The most well-characterized of the deorphanized GPCRs, GPR55 (Ryberg et al., 2007), is expressed most highly in brain, adrenal gland, and digestive tract (although at reportedly low levels relative to that of CB1). When expressed in HEK293 cells, specific binding of the synthetic CB1 agonist CP55940 is observed, but not of the CB1/CB2 synthetic agonist WIN55212-2. Interestingly, a very small amount of specific GPR55 binding was observed using 50 nM of the CB1-selective antagonist/inverse agonist SR141716A (SR).

Using the same HEK293 cell line for GTPγS functional assays, the endogenous agonist 2-arachidonylglyerol (2-AG) potently activates GPR55 (EC50 = 3 nM), although virodhamine (EC50 = 12 nM) is a more complete agonist with about 50% greater efficacy. Notably, in a separate assay of intracellular calcium release, 2-AG and virodhamine showed no GPR55 agonism, suggesting this receptor is subject to agonist-dependent functional selectivity (Lauckner et al., 2008). Other potent agonists discovered in this initial screen of GPR55 efficacy include; palmitoylethanolamide (PEA), CP55940, Δ⁹-tetrahydrocannabinol (THC), noladin ether, and anandamide with EC50s of 4, 5, 8, 10, and 18 nM respectively, and efficacies similar to that of 2-AG. Interestingly, the non-psychoactive phytocannabinoid cannabidiol (CBD) was found to antagonize GPR55 activity at physiologically relevant concentrations.

GPR55 primarily couples through the relatively obscure Gα13. This interesting G-protein activates a cascade involving RhoA that, among other effects, ultimately alters actin polymerization and stability, implicating GPR55 signaling in processes related to neuronal morphology (Worzfeld et al., 2008). This potential role is further supported by effects to promote neurite retraction (Obara et al., 2011). More recently, evidence in transfected HEK293 cells indicates that signaling through G_q also occurs (Lauckner et al., 2008).

In ERK activation assays with GPR55-expressing HEK293 cells, lysophosphatidylinositol (LPI) is implicated as an important endogenous agonist (Oka et al., 2007). Notably, 2-AG and virodhamine were not effective in stimulating ERK in transfected HEK293 cells, possibly indicating ligand-dependent functional selectivity for different signal transduction pathways. LPI was also found to dose-dependently increase intracellular calcium in these cells with an EC50 ~ 1 μM, an effect possibly related to GPR55 activation of G_q. System-dependent efficacy of GPR55 signaling may also involve interaction with other receptors including CB1. GPR55 and CB1 have been shown to heterodimerize within rat and macaque striatum, possibly related to a role in motor behaviors. When co-expressed in HEK293 cells, CB1 and GPR55 antagonize respective agonist effects to promote ERK1/2 activation (Martínez-Pinilla et al., 2014).

The physiological significance of GPR55 expression in brain remains an open question; although, some evidence indicates a role in controlling ingestive behaviors (reviewed by Liu et al., 2015). A role in promoting appetite makes sense as it has been reported that GPR55 regulates peripheral metabolism and energy homoeostasis (reviewed by Simcocks et al., 2014). Deletion of GPR55 in mice has subtle effects on motor coordination, but it has shown no significant effect on several learning and memory tests, in contrast to the role of CB1 signaling, and despite dense GPR55 expression in hippocampus, striatum, and cortex (Wu et al., 2013). Spinal cord expression of GPR55 is upregulated in a chronic constriction injury model of neuropathic pain; this suggests that it may mediate some of the analgesic effects of N-arachidonoyl-serotonin (AA-5-HT, Malek et al., 2016). GPR55 activation by some agonists increases calcium release from intraneuronal stores and inhibits M-type potassium current, both tending to promote neuronal activity (Lauckner et al., 2008). These effects may be important in the context of CBD's ability to antagonize the receptor, as this non-psychoactive phytocannabinoid has recently been shown effective for the treatment of Dravet syndrome, a formerly intractable form of childhood epilepsy (Devinsky et al., 2017). Whether the efficacy of CBD in this syndrome involves GPR55 antagonism, or perhaps, involves some combination of the many other established targets of this drug (see Table 2) remains to be resolved. Other evidence for potential roles for GPR55 signaling includes: distinct expression in rod cells of primate retina suggesting a role in low-light vision (Bouskila et al., 2013) and; expression in microglia that, following LPI activation, acts to protect hippocampal neurons from excitotoxicity (Kallendrusch et al., 2013).

Despite the numerous studies illuminating the physiological functions of GPR55 in the CNS described above (section GPR55 in CNS), limited information is available about the role of this receptor in cancer. GPR55 is expressed in different cancer cell types raising the possibility that it may be a target for cancer chemotherapeutic agent development (reviewed by Falasca and Ferro, 2016). Much of the research conducted thus far indicates that GPR55 promotes tumor formation. GPR55 expression was examined in human tumor biopsy samples from breast, pancreatic, and glioblastoma patients (Andradas et al., 2011). In this study, high levels of GPR55 were strongly correlated with the aggressiveness of the malignancy. Additional evidence for GPR55-promotion of malignancy includes; (1) elevated levels of its endogenous ligand lysophosphatidylinositol (LPI) in ovarian cancers (Xiao et al., 2001) and; (2) exogenous LPI promotion of proliferation and migration of various cancer cell lines (Ford et al., 2010; Piñeiro et al., 2011). However, when GPR55 is activated by the endocannabinoid, AEA, cholangiocarcinoma cell survival is inhibited. AEA induces cholangiocarcinoma cell apoptosis by recruiting Fas death receptors into lipid rafts and activating the JNK signaling pathway (DeMorrow et al., 2007; Huang et al., 2011). Genetic disruption of GPR55 receptor expression using shRNA blocked the antiproliferative effects of AEA in vitro and in vivo (Huang et al., 2011). Of note, AEA-induced cell death was not reversed in the presence of CB1- (SR141716A) or CB2- (SR144528) selective antagonists although each cannabinoid receptor was expressed (DeMorrow et al., 2007). These findings suggest that AEA elicits antiproliferative effects on cholangiocarcinoma that are GPR55-dependent and CB receptor-independent. Other deorphanized GPCRs, including GPR18 and GPR35, have been identified as modulators of tumorigenesis (Okumura et al., 2004; Qin et al., 2011); however, the role of cannabinoid ligands in these systems is unclear. Examination of the deorphanized cannabinoid receptors in cancer will provide greater insight into their impact on tumor development and progression. In addition, determining whether classical cannabinoids mediate their effects through the deorphanized receptors will shed light on mechanisms of their antitumor activity.

Interestingly, microglia migration is stimulated by activation of a second formerly orphan cannabinoid-related receptor, GPR18 (McHugh et al., 2010). This G_i/o-coupled receptor was originally reported to be highly-expressed in testes and spleen (Gantz et al., 1997); yet, it clearly is also functional within brainstem (Penumarti and Abdel-Rahman, 2014b). GPR18 is activated by N-arachidonoyl glycine (NAGly, Kohno et al., 2006) that is a product of anandamide metabolism (Aneetha et al., 2009; Bradshaw et al., 2009). It is also activated by abnormal cannabidiol (abn-CBD) that, when infused into regions of the brainstem, causes reduction in blood pressure in a manner that involves nitric oxide synthase and adiponectin signaling (Penumarti and Abdel-Rahman, 2014a). As with GPR55 discussed above (section GPR55 in CNS), GPR18 receptor expression is upregulated following spinal cord injury; this suggests that it may mediate some of the analgesic effects of N-arachidonoyl-serotonin (AA-5-HT, Malek et al., 2016). Notably, the principal active constituent of Cannabis, THC, is a potent agonist of GPR18 when expressed in HEK293 cells (McHugh et al., 2012). The lesser Cannabis constituent, CBD, along with synthetic CB agonists CP55940 and WIN55212-2, have no activity at physiologically-relevant concentrations. Potent GPR18 activation by THC and NAGly has been suggested to underlie efficacy to resolve inflammatory pain (Burstein et al., 2011; McHugh et al., 2014; Crowe et al., 2015). Some controversy about GPR18 signal transduction was raised when NAGly failed to inhibit Ca⁺⁺ currents following heterologous expression and agonist stimulation in rat superior cervical ganglion neurons (Lu et al., 2013). Whether this is a special case of system-dependent functional selectivity, or an indication that NAGly modulates neuronal activity via other receptor systems, remains an open question.

A third relevant G-protein-coupled receptor, GPR119, has been deorphanized. In humans, GPR119 was initially reported to be expressed only outside of CNS, most notably in pancreas, suggesting a peripheral metabolic function (Fredriksson et al., 2003). A possible CNS expression difference between human and rodent species is suggested in a patent application that indicates GPR119 is also expressed at high density in rat and mouse brain (Bonini et al., 2002). Mouse CNS expression is confirmed by high-affinity GPR119 binding and potent efficacy to reduce seizure-related hippocampal activity (Scott et al., 2014). At micromolar concentrations, the endogenous peroxisome proliferator-activated receptor alpha (PPARα) agonist oleoylethanolamide (OEA) activates GPR119, while anandamide appears to have partial efficacy in yeast cells expressing the receptor (Overton et al., 2006). Notably, 2-AG, CP55940, WIN55212-2, methanandamide, and JWH-133 did not activate GPR119. Supporting CNS activity, and a role in controlling ingestive behaviors, OEA and a synthetic GPR119 agonist with similar efficacy, but four-fold greater potency, PSN632408, were found to reduce food consumption and body weight in Sprague-Dawley rats (Overton et al., 2006). More recent evidence suggests that PSN632408 may lack selectivity for GPR119 in peripheral tissues (Ning et al., 2008). Despite evidence of GPR119 CNS activity, this receptor is being most closely studied in as a target for treating diabetes and other metabolic disorders (Ansarullah et al., 2013; Cornall et al., 2013, 2015).

A final G_i/o-coupled deorphanized receptor, GPR35, has been reported to transduce effects of 2-arachidonyl lysophosphatidic acid (2A-LPA) and kynurenic acid, both of which are present at relevant concentrations within neuronal tissues. 2A-LPA is a product of metabolism of the principal brain endocannabinoid, 2-arachidonyl glycerol (2-AG, Oka et al., 2010). Kynurenic acid is the product of tryptophan metabolism, and is most well-characterized as an endogenous antagonist of receptors for the principal excitatory neurotransmitter, NMDA (Stone et al., 2013). A very recent structure-activity study has led to identification of several potent GPR35 agonists and three structures critical for efficacy (Abdalhameed et al., 2017). GPR35 is expressed most predominantly in the gastrointestinal tract and in leukocytes (Wang et al., 2006). Low-level expression of GPR35 in brain reduces the likelihood that it significantly modulates neuronal activity within this region of the CNS. Within spinal cord, on the other hand, convincing evidence demonstrates functionally-relevant expression of GPR35, with distinct enrichment within murine dorsal horn ganglia (Cosi et al., 2011). In an acetic acid-induced pain assay, both kynurenic acid, and the phosphodiesterase inhibitor, zaprinast, were found to produce significant analgesia with EC50s of about 100 and 1 mg/kg, respectively. Note zaprinast is an effective agonist of GPR35 with a particularly high potency at rat vs. human forms of the receptor (Taniguchi et al., 2006). Homology between GPR35, and other LPA receptors, may suggest that 2A-LPA is more relevant than kynurenic acid as an endogenous modulator (Zhao and Abood, 2013). Clearly, more needs to be learned and understood about GPR35 signaling.

As with other systems, evidence for cannabinoid modulation of serotonin signaling was obtained soon after CB1 receptors were identified. This evidence began to accumulate through studies of the rat nodose ganglion which conducts afferent transmission from GI, heart, and lung. Activity in this ganglion is increased by the 5-HT3 subtype of serotonin-gated cation channels. Using the synthetic agonists CP55940 and WIN, and the most well-established endocannabinoid at the time, anandamide, it was discovered that each of these agonists non-competitively antagonizes serotonergic activation of 5-HT3 receptors at nanomolar concentrations (Fan, 1995). The inability to modulate cannabinoid effects on 5-HT3 activity with guanyl nucleotides suggested a non-G-protein dependent mechanism, and possible direct interaction with the ion channel. This finding raised speculation that analgesic and antiemetic efficacy of cannabinoid agonists may, at least in part, be attributable to 5-HT3 antagonism.

Effects in the isolated tissue preparation described above were corroborated by results of experiments using a human 5-HT3A-expressing CHO cell line (Barann et al., 2002). In patch clamp studies it was found that a series of synthetic, endogenous, and phytocannabinoid agonists potently inhibited serotonin activation of 5-HT3 cation channels. Notably, the phytocannabinoid THC was the most potent compound evaluated with EC50 ~ 40 nM. Cannabinoid antagonism was not inhibited by the CB1-selective antagonist SR. Specific binding of a 5-HT3-selective radioligand wasn't displaced by the cannabinoids employed, suggesting interaction with an allosteric site that doesn't influence serotonin affinity.

The 5-HT1A subtype of serotonin receptors are notably associated with presynaptic distribution, where, similar to CB1 receptors, their activation reduces probability of neurotransmitter release. Although this type of presynaptic autoreceptor mechanism is well-characterized for 5-HT1A, the distribution of these receptors is wide, and it includes both excitatory and inhibitory terminals, making prediction of effects produced by modulators difficult.

Multiple lines of evidence suggest that the enigmatic non-psychotropic phytocannabinoid CBD exerts at least some of its actions through agonism of 5-HT1A autoreceptors (McPartland et al., 2015). First, CBD displaces specific binding of the 5-HT1A-selective radioligand [³H]8-OH-DPAT, activates GTPγS binding and inhibits adenylyl cyclase activity in heterologously expressing CHO cell cultures (Russo et al., 2005). Second, 5-HT1A receptor expression is upregulated under conditions of neuropathic pain in a manner that is reduced by cannabinoid agonism (Palazzo et al., 2006). Finally, microinfusion of CBD into the bed nucleus of the stria terminalis (BNST) dose-dependently reduces anxiety as measured by both elevated plus maze and Vogel conflict tests (Gomes et al., 2011).

These anxiolytic CBD responses are reversed by pretreatment with the 5-HT1A-selective agent WAY-100635 (WAY) that has been reported to be a neutral antagonist, although agonism at D₄ dopamine receptors has also been reported (Chemel et al., 2006). Recently, WAY-reversible anxiolytic effects of CBD as measured in the elevated plus maze have been confirmed by others (Fogaça et al., 2014). CBD mitigation of acute restraint stress was also found, although, perhaps importantly, this effect was more resistant to WAY antagonism, possibly suggesting involvement of another target in stress vs. anxiety responses. Interestingly, anxiolytic effects of both CB1 and 5-HT1A agonists have been reported in a non-mammalian teleost fish, suggesting a conserved relationship between cannabinoid and serotonergic signaling in responding to stress (Connors et al., 2014).

Extending studies of CBD anxiolysis that are reversed by the 5-HT1A antagonist WAY, mitigation of nausea as measure in the rat gaping model (Rock et al., 2014), reduction of both neuropathic pain (Ward et al., 2014) and panicolytic effects (Twardowschy et al., 2013) have all been reported. In the context of drug abuse, CBD antagonizes effects of morphine to reduce threshold intracranial self-stimulation responding—a measure of drug reward. CBD antagonism of morphine reward was reversed by microinjection of WAY into dorsal raphe, further implicating 5-HT1A involvement (Katsidoni et al., 2013). More recently, CBD has been reported to have rapid-onset antidepressive efficacy that is dependent upon 5-HT1A activation (Linge et al., 2016).

Physiological interactions, where activity of one signaling system influences activity of another, have been documented to occur between cannabinoid and serotonergic signaling in models of chronic pain (Campos et al., 2013); epilepsy (Devinsky et al., 2014); stress-induced analgesia (Yesilyurt et al., 2015) and; tic disorders (Ceci et al., 2015). These studies reinforce the possibility of using inhibitors of endocannabinoid uptake and metabolism therapeutically.

Given general sedative effects of cannabinoid agonists, it isn't surprising that these agents act, at many sites, to reduce release of the principal excitatory neurotransmitter, NMDA. This was found to clearly be the case in rat brain slice preparations for synthetic agonists (Shen et al., 1996) and both the endocannabinoid anandamide and phytocannabinoid THC. Inhibitory effects of THC and anandamide were reversed in the presence of both the CB1-selective antagonist SR and G_i inhibitor pertussis toxin implicating cannabinoid-receptor involvement (Hampson et al., 1998). But, in the presence of SR, anandamide potentiation of NMDA-induced currents were noted. This stimulatory effect of anandamide was also observed in Xenopus oocytes expressing NMDA receptor (NMDAR) subunits, suggesting a direct agonist interaction with these cation channel proteins.

With similarities to results of experiments done in slice preparations, additional evidence for anandamide interaction with NMDA receptors derives from studies of the complex cannabinoid modulation of blood pressure control in rats (Malinowska et al., 2010). In this system, it was discovered that anandamide produces a pressor effect in the presence of the CB1-selective antagonist, SR. This increase in pressure was partially reduced by the NMDA receptor-selective antagonist MK-801, suggesting a possible direct interaction with relevance to central control of blood pressure.

More recently, the interaction of anandamide and NMDAR activation has been more elaborately studied through a series of electrophysiology studies in rat hippocampal sections and dissected cells (Yang et al., 2014). These experiments demonstrated that CA1 pyramidal cell NMDAR activation by anandamide was reversed in the presence of the TRPV1 antagonist capsazepine, raising the possibility of vanilloid receptor involvement in the anandamide effects discussed above. 2-AG was also studied in these experiments and found to produce a similar potentiation of NMDAR activation, although distinct in that it was not blocked by TRPV1 antagonism. Interestingly, the efficacy of 2-AG was increased when delivered intracellularly, suggesting interaction with NMDAR or other protein domains inside the neuron.

It has long been suspected that Cannabis abuse increases risk of psychoses (Colizzi et al., 2016). In a mouse model of NMDAR antagonist-precipitated psychosis (MK-801), the CB1-selective antagonist AM 251 reduced behavioral symptoms, including hyperactivity (Kruk-Slomka et al., 2016). Interestingly, CB1-antagonism also improved psychosis-related memory impairments in this model, suggesting a possible role for endocannabinoid signaling in the memory deficits associated with schizophrenia and bipolar disorder.

An early Xenopus oocyte expression study examining sensitivity of various AMPA receptor subunit compositions demonstrated that high concentrations of anandamide (EC50 > 100 μM) effectively inhibit/s AMPA agonist-initiated currents (Akinshola et al., 1999). Anandamide inhibition was not reversed by SR, as expected in the CB1-deficient expression system, and similar efficacy was not observed for the synthetic agonist WIN. Enhancement of anandamide's effect was produced by addition of forskolin, a direct adenylyl cyclase activator, and partially reversed by cyclase inhibitors, implicating this enzyme in mediating the effect.

An interesting physiological interaction between endocannabinoid and AMPA receptor signaling has been reported in chicken embryo spinal cord (Gonzalez-Islas et al., 2012). In this system, a basal endocannabinoid tone inhibits activity of motor neurons which decreases spontaneous activity. Reversal of endocannabinoid tone via application of the CB1-selective antagonist, AM 251 resulted in an increase of spontaneous activity that, over 2 days, resulted in AMPA receptor desensitization, implicating endocannabinoid signaling in regulation of motor activity during development.

Physiological interaction of metabotropic glutamate receptors (mGluR) and endocannabinoid signaling has been reported in striatal and hippocampal slice preparations (Jung et al., 2005). In this system, activation of the mGluR5 receptor subtype resulted in release of the endocannabinoid 2-AG but not anandamide. This finding implicates 2-AG as the endocannabinoid involved in mediating AMPA receptor-induced plasticity important to processes of learning and memory.

In the case of cannabinoid receptor-independent effects on GABAergic signaling, in hippocampal slice preparations it has been found that WIN55212-2 and anandamide (but not 2-AG) potentiate GABA release within rat dentate gyrus. This release promoted measurable inhibitory post-synaptic currents (IPSCs) measured by electrophysiology. The fact these WIN55212-2 and anandamide-promoted currents were resistant to both CB1- and CB2-selective antagonists in tissues taken from CB1 deficient mice, is evidence that they are the result of interaction with a yet uncharacterized target (Hofmann et al., 2011). Interestingly, the agonist specificity (WIN55212-2 and anandamide) of this IPSC effect is similar to that discovered earlier in GTPγS binding assays employing mouse tissue (Breivogel et al., 2001) suggesting that the same target is responsible.

In the context of behavior, 2-AG has been shown to reduce locomotor activity in CB1/CB2 double knockout mice (Sigel et al., 2011). Unfortunately only endocannabinoids were investigated in this study leaving open the question of WIN55212-2 activity that is implicated by earlier studies mentioned above. Hypermotility was observed in mice deficient for the GABA_A B₂ subunit. When heterologously expressed in Xenopus oocytes, it was found that electrophysiological effects of 2-AG were only observed in systems producing B₂ protein, suggesting this subunit is required for 2-AG interaction with GABA_A. B₂/2-AG interaction has been further supported by modeling and studies of the effect of amino acid substitutions (Baur et al., 2013). This work has recently been extended to a slice preparation model that demonstrates 2-AG influences GABA_A signaling under physiologically-relevant conditions (Golovko et al., 2014). This physiological modulation involves both synaptic and extrasynaptic populations of GABA_A receptors enhancing the former and inhibiting the latter in a manner to potentiate overall GABAergic inhibition (Brickley and Mody, 2012).

Endocannabinoid regulation of spinal nociceptive vs. non-nociceptive transmission has been established in vertebrate species (Pernía-Andrade et al., 2009). This work has been extended to demonstrate that the endocannabinoid 2-AG produces similar effects in a non-cannabinoid receptor-expressing invertebrate species of leech (Higgins et al., 2013). The target of 2-AG in this animal appears to be a TRPV channel, as a selective antagonist blocked the effect. Interestingly, the 2-AG effect to potentiate non-nociceptive vs. nociceptive transmission was antagonized by the GABA_A-selective agent, bicuculline, suggesting a potential TRPV-GABAergic relationship in this physiological system.

Evidence for direct interaction of the endocannabinoids anandamide and 2-AG and glycine receptors were first reported from electrophysiological studies employing primary neuronal cell cultures and hippocampal slice preparations (Lozovaya et al., 2005). These experiments demonstrated that anandamide, 2-AG and the synthetic agonist WIN55212-2 inhibit glycine receptor conductance post-synaptically, and accelerate desensitization. This effect contrasts with the retrograde, presynaptic endocannabinoid mechanism that has been better characterized to date. Glycine receptor inhibition was observed in the presence of CB1, CB2, and vanilloid receptor antagonists, suggesting a direct agonist mechanism.

Complicating the picture, in a different Xenopus oocyte expression system, anandamide agonism of glycine receptors was discovered (Hejazi et al., 2006). These studies extended the effect to include the phytocannabinoid, THC. In an additional heterologous expression system, using HEK293 cell cultures expressing various glycine receptor subunits, an even more complex picture emerged (Yang et al., 2008). In these studies, anandamide and HU210 potently activated α1 subunit-containing glycine receptors. In contrast, the synthetic agonists HU210, HU308, and WIN55212-2 each potently (EC50 38—3030 nM) inhibited the α2 subunit-mediated currents, while the endocannabinoid NAGly (discussed above as a GPR18 agonist) activated it at high concentration. Efficacy and potency of glycine receptor inhibition varied with differential subunit expression. These types of apparent conflicts in pharmacological literature are common and, as we see with glycine receptors, are often attributable to different physiological/expression systems employed (Urban et al., 2007).

PPARs are a superfamily of nuclear receptors that play an important role in the regulation of lipid metabolism, glucose homeostasis, cell differentiation and tumorigenesis (Vamecq and Latruffe, 1999). PPAR transcription factors form heterodimers with retinoid X receptor (RXR) that then bind to peroxisome proliferator responsive elements (PPRE) and regulate transcription of target genes. PPARs are classified into three subtypes: PPARα, PPARδ, and PPAR⋎. In cancer cells, targeting PPAR⋎ with selective agonists inhibits cell proliferation, induces programmed cell death (apoptosis and autophagy) and promotes cell differentiation in multiple in vitro and in vivo studies (Campbell et al., 2008).

Cannabinoids increase PPAR⋎ transcriptional activity by diverse mechanisms (reviewed in O'Sullivan, 2007). Cannabinoids can bind and activate cell surface cannabinoid receptors which promotes MAPK signal transduction resulting in downstream PPARγ activation. Cannabinoid-induced PPARγ activation can also occur independent of cannabinoid receptors by direct and indirect mechanisms. Cannabinoids (e.g., ajulemic acid) and endocannabinoids (e.g., AEA and 2-AG) can directly bind, and increase the transcriptional activity of, PPARγ. Indirect PPARγ activation occurs as a consequence of the metabolism of endocannabinoids (2-AG, AEA) to PPARγ agonists or by increasing the synthesis of endogenous PPAR agonists (Burstein, 2005; O'Sullivan, 2007).

Several studies have shown that PPAR⋎ mediates the antitumor activity of cannabinoids independent of CB1 and CB2 receptors. In lung cancer cells, PPARγ activation was required for CBD-induced apoptosis (Ramer et al., 2013). This effect of CBD on PPARγ was mediated by production of the prostaglandins (PGs), PGD₂ and 15deoxy, Δ^{12, 14} PGJ2 (15d-PGJ₂) which are PPARγ activators. Prevention of PG synthesis with a selective inhibitor of COX-2 activity (NS-398) or siRNA directed toward COX-2 reduced CBD-induced PPARγ nuclear translocation and cell death. However, the use of CB1 (AM-251), CB2 (AM-630), or TPRV1 (capsazepine) antagonists did not alter CBD-mediated apoptotic cell death. Furthermore, in human cervical carcinoma cells treated with the non-hydrolysable AEA derivative, met-AEA, apoptosis was reliant upon the production of PGD₂ and PGJ₂ as well as the activation of PPARγ (Eichele et al., 2009). These findings indicate that cannabinoid-induced prostaglandin synthesis and the engagement of these prostaglandins with PPARγ represents an important pathway by which cannabinoids regulate cellular fate independent of the cannabinoid receptors.

In the case of the synthetic cannabinoid, ajulemic acid that is structurally-related to THC, patch clamp studies of HEK293 and ND7/23 cells expressing various types of voltage-gated sodium channels demonstrated inhibition with potencies ranging from 1 to 10 μM (Foadi et al., 2014). These results suggest that the efficacy of ajulemic acid to reduce neuropathic pain may involve sodium channel inhibition.

TRP channels are a superfamily of cation channels located on the cell plasma membrane that control inward movement of monovalent or divalent cations notably including Ca⁺⁺. These channels are largely sensory-related, and they are particularly important to perception of temperature and mechanical stimulation (Voets et al., 2005). TRP channels have already been mentioned above (in the context of anandamide interaction with NMDA receptors) (section NMDA Receptors in CNS). In addition to a role in mediating peripheral afferent signaling, TRP channels are physiologically relevant to the extraneuronal function of vascular smooth muscle (Fernandes et al., 2012) and are also expressed within CNS (Vennekens et al., 2012). Several members of this family may represent “ionotropic cannabinoid receptors” moving cannabinoid signaling into line with most other CNS signaling systems that include both metabotropic and ion channel targets (Akopian et al., 2009).

Several members of this family, including TRP ankyrin type 1 (TRPA1), the vanilloid receptors types 1 and 2 (TRPV1 and TRPV2), and TRP melastatin type 8 (TRPM8), have been identified through a remarkably ambitious and thorough screening study as targets of an array of compounds isolated from Cannabis (De Petrocellis et al., 2011). These phytocannabinoids include cannabichromene (CBC), CBD, cannabidiol acid (CBDA), cannabidivarin (CBDV), cannabidivarin acid (CBDVA), cannabigerol (CBG), cannabigerol acid (CBGA), cannabigivarin (CBGV), cannabinol (CBN), THC, THC acid (THCA), and tetrahydrocannabivarin acid (THCVA). Each of these compounds are effective agonists for TRP1A with EC50s ranging from 90 nM to 8.4 μM and a rank order of potency of CBC > CBD > CBN > CBDV > CBG > THCV > CBGV > THCA > CBDA > CBGA > THCVA. At TRPV1; CBD, CBG, CBN, CBDV, CBGV, and THCV have agonist activity with EC50 < 10 μM (the most potent compound being CBD with EC50 = 1 μM). At TRPV2, several of the compounds had agonist activity with a rank order of potency = THC > CBD > CBGV > CBG > THCV > CBDV > CBN (EC50 ranging from 650 nM to 19 μM). Finally, at TRPM8, each of the compounds were found to effectively antagonize icilin-stimulated Ca⁺⁺ conductance with a rank order of potency = CBD > CBG > THCA > CBN > THCV > CBDV > CBGA > THCVA > CBGV > CBDA and IC50s ranging from 60 nM to 4.8 μM.

Interestingly, the synthetic cannabinoid compounds AM251 and AM630, that are CB1- and CB2-selective antagonists/inverse agonists, respectively, have been found to activate Ca⁺⁺ channels in primary cultures of trigeminal nerve neurons (Patil et al., 2011). When evaluated in CHO cell culture systems heterologously expressing TRP1A and TRPV1 channels, it was found that both CB receptor antagonists have agonist activity at TRP1A but not TRPV1. These findings suggest that in vivo AM251 and AM630 may activate sensory neurons via TRP1A agonism.

A growing body of evidence implicates members of the TRP family, including TRPV1, TRPV2, and TRPM8, in calcium-mediated signal transduction that regulates proliferation, migration, and metastasis of cancer cells (reviewed by Déliot and Constantin, 2015; Yee, 2015). As discussed previously, numerous cannabinoids bind to, and modify, the activity of TRP channels. We review TRP-mediated activity of several cannabinoids in cancer below.

The elaborate study of phytocannabinoids discussed above in the context of TRP channels [section Transient Receptor Potential (TRP) Cation Channels in CNS] in CNS also included evaluation of the same series of compounds for effects on activity of enzymes related to endocannabinoid signaling—including those responsible for endocannabinoid production (DAGLα), metabolism [monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), and N-acylethanolamine acid amide hydrolase (NAAA)] and uptake (anandamide cellular uptake [ACU]). Results demonstrated that a few of the compounds inhibit DAGLα with a rank order of potency CBDV = CBDA > CBGA = THCA = CBDVA (EC50s ranged from 17 to 35 μM, De Petrocellis et al., 2011). Both CBC and CBG partially inhibit MAGL activity with efficacies of ~50% and IC50s = 50 and 95 μM, respectively. THCA is a more fully-effective MAGL inhibitor with IC50 = 46 μM. Only CBD was found to inhibit FAAH at concentrations under 50 μM (IC50 = 15 μM). Only CBDA inhibited NAAA, the enzyme responsible for degradation of the endocannabinoid-like compound N-palmitoylethanolamine (PEA), doing so with IC50 = 23 μM.

FAAH is a membrane-bound serine hydrolase that catabolizes naturally occurring fatty acid amides including AEA, OEA, and PEA to fatty acids plus ethanolamine (Cravatt et al., 1996, 2001). FAAH regulates the levels of its main cannabinoid substrate AEA, and it is overexpressed in different types of cancer. FAAH expression was elevated in lung adenocarcinoma compared to non-malignant respiratory epithelial cells (Ravi et al., 2014). In prostate cancer cells, FAAH was overexpressed, and the upregulated FAAH levels correlated with poor patient prognosis (Thors et al., 2010). In addition, AEA levels and FAAH expression and activity were elevated in human colorectal cancer tissue compared to non-tumor colon tissue (Chen et al., 2015). Consistent with findings demonstrating that FAAH is overexpressed in cancer, in vitro and in vivo studies revealed that the antitumor activity of endocannabinoids was increased by inhibiting FAAH activity. AEA-mediated death in HT29 colorectal cancer cells was enhanced by its co-administration with the FAAH inhibitor, MAFP (Patsos et al., 2005). Similarly, the apoptotic effect of AEA was increased by co-exposure to the FAAH inhibitor URB597 in non-melanoma skin cancer cells (Kuc et al., 2012). In addition, FAAH inhibition with URB597 prevented AEA degradation and increased its cytotoxicity in neuroblastoma cells (Hamtiaux et al., 2011). The reduction in cell viability caused by the co-administration of AEA and URB597 was found to be independent of CB1, TRPV1, PPAR-α, PPAR-γ, and GPR55 receptor activity (Hamtiaux et al., 2011). These findings suggest that increasing cellular levels of AEA, by decreasing FAAH activity, allows greater quantities of this endocannabinoid to interact with its molecular targets (thereby enhancing its antitumor activity).

MAGL is a membrane-associated serine hydrolase that metabolizes monoacylglycerols (MAG) to free fatty acids (FFA) plus glycerol. MAGL is a prominent regulator of the levels of the endocannabinoid, 2-AG. MAGL was found to be elevated in aggressive cancer cells (compared to non-aggressive cells) (Nomura et al., 2010). MAGL increased FFA production to promote tumor growth, tumor cell migration, and tumor invasion. Inhibiting MAGL expression with shRNAs, or blocking its activity with JZL184, reduced tumor cell migration in a manner that was not dependent on the CB1 or CB2 receptors (Nomura et al., 2010). These data indicate that the tumor promoting metabolic products of 2-AG increase cancer cell survival. As such, examining MAGL, 2-AG and FFA levels in different tumor types, and identifying cellular targets of FFAs, will be an important step toward understanding whether this pathway can be exploited for therapeutic benefit.

COX-2 oxygenates arachidonic acid (AA) to prostaglandin H₂ (PGH₂) that is further metabolized by PG synthase-E, -F_2α, and -D to PGE₂, PGF_2α, and PGD₂, respectively (Rouzer and Marnett, 2009). PGD₂ is then dehydrated to J-series PGs. Substantial evidence points to a role for COX-2 and its products, notably PGE₂, in the growth of epithelial tumors of the colon, lung, breast, skin, and other organs (Jiao et al., 2014; Ma et al., 2015; Majumder et al., 2015; Kiraly et al., 2016). Pro-inflammatory COX-2 and PGE₂ promote cell proliferation, angiogenesis, and cell migration by activating PGE₂ receptors which increase oncogene and cytokine activity (Rundhaug et al., 2007, 2011). In contrast, cannabinoids possess anti-inflammatory activity (Burstein and Zurier, 2009). The mechanism by which cannabinoids suppress inflammatory cascades is a matter of debate; although, some studies suggest that cannabinoids block cyclooxygenase activity. Ruhaak et al. reported that several cannabinoids isolated from Cannabis sativa inhibited COX-2 activity and reduced prostaglandin generation (Ruhaak et al., 2011). Tetrahydrocannabinolic acid (THC-A), cannabigerol (CBG), and cannabigerolic acid (CBGA) inhibited the activity of COX-2 in a cell-free enzyme assay with IC50 values of 6.3 × 10⁻⁴, 2.7 × 10⁻⁴, and 2.0 × 10⁻⁴ M, respectively. However, in TNFα stimulated cancer cells, a significant reduction in COX-2 activity was not observed (Ruhaak et al., 2011). In a different study, CBDA was a potent and selective inhibitor of COX-2 activity with an IC50 value of 2.2 μM and an IC50 value of 20 μM for COX-1 (Takeda et al., 2008). These authors suggested that the inhibitory effect of cannabinoids on COX-2 activity was due to the presence of the salicylic acid moiety in the chemical structure of CBDA. Additional studies are needed to uncover the biological and clinical relevance of cannabinoid-mediated cyclooxygenase inhibition.

In contrast to the inhibitory effects of cannabinoids on COX-2 activity mentioned previously, other reports indicate that cannabinoids upregulate COX-2 expression. A series of studies by Hinz et al. demonstrated that cannabinoid-induced COX-2 expression increased the synthesis of arachidonic acid-derived prostaglandins that promoted cell death (Hinz et al., 2004a,b). In lung cancer cells, CBD increased COX-2 expression and the synthesis of D- and J- series prostaglandins which initiated tumor cell apoptosis (Ramer et al., 2013). Met-AEA also increased COX-2 expression in a manner that was dependent on lipid rafts, ceramide, and the activation of p38 and p42/44 MAPK (Ramer et al., 2003; Hinz et al., 2004a). However, Met-AEA-induced apoptosis was not reversed by CB1 (AM251), CB2 (AM630), or TRPV1 (capsazepine) receptor antagonists in human neuroglioma cells (Hinz et al., 2004b). It was also reported that Met-AEA increased ceramide synthesis, COX-2 expression and the production of proapoptotic PGD₂ in human cervical carcinoma cells (Eichele et al., 2009). In this study, inhibiting the production of D-series PGs using siRNA directed against lipocalin-PGDS or siRNA against PPARγ prevented Met-AEA-induced apoptosis. A reversal in Met-AEA-induced apoptosis, however, was not observed in the presence of the receptor antagonists, AM251, AM630 or capsazepine (Eichele et al., 2009). In contrast, Garder et al. determined that Met-AEA increased lung tumor growth in vitro and in vivo (Gardner et al., 2003). This pro-tumorigenic effect was blocked by pharmacological inhibitors of COX-2, p38, and p42/44, but it was not affected by the CB1 (SR141716) and CB2 (SR144528) receptor antagonists (Gardner et al., 2003).

AEA is also a substrate of COX-2, and, as a result, is cytotoxic in tumor cells that overexpress COX-2. AEA has an unmodified arachidonate backbone, and it is therefore susceptible to oxidative metabolism by COX-2 to form prostaglandin-ethanolamines, also known as prostamides (PM) (Yu et al., 1997; Kozak et al., 2002; Soliman et al., 2016). The metabolic products of AEA, prostamides-E₂, F_2α, and -D₂, do not bind to prostaglandin receptors, and they are metabolically stable relative to prostaglandins derived from arachidonic acid (Kozak et al., 2001; Matias et al., 2004). Recently, we demonstrated that AEA was also metabolized by COX-2 to novel J series prostamides that initiated tumor cell apoptosis (Kuc et al., 2012; Ladin et al., 2017). Because epithelial cancer cells typically overexpress COX-2, AEA was metabolized to pro-apoptotic J-series prostamides in tumor cells, but J-series prostamides were not detected in non-tumor cells which had low endogenous levels of COX-2 (Soliman et al., 2016). Blockade of AEA degradation with the FAAH inhibitor, URB597, increased J-series prostaglandin synthesis and apoptosis; however, inhibition of CB1, CB2, and TRPV-1 receptors using selective antagonists did not reverse this effect (Soliman and Van Dross, 2016). Furthermore, cell treatment with exogenous 15-deoxy, Δ^{12, 14} prostamide J₂ (15d-PMJ₂), the most abundant J-series product of AEA metabolism by COX-2, also caused cell death in vitro and in vivo (Ladin et al., 2017). Reports by Pastos et al. also demonstrated that COX-2 was required to induce death in cancer cells treated with AEA (Patsos et al., 2005, 2010). Prostamides E₂ and D₂ were noted to cause tumor cell death while blockade of COX-2 activity or expression partially reversed this antiproliferative effect. However, AEA-mediated cell death was not blocked by CB1, CB2, or TRPV1 receptor antagonists (Patsos et al., 2010). Thus, COX-2 is a critical regulator of CB receptor-independent cannabinoid activity in cancer cells. These studies demonstrate that cannabinoid metabolism and signal transduction is modulated by COX-2 and that COX-2 is also inhibited by molecules within the cannabinoid family. This implies that a complex relationship exists between cannabinoids, the endocannabinoid system, and cyclooxygenases in cancer and potentially in other organ systems.

The restoration of phosphatase expression and activity is a desirable effect of chemotherapeutic agents, as these enzymes dephosphorylate kinases, and other pro-tumorigenic proteins, that are often constitutively active in cancer (Perrotti and Neviani, 2013). The anticancer activity of WIN55212-2 was reported to be mediated by phosphatases in a cannabinoid receptor-independent manner (Sreevalsan et al., 2011; Sreevalsan and Safe, 2013). WIN55212-2 increased the expression of several phosphatases including dual specificity phosphatases 1 and 10 (DUSP1, DUSP10) and protein tyrosine phosphatase, non-receptor type 6 (PTPN6) in LNCaP prostate cancer cells (Sreevalsan et al., 2011). WIN55212-2 also increased PTPN6 expression and protein phosphatase A2 (PPA2) activity in SW480 colon cancer cells (Sreevalsan et al., 2011; Sreevalsan and Safe, 2013) and reduced the expression of the pro-tumorigenic transcription factors, specificity protein 1, 3, and 4 (SP1, SP3, and SP4). The effect of WIN55212-2 on SP expression and apoptosis was dependent on phosphatase PPA2, a prominent serine-threonine phosphatase in eukaryotic cells that regulates the cell cycle and apoptosis. Specifically, it was determined that WIN55212-2 increased the activity of PPA2 and activation of the Sp repressor, ZBTB10, thereby inhibiting Sp expression through a pathway that was regulated by micro RNA-27a (miR-27a). However, this cytotoxic activity was not prevented by blockade of CB1 or CB2 receptor activity (Sreevalsan and Safe, 2013). These reports suggest that cannabinoid activity is modulated by phosphatases independent of the cannabinoid receptors. Phosphatases have significant impacts on cellular behavior, because these enzymes control kinase activity. Therefore, examination of the role of phosphatases in cannabinoid activity may allow these proteins to be targeted to improve the action of cannabinoids.

Lipid rafts are glycoprotein microdomains enriched in cholesterol and sphingolipids. Lipid rafts serve as organization centers that promote interactions between proteins to aid in intracellular signal transduction (Simons and Ikonen, 1997). Ceramide is a membrane lipid that displaces cholesterol from lipid rafts, enhances membrane rigidity, stabilizes rafts and induces formation of large raft domains (“platforms”) in plasma membranes (Simons and Ikonen, 1997; Megha and London, 2004). It has been reported that the antitumor activity of cannabinoids is regulated by ceramide and lipid rafts which control CB1 and/or CB2-mediated signal transduction (Bari et al., 2005; Sarnataro et al., 2006). However, other studies demonstrate that ceramide and lipid rafts transmit lethal cannabinoid signals independent of CB1 and CB2. DeMorrow et al., found that AEA increased ceramide synthesis and localization of the death receptor/ligand, Fas/FasL, to lipid rafts leading to cholangiocarcinoma cell death (DeMorrow et al., 2007; Huang et al., 2011). In these studies, selective antagonists of CB1 and CB2 did not inhibit the cytotoxicity of AEA. In a different report, lipid raft disruptors completely attenuated AEA-mediated cell death in neuroblastoma cells (Hamtiaux et al., 2011). This cytotoxicity was not blocked by pharmacological antagonism of CB1, CB2, GPR55, TRPV1, or PPAR⋎. Similarly, the disruption of lipid rafts, but not the antagonism of CB1, CB2, or TRPV1 receptors, prevented AEA cytotoxicity in cutaneous melanoma cells (Adinolfi et al., 2013). In addition, Sarker and Maruyama found that disruption of lipid rafts blocked AEA-induced oxidative stress and apoptosis independent of the CB1, CB2, or TRPV1 receptors in different cell lines (Sarker and Maruyama, 2003). Consistent with these findings, Met-AEA-induced apoptosis was inhibited by the use of ceramide synthase inhibitors and lipid raft disruptors but not by CB1, CB2, or TRPV1 receptor antagonists in human neuroglioma and cervical carcinoma cells (Hinz et al., 2004b; Eichele et al., 2009). Moreover, WIN55212-2, caused lipid raft-mediated cell death in cultured melanoma cells in a CB1/CB2 receptor-independent manner (Scuderi et al., 2011). Collectively, these findings suggest that cannabinoids reduce tumor cell viability by modulating lipid rafts through cannabinoid receptor-dependent and -independent pathways.

Reactive oxygen species (ROS) are second messenger signal transduction molecules in eukaryotic cells. Low levels of ROS are needed for physiological processes such as immune defense against pathogens, mitogenic responses, and maintenance of cellular homeostasis (Finkel, 1998; Kim et al., 1998; Dröge, 2002). Physiologic levels of ROS are maintained by a balance between antioxidant and pro-oxidant systems in the cell. However, when antioxidant systems become overwhelmed, oxidative stress occurs. Cancer cells contain supraphysiological levels of ROS that activate signaling pathways which promote cell proliferation, survival, angiogenesis, and metastasis (Nourazarian et al., 2014). However, excessively high ROS levels causes oxidative damage to cellular proteins, lipids, and DNA that triggers cell cycle arrest and cell death (Kaur et al., 2014). Structurally distinct cannabinoids can inhibit tumor cell survival by generating high levels of ROS. CBD decreased glutathione (GSH) levels and increased GSH reductase and GSH peroxidase content thereby causing cytotoxic oxidative stress in glioma cells (Massi et al., 2006). The cytotoxicity of CBD was abrogated by the antioxidant, α-tocopherol; however, blockade of CB receptor activity or ceramide synthesis did not significantly alter CBD cytotoxicity (Massi et al., 2004). Moreover, CBD-induced oxidative stress and cell death occurred in tumorigenic U87 glioma cells but not in non-tumorigenic primary glial cells (Massi et al., 2004), suggesting ROS elicits tumor-selective cytotoxicity. Ligresti et al. also found that antioxidants (α-tocopherol and vitamin C), but not CB receptor antagonists, blocked the antiproliferative activity of CBD in breast adenocarcinoma cells (Ligresti et al., 2006). Similarly, Shrivastava et al. determined that CBD increased ROS production that initiated autophagy and apoptosis independent of CB1, CB2, or TRPV1 in breast cancer cells (Shrivastava et al., 2011).

In several reports, the endocannabinoid, AEA, induced ROS-dependent, CB receptor-independent cell death. Oxidative stress and apoptosis in AEA treated non-melanoma skin cancer cells were prevented by the antioxidant, N-acetyl cysteine (NAC) (Van Dross, 2009). Blockade of CB1 and CB2 receptor activity did not rescue cells from AEA-mediated oxidative stress or apoptosis (Soliman and Van Dross, 2016). Similarly, in colon cancer cells, AEA-induced cell death and apoptosis was reversed by the use of antioxidants but not by CB1 or CB2 receptor antagonists (Gustafsson et al., 2009; Soliman, 2015). These findings suggests that oxidative stress regulates cannabinoid activity through pathways that circumvent the cannabinoid receptors.

ER stress occurs when the capacity of the cell to fold proteins is exceeded by the protein folding load resulting in the accumulation of unfolded proteins in ER (the unfolded protein response, UPR) (Holcik and Sonenberg, 2005). The UPR is an integrated intracellular signal transduction pathway that is regulated by three ER-resident stress sensors: double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), activating transcription factor-6 (ATF6), and inositol requiring kinase-1 (IRE1) (Lin et al., 2008; Chakrabarti et al., 2011). Activated PERK phosphorylates the α subunit of eukaryotic initiation factor 2α (eIF2α), resulting in a reduction in global translation with a preferential increase in the expression of activated transcription factor 4 (ATF4)-regulated genes (reviewed in Marciniak et al., 2006). Activation of PERK, ATF6, and IRE1 promotes cell survival by decreasing protein synthesis and by increasing protein folding and degradation to reestablish ER homeostasis. However, excessive or prolonged ER stress activates cell death pathways primarily by increasing expression of the transcription factor, C/EBP homologous protein 10 (CHOP10, also known as the growth arrest and DNA damage-inducible gene 153 [GADD153]) which transcribes pro-apoptotic genes (reviewed in Marciniak and Ron, 2006). Several studies have shown that the ER stress pathway mediates the anticancer activity of cannabinoids. The phytocannabinoid, THC, increased phosphorylation of eIF2α and the expression of the CHOP10 transcriptional product, TRB3, in hepatocellular carcinoma cells (HEPG2), human glioma cells, and RasV12/E1A-transformed mouse embryonic fibroblasts (MEF, Salazar et al., 2009, 2013; Vara et al., 2013). TRB3 is known to inhibit cancer cell proliferation by inactivating AKT/ mammalian target of the rapamycin complex 1 (mTORC) signal transduction (Ohoka et al., 2005). In human glioma cells treated with THC, it was determined that TRB3 was required for inhibition of Akt/mTORC1 signaling and the induction of autophagy and apoptosis (Salazar et al., 2009). Furthermore, unlike Trib3^+/+ (also known as TRB3) MEF cells, Trib3^−/− MEFs were resistant to cell death caused by THC (Salazar et al., 2013). Similar results were observed when the two cell types were injected subcutaneously in nude mice that were subsequently treated with THC. THC reduced the growth of Trib3^+/+ xenograft tumors; however, this effect was not seen in mice engrafted with Trib3^−/− cells (Salazar et al., 2013). WIN55212-2 also induced ER stress and increased the expression of CHOP10 and TRB3 in cancer cells (Wasik et al., 2011; Notaro et al., 2014; Pellerito et al., 2014). In addition, WIN55212-2 reduced the viability of osteosarcoma cells by initiating cytotoxic autophagy (Notaro et al., 2014). Genetic ablation of CHOP10 using siRNA prevented WIN-induced autophagic cell death. WIN55212-2 also caused autophagy and apoptosis in human colorectal cancer cells that was prevented by reducing the expression of CHOP10 with siRNA (Pellerito et al., 2014). Interestingly, Wasik et al. reported that WIN55212-2-induced autophagic cell death in Mantle cell lymphoma (MCL) cells occurred independent of the CB1 and CB2 receptors (Wasik et al., 2011). Consistent with these observations, the ER stress inhibitors, phenylbutyric acid (PBA) and salubrinal, prevented apoptosis in non-melanoma skin cancer cells treated with AEA. However, selective antagonism of the CB1 and CB2 receptor failed to inhibit AEA-induced ER stress or apoptosis (Soliman and Van Dross, 2016; Soliman et al., 2016). Structurally diverse cannabinoids initiate cytotoxic ER stress in cancer cells independent of the cannabinoid receptors. Because the ER stress pathway is a primary regulator of protein folding and synthesis in cells, understanding the impact of cannabinoids on the ER stress pathway is of vital importance for development of effective therapeutic agents.