannabinoid receptor crosstalk and simultaneous presence in the cancer microenvironment of both molecules, their combined activity has never been studied. We evaluated the effect of LPI on the AEA activity in six human breast cancer cell lines of different carcinogenicity (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, MDA-MB-231) using resazurin and LDH tests after a 72 h incubation. AEA exerted both anti-proliferative and cytotoxic activity with EC50 in the range from 31 to 80 µM. LPI did not significantly affect the cell viability. Depending on the cell line, the response to the LPI-AEA combination varied from a decrease in AEA cytotoxicity to an increase in it. Based on the inhibitor analysis of the endocannabinoid receptor panel, we showed that for the former effect, an active GPR18 receptor was required and for the latter, an active CB2 receptor. The data obtained for the first time are important for the understanding the manner by which endocannabinoid receptor ligands acting simultaneously can modulate cancer growth at different stages.

Effect of MCH1, a fatty-acid amide hydrolase inhibitor, on the depressive-like behavior and gene expression of endocannabinoid and dopaminergic-signaling system in the mouse nucleus accumbens.

MCH1 is a synthetic macamide that has shown in vitro inhibitory activity on fatty acid amide hydrolase (FAAH), an enzyme responsible for endocannabinoid metabolism. This inhibition can modulate endocannabinoid and dopamine signaling in the nucleus accumbens (NAc), potentially having an antidepressant-like effect. The present study aimed to evaluate the effect of the in vivo administration of MCH1 (3, 10, and 30 mg/kg, ip) in 2-month-old BALB/c male mice (n=97) on forced swimming test (FST), light-dark box (LDB), and open field test (OFT) and on early gene expression changes 2 h after drug injection related to the endocannabinoid system (Cnr1 and Faah) and dopaminergic signaling (Drd1 and Drd2) in the NAc core. We found that the 10 mg/kg MCH1 dose reduced the immobility time compared to the vehicle group in the FST with no effect on anxiety-like behaviors measured in the LDB or OFT. However, a 10 mg/kg MCH1 dose increased locomotor activity in the OFT compared to the vehicle. Moreover, RT-qPCR results showed that the 30 mg/kg MCH1 dose increased Faah gene expression by 2.8-fold, and 10 mg/kg MCH1 increased the Cnr1 gene expression by 4.3-fold compared to the vehicle. No changes were observed in the expression of the Drd1 and Drd2 genes in the NAc at either MCH1 dose. These results indicated that MCH1 might have an antidepressant-like effect without an anxiogenic effect and induces significant changes in endocannabinoid-related genes but not in genes of the dopaminergic signaling system in the NAc of mice.

Endocannabinoid regulation in the cervix during pregnancy: insights into molecular mechanisms of premature labor.

In brief: The cervix plays a crucial role not only in the maintenance of pregnancy but also during delivery, when it undergoes extensive changes. This study highlights the involvement of the endocannabinoidome in cervical remodeling, emphasizing its relevance in the shift from a nonpregnant to pregnant state and its potential contribution to preterm delivery in inflammatory contexts. Abstract: During pregnancy, the main role of the cervix is to isolate the fetus from outside pathogens and maintain the relatively closed system of uterine gestation. Conversely, toward the end of pregnancy, the cervix must be remodeled to increase flexibility and allow the delivery. This process is called cervical remodeling and dysregulation of the process plays a role in premature delivery. The endocannabinoidome plays an important role in several reproductive events; however, its function on cervical tissue throughout pregnancy is poorly understood. The goal of this study was to evaluate the presence and participation of the endocannabinoidome in lipopolysaccharide (LPS)-induced cervical changes. Therefore, we evaluated key components of the endocannabinoidome in cervical tissue from nonpregnant mice and pregnant mice with and without LPS treatment. Using mass spectrometric analysis, we found an increase in anandamide and 2-arachidonoylglycerol in the cervix of pregnant mice when compared to nonpregnant mice. We have also found a reduction in FAAH protein expression in these tissues. Furthermore, when treated with LPS, we observed a reduction in the cervical immunostaining with anti-CB1 and anti-CB2 antibodies. Likewise, using cervix explants from pregnant mice, we found that LPS significantly increased cervical metalloprotease activity and cyclooxygenase 2, which were subsequently modulated by cannabinoid receptor antagonists. Collectively, our findings suggest that an LPS-induced imbalance of cervix endocannabinoidome likely contributes to premature cervical remodeling, which is part of the key components that contribute to premature delivery.

Astrocytic Regulation of Endocannabinoid-Dependent Synaptic Plasticity in the Dorsolateral Striatum.

Astrocytes are pivotal for synaptic transmission and may also play a role in the induction and expression of synaptic plasticity, including endocannabinoid-mediated long-term depression (eCB-LTD). In the dorsolateral striatum (DLS), eCB signaling plays a major role in balancing excitation and inhibition and promoting habitual learning. The aim of this study was to outline the role of astrocytes in regulating eCB signaling in the DLS. To this end, we employed electrophysiological slice recordings combined with metabolic, chemogenetic and pharmacological approaches in an attempt to selectively suppress astrocyte function. High-frequency stimulation induced eCB-mediated LTD (HFS-LTD) in brain slices from both male and female rats. The metabolic uncoupler fluorocitrate (FC) reduced the probability of transmitter release and depressed synaptic output in a manner that was independent on cannabinoid 1 receptor (CB1R) activation. Fluorocitrate did not affect the LTD induced by the CB1R agonist WIN55,212-2, but enhanced CB1R-dependent HFS-LTD. Reduced neurotransmission and facilitated HFS-LTD were also observed during chemogenetic manipulation using Gi-coupled DREADDs targeting glial fibrillary acidic protein (GFAP)-expressing cells, during the pharmacological inhibition of connexins using carbenoxolone disodium, or during astrocytic glutamate uptake using TFB-TBOA. While pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) failed to prevent synaptic depression induced by FC, it blocked the facilitation of HFS-LTD. While the lack of tools to disentangle astrocytes from neurons is a major limitation of this study, our data collectively support a role for astrocytes in modulating basal neurotransmission and eCB-mediated synaptic plasticity.

GPR55 inhibits the pro-adipogenic activity of anandamide in human adipose stromal cells.

The endocannabinoid anandamide (AEA) stimulates adipogenesis via the cannabinoid receptor CB1 in adipose stromal cells (ASCs). However, AEA interacts also with nonclassical cannabinoid receptors, including transient receptor potential cation channel (TRPV)1 and G protein-coupled receptor (GPR)55. Their roles in AEA mediated adipogenesis of human ASCs have not been investigated. We examined the receptor-expressions by immunostaining on human ASCs and tested their functionality by measuring the expression of immediate early genes (IEGs) related to the transcription factor-complex AP-1 upon exposition to receptor agonists. Cells were stimulated with increasing concentrations of specific ligands to investigate the effects on ASC viability (proliferation and metabolic activity), secretory activity, and AEA mediated differentiation. ASCs expressed both receptors, and their activation suppressed IEG expression. TRPV1 did not affect viability or cytokine secretion. GPR55 decreased proliferation, and it inhibited the release of hepatocyte growth factor. Blocking GPR55 increased the pro-adipogenic activity of AEA. These data suggest that GPR55 functions as negative regulator of cannabinoid mediated pro-adipogenic capacity in ASCs.

Function of Presynaptic Inhibitory Cannabinoid CB1 Receptors in Spontaneously Hypertensive Rats and Its Modification by Enhanced Endocannabinoid Tone.

We studied whether the function of presynaptic inhibitory cannabinoid CB1 receptors on the sympathetic nerve fibres innervating resistance vessels is increased in spontaneously hypertensive rats (SHR) like in deoxycorticosterone (DOCA)-salt hypertension. An increase in diastolic blood pressure (DBP) was induced by electrical stimulation of the preganglionic sympathetic neurons or by phenylephrine injection in pithed SHR and normotensive Wistar-Kyoto rats (WKY). The electrically (but not the phenylephrine) induced increase in DBP was inhibited by the cannabinoid receptor agonist CP55940, similarly in both groups, and by the endocannabinoid reuptake inhibitor AM404 in SHR only. The effect of CP55940 was abolished/reduced by the CB1 receptor antagonist AM251 (in both groups) and in WKY by endocannabinoid degradation blockade, i.e., the monoacylglycerol lipase (MAGL) inhibitor MJN110 and the dual fatty acid amide hydrolase (FAAH)/MAGL inhibitor JZL195 but not the FAAH inhibitor URB597. MJN110 and JZL195 tended to enhance the effect of CP55940 in SHR. In conclusion, the function of presynaptic inhibitory CB1 receptors depends on the hypertension model. Although no differences occurred between SHR and WKY under basal experimental conditions, the CB1 receptor function was better preserved in SHR when the endocannabinoid tone was increased by the inhibition of MAGL or the endocannabinoid transporter.

ß-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.

AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) reduces the viability of the various melanoma cells, but it exerts significant cytotoxic effects on healthy cells: an in vitro study based on isobolographic analysis.

BACKGROUND: Despite great advances in our understanding of the impact of cannabinoids on human organism, many of their properties still remain undetermined, including their potential antineoplastic effects. This study was designed to assess the anti-proliferative and cytotoxic effects of AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) administered alone and in combinations with docetaxel (DOCX), paclitaxel (PACX), mitoxantrone (MTX) and cisplatin (CDDP) on various human malignant melanoma A375, FM55P, SK-MEL 28 and FM55M2 cell lines. MATERIALS: In the MTT, LDH, and BrdU assays, the potency and safety of AM1172 when administered alone and in combinations with DOCX, PACX, MTX, and CDDP were determined. RESULTS: The isobolographic analysis revealed that combinations of AM1172 with PACX, DOCX, MTX, and CDDP exerted additive interactions, except for a combination of AM1172 with PACX in primary melanoma A375 cell line, for which synergy was observed (*p<0.05). Nevertheless, AM1172 when administered alone produced cytotoxic effects on healthy human melanocytes (HEMa-LP) and human keratinocytes (HaCaT), which unfortunately limits its potential therapeutic utility. CONCLUSIONS: AM1172 cannot be used separately as a chemotherapeutic drug, but it can be combined with PACX, DOCX, MTX, and CDDP, offering additive interactions in terms of the anti-proliferative effects in various malignant melanoma cell lines.

ENDOCANNABINOID SYSTEM IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS MODULATES AUTONOMIC AND CARDIOVASCULAR CHANGES BUT NOT VASOPRESSIN RESPONSE IN A RAT HEMORRHAGIC SHOCK MODEL.

ABSTRACT: We evaluated the participation of the endocannabinoid system in the paraventricular nucleus of the hypothalamus (PVN) on the cardiovascular, autonomic, and plasma vasopressin (AVP) responses evoked by hemorrhagic shock in rats. For this, the PVN was bilaterally treated with either vehicle, the selective cannabinoid receptor type 1 antagonist AM251, the selective fatty acid amide hydrolase amide enzyme inhibitor URB597, the selective monoacylglycerol-lipase enzyme inhibitor JZL184, or the selective transient receptor potential vanilloid type 1 antagonist capsazepine. We evaluated changes on arterial pressure, heart rate, tail skin temperature (ST), and plasma AVP responses induced by bleeding, which started 10 min after PVN treatment. We observed that bilateral microinjection of AM251 into the PVN reduced the hypotension during the hemorrhage and prevented the return of blood pressure to baseline values in the posthemorrhagic period. Inhibition of local 2-arachidonoylglycerol metabolism by PVN treatment with JZL184 induced similar effects in relation to those observed in AM251-treated animals. Inhibition of local anandamide metabolism via PVN treatment with URB597 decreased the depressor effect and ST drop induced by the hemorrhagic stimulus. Bilateral microinjection of capsazepine mitigated the fall in blood pressure and ST. None of the PVN treatments altered the increased plasma concentration of AVP and tachycardia induced by hemorrhage. Taken together, present results suggest that endocannabinoid neurotransmission within the PVN plays a prominent role in cardiovascular and autonomic, but not neuroendocrine, responses evoked by hemorrhage.

Anxiogenic doses of rapamycin prevent URB597-induced anti-stress effects in socially defeated mice.

Repeated exposure to psychosocial stress modulates the endocannabinoid system, particularly anandamide (AEA) signaling in brain regions associated with emotional distress. The mTOR protein regulates various neuroplastic processes in the brain disrupted by stress, including adult hippocampal neurogenesis. This kinase has been implicated in multiple effects of cannabinoid drugs and the anti-stress behavioral effects of psychoactive drugs. Therefore, our hypothesis is that enhancing AEA signaling via pharmacological inhibition of the fatty acid amide hydrolase (FAAH) enzyme induces an anti-stress behavioral effect through an mTOR-dependent mechanism. To test this hypothesis, male C57Bl6 mice were exposed to social defeat stress (SDS) for 7 days and received daily treatment with either vehicle or different doses of the FAAH inhibitor, URB597 (0.1; 0.3; 1 mg/Kg), alone or combined with rapamycin. The results suggested that URB597 induced an inverted U-shaped dose-response curve in mice subjected to SDS (with the intermediate dose of 0.3 mg/kg being anxiolytic, and the higher tested dose of 1 mg/Kg being anxiogenic). In a second independent experiment, rapamycin treatment induced an anxiogenic-like response in control mice. However, in the presence of rapamycin, the anxiolytic dose of URB597 treatment failed to reduce stress-induced anxiety behaviors in mice. SDS exposure altered the hippocampal expression of the mTOR scaffold protein Raptor. Furthermore, the anxiogenic dose of URB597 decreased the absolute number of migrating doublecortin (DCX)-positive cells in the dentate gyrus, suggesting an anti-anxiety effect independent of newly generated/immature neurons. Therefore, our results indicate that in mice exposed to repeated psychosocial stress, URB597 fails to counteract the anxiogenic-like response induced by the pharmacological dampening of mTOR signaling.

Targeting Fatty Acid Amide Hydrolase Counteracts the Epithelial-to-Mesenchymal Transition in Keratinocyte-Derived Tumors.

The endocannabinoid system regulates physiological processes, and the modulation of endogenous endocannabinoid (eCB) levels is an attractive tool to contrast the development of pathological skin conditions including cancers. Inhibiting FAAH (fatty acid amide hydrolase), the degradation enzyme of the endocannabinoid anandamide (AEA) leads to the increase in AEA levels, thus enhancing its biological effects. Here, we evaluated the anticancer property of the FAAH inhibitor URB597, investigating its potential to counteract epithelial-to-mesenchymal transition (EMT), a process crucially involved in tumor progression. The effects of the compound were determined in primary human keratinocytes, ex vivo skin explants, and the squamous carcinoma cell line A431. Our results demonstrate that URB597 is able to hinder the EMT process by downregulating mesenchymal markers and reducing migratory potential. These effects are associated with the dampening of the AKT/STAT3 signal pathways and reduced release of pro-inflammatory cytokines and tumorigenic lipid species. The ability of URB597 to contrast the EMT process provides insight into effective approaches that may also include the use of FAAH inhibitors for the treatment of skin cancers.

Investigating the Effects of Exogenous and Endogenous 2-Arachidonoylglycerol on Retinal CB1 Cannabinoid Receptors and Reactive Microglia in Naive and Diseased Retina.

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

FAAH inhibition ameliorates breast cancer in a murine model.

Breast cancer is the leading cancer among females worldwide. Disease outcome depends on the hormonal status of the cancer and whether or not it is metastatic, but there is a need for more efficacious therapeutic strategies where first line treatment fails. In this study, Fatty Acid Amide Hydrolase (FAAH) inhibition and endocannabinoids were examined as therapeutic alternatives. FAAH is an integral membrane enzyme that hydrolyzes endocannabinoids, rendering them inactive, and FAAH inhibition is predicted to increase cancer cell death. To test this, breast cancer cells were probed for FAAH expression using Western blot analysis, treated with FAAH inhibitors, exogenous endocannabinoids, and combinations of the two treatments, and assessed for viability. High levels of FAAH were observed in different breast cancer cell lines. FAAH inhibition was more effective than exogenous endocannabinoid treatment, and the combination of FAAH inhibitors and endocannabinoids was the most effective in inducing apoptosis of breast cancer cells in vitro. In addition, in vivo FAAH inhibition reduced breast cancer growth in immunodeficient mice. FAAH inhibition is a promising approach, and tremendous progress has been made in the field to validate this mechanism as an alternative to chemotherapy. Further research exploring the therapeutic potential and impact of FAAH expression on cancer cells is warranted.

Tumor necrosis factor α, and agonist and antagonists of cannabinoid receptor type 1 and type 2 alter the immunophenotype of stem cells from human exfoliated deciduous teeth.

OBJECTIVE: To verify the involvement of the endocannabinoid system in the immunomodulatory profile of stem cells from human exfoliated deciduous teeth, in the presence or absence of TNF-α, and agonist and antagonists of CB1 and CB2. METHODS: Stem cells from human exfoliated deciduous teeth were cultured in the presence or absence of an agonist, anandamide, and two antagonists, AM251 and SR144528, of CB1 and CB2 receptors, with or without TNF-α stimulation. For analysis of immunomodulation, surface molecules linked to immunomodulation, namely human leukocyte antigen-DR isotype (HLA-DR), and programmed death ligands 1 (PD-L1) and 2 (PD-L2) were measured using flow cytometry. RESULTS: The inhibition of endocannabinoid receptors together with the proinflammatory effect of TNF-α resulted in increased HLA-DR expression in stem cells from human exfoliated deciduous teeth, as well as, in these cells acquiring an anti-inflammatory profile by enhancing the expression of PD-L1 and PD-L2. CONCLUSION: Stem cells from human exfoliated deciduous teeth respond to the endocannabinoid system and TNF-α by altering key immune response molecules. Inhibition of endocannabinoid receptors and TNF-α led to an increase in HLA-DR, PD-L1, and PD-L2 levels in stem cells from human exfoliated deciduous teeth. This study shows the interaction between mesenchymal stromal cells and the immune and endocannabinoid systems.

The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.