Tumour cell growth and viability

The first study monitoring the effects of phytocannabinoids on cancer regression in animal experiments was published by Munson et al. (1975), who showed suppression of tumour growth by Δ8‐tetrahydrocannabinol, Δ9‐tetrahydrocannabinol (THC) and cannabinol, long before the discovery of cannabinoid receptors and endocannabinoids. Meanwhile, cannabinoid compounds were revealed as potent inhibitors of cancer progression on different levels of cancer cell growth and spreading in numerous preclinical investigations.

During the last few decades, a large body of evidence has accumulated to suggest endocannabinoids, phytocannabinoids and synthetic cannabinoids exert an inhibitory effect on cancer growth via blockade of cell proliferation and induction of apoptosis. In the late 1990s, De Petrocellis et al. (1998), addressing the role of cannabinoid receptors in the growth inhibitory action of several cannabinoid compounds, reported AEA to reduce the proliferation of breast cancer cell lines at IC 50 values of 0.5 μM (MCF‐7 cells) and 1.5 μM (EFM‐19 cells) via a CB 1 receptor‐dependent mechanism. In their study, the synthetic cannabinoid HU‐210, likewise, elicited a comparable antiproliferative action on EFM‐19 cells but with less potency. Notably, although the authors did not mention the IC 50 levels for the antiproliferative effects of HU‐210, half maximal inhibition of proliferation can be estimated to be above 5 μM. Two years later, the first study demonstrating that THC and the synthetic cannabinoid WIN 55,212‐2 caused regression of glioma growth in Wistar rats and in Rag2−/− mice was published (Galve‐Roperh et al., 2000). In that study, rats, treated with THC or WIN 55,212‐2, were shown to live significantly longer than vehicle‐treated animals. These experiments were supplemented by in vitro investigations showing that THC, as well as the synthetic cannabinoids WIN 55,212‐2, CP55940 and HU‐210 inhibited glioma cell growth, associated with CB 1 ‐dependent and CB 2 ‐dependent sustained ceramide accumulation and p42/44 MAPK activation. Subsequently, the selective CB 2 receptor agonist JWH‐133 also inhibited growth of glioma cells (Sánchez et al., 2001). Following these and other pioneering studies (see Caffarel et al., 2012), a large number of investigations on anticancerogenic cannabinoid actions have been published.

In terms of the intracellular mechanisms of the antiproliferative actions of cannabinoids, several studies have demonstrated cannabinoid compounds to modulate cell cycle checkpoints. As such, cannabinoid receptor activation induced melanoma cell cycle arrest via inhibition of Akt and hypophosphorylation of the retinoblastoma‐associated protein (Rb) (Blázquez et al., 2006). While the latter and other studies reported cannabinoids to confer cell cycle arrest at the G 1 –S transition, another study using breast cancer cells showed that THC arrested the G 2 –M transition, via down‐regulation of cyclin‐dependent kinase 1 [CDK1/cell division cycle (Cdc)2] and induction of p21, a CDK inhibitor that suppresses Cdc2–cyclin B activation (Caffarel et al., 2006). A further investigation addressing the antiproliferative impact of a stable AEA analogue, 2‐methyl‐2′‐F‐anandamide (Met‐F‐AEA), on breast cancer cells, found a transient and delayed cell cycle checkpoint response (Laezza et al., 2006). Accordingly, Met‐F‐AEA caused an increase of p21waf and p27kip1 and a decrease of cyclins A and E. As upstream events of cyclin degradation, the authors demonstrated a rapid activation of checkpoint kinase 1 that induced downstream degradation of Cdc 25 homologue A and Cdk2 inactivation. As a delayed response, Met‐F‐AEA activated the p21waf cascade that additionally resulted in Cdk2 inhibition. These effects were further associated with a reduction of Rb activity, a prominent target of Cdk2 activity.

As a mechanism of glioma cell death in response to THC treatment, a CB 2 receptor‐mediated up‐regulation of the stress‐associated transcriptional co‐activator p8 mediated a proapoptotic action via up‐regulation of the endoplasmic reticulum stress‐related genes activating transcription factor (ATF)‐4 and the pseudo‐kinase tribbles homolog (TRB)3 (Carracedo et al., 2006). A contribution of p8 induction to cannabinoid‐induced apoptosis was later substantiated for THC and HU‐210 in rhabdomyosarcoma cells. In that study, cannabinoid‐induced apoptosis was associated with inhibition of Akt signalling and, as shown for HU‐210, restored by a CB 1 receptor antagonist (Oesch et al., 2009). Another investigation reported THC and the selective CB 2 receptor agonist, JWH‐133, to inhibit the growth of highly aggressive ErbB2‐positive breast cancers, associated with inhibition of the pro‐tumourigenic Akt pathway (Caffarel et al., 2010).

A panel of investigations reported cannabinoid‐induced impaired cancer cell viability via mechanisms bypassing activation of cannabinoid receptors. For example, CP55940, JW015 and the FAAH inhibitor, N‐arachidonoyl serotonin (AA‐5HT), inhibited proliferation of rat glioma cells independently of both CB receptors and TRPV1 channel activation (Jacobsson et al., 2001). In the same investigation, however, AEA and 2‐AG exerted antiproliferative actions via cannabinoid receptor‐dependent and TRPV1‐dependent oxidative stress and calpain activation. Furthermore, R(+)‐methanandamide induced a cannabinoid receptor‐ and TRPV1‐independent apoptosis in human neuroglioma cells by de novo synthesis of ceramide (Hinz et al., 2004). In the latter type of cells, the proapoptotic mechanism of R(+)‐methanandamide was based on a ceramide‐dependent up‐regulation of COX‐2 expression (Ramer et al., 2003) and increased synthesis of proapoptotic PGE 2 (Hinz et al., 2004).

Notably, reports on anticancer effects of CBD, a non‐psychoactive cannabinoid with low affinity to CB receptors, revealed proapoptotic effects, without CB receptor activation. Thus, CBD suppressed proliferation of glioma cells via decreased activity and content of 5‐lipoxygenase and of its end product LTB 4 (Massi et al., 2008). Another study found CBD to induce PPARγ‐dependent toxicity by upstream induction of COX‐2‐dependent PGs of the D and J series in lung cancer cells (Ramer et al., 2013). A further investigation addressing inhibition of glioma stem cell growth demonstrated CBD as a potential ‘redox therapeutic’ that inhibited glioma stem cell survival, associated with Akt phosphorylation via activation of the p38 MAPK pathway, as well as down‐regulation of stem cell marker proteins such as sex‐determining region Y‐box (Sox)2 and inhibitor of DNA binding (Id)‐1, an inhibitor of basic helix–loop–helix transcription factors (Singer et al., 2015). Here, growth inhibition by CBD was mediated via production of ROS that induced a regrowth via a counter‐regulated induction of the antioxidant protein SLC7A11 (xCT, cystine‐glutamate transporter). The antineoplastic action of CBD was further substantiated by McAllister et al. (2011), who demonstrated inhibition of growth and spread of breast cancer cells via mitochondrial damage, increased levels of ROS and down‐regulation of Id‐1. In breast cancer cells, CBD induced intrinsic apoptosis associated with autophagic pathways as indicated by decreased levels of a phosphorylated mechanistic target of rapamycin (mTOR), eukaryotic translation initiation factor (eIF) 4E‐binding protein 1 and cyclin D1 (Shrivastava et al., 2011).

Recent research concerning induction of cancer cell death has focused on autophagy as an underlying mechanism of cannabinoid‐induced antineoplastic action. In this context, THC induces ceramide accumulation, leading to downstream phosphorylation of eIF2α and subsequent endoplasmic reticulum stress associated with autophagy in glioma cells (Salazar et al., 2009). This autophagic effect was, likewise, associated with up‐regulation of ATF‐4 and TRB3, conferring downstream inhibition of the prosurvival kinase Akt with subsequent inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and thus induction of autophagic cell death. A contribution of the Akt/mTORC1 axis to a CB 2 receptor‐dependent autophagy by THC and the CB 2 receptor agonist JWH015 was further confirmed in experiments using hepatocellular carcinoma in vitro and in vivo (Vara et al., 2011). In addition to the death‐inducing Akt/mTORC1 modulation by cannabinoids, the latter investigation revealed a separate pathway leading to autophagy that encompasses a calmodulin‐activated kinase kinase subtype, which subsequently phosphorylates AMP‐activated kinase, thereby conferring autophagy as a response to cannabinoid treatment. Another study was able to demonstrate that cannabinoid‐induced TRB3 expression was associated with an up‐regulation of PPARγ that appeared essential for an appropriate autophagosome operation, thereby serving as a causal link to cannabinoid‐induced autophagic cell death (Vara et al., 2013). Noteworthy, results from these studies suggested autophagy, as a response to cannabinoid treatment, to be involved in the upstream activation of apoptosis rather than acting as apoptosis‐alternative pathway leading to cell death (Salazar et al., 2009; Vara et al., 2011). In this context, a recent investigation revealed TRPV2 as a cannabinoid target, involved in CBD‐induced autophagy (Nabissi et al., 2015).

Within the past years, several cannabinoids were found to modulate GPR55, another important key player of cancer progression. GPR55 was found to promote carcinogenesis and was up‐regulated in human carcinomas compared with corresponding healthy tissues (Pérez‐Gómez et al., 2013). Although the complex network of cannabinoid action on GPR55 signalling requires more data, a recent investigation led authors to pursue the hypothesis of a negative crosstalk between GPR55 and CB 2 receptors and a bidirectional cross‐antagonism between both receptors (Moreno et al., 2014).

Besides these antineoplastic effects of exogenously added cannabinoid compounds, a large body of evidence suggests endocannabinoids are similarly able to inhibit cancer cell growth (see Ramer and Hinz, 2016). In agreement with the proposed role of endocannabinoids as cancer‐repressive substances, a panel of investigations provided evidence for inhibitors of endocannabinoid turnover to exert comparable effects on cancer cells. In the first publication that revealed a contribution of cannabinoid receptors to the antiproliferative action of AEA, arachidonoyl‐trifluoromethylketone, an inhibitor of FAAH, enhanced the antiproliferative effects of exogenously added AEA, while arachidonoyl‐trifluoromethylketone alone did not affect the proliferation of breast carcinoma cells (De Petrocellis et al., 1998). The concept of inhibition of FAAH as an anticancer strategy was later supported by reports of the FAAH inhibitor, AA‐5HT, inhibiting the growth of xenografts generated from thyroid cancer cells (Bifulco et al., 2004) and reducing colonic carcinogenesis (Izzo et al., 2008). Furthermore, targeting the 2‐AG‐degrading MAGL has revealed a possible option for suppression of cancer progression. Knockdown of MAGL, using small hairpin RNA and pharmacological inhibition of MAGL with the MAGL inhibitor JZL184, suppressed the growth of prostate carcinoma in vivo via partial involvement of CB 1 receptors (Nomura et al., 2011). Using breast, ovarian and melanoma cancer cells, another study found MAGL inhibition suppressed cancer aggressiveness, without cannabinoid receptor activation, through a decrease of free fatty acids resulting in less cancer‐promoting PGs and lysophosphatidic acid (Nomura et al., 2010). In the latter study, inhibition of cancer growth by MAGL inhibition was reversed by adding back free fatty acids.

Inhibition of cancer growth in a murine xenograft model has further been demonstrated for colorectal carcinoma cells (Ye et al., 2011). Here, xenograft growth was inhibited in mice that received colorectal cancer cells transfected with MAGL siRNA or treatment with JZL184. In vivo experiments of this investigation revealed knockdown of MAGL, as well as treatment with JZL184 inhibiting cancer cell proliferation and invasion associated with down‐regulation of cyclin D1 and B‐cell lymphoma 2 (Bcl‐2). Similar results using colorectal carcinoma cells were reported by Ma et al. (2016). The latter study found an apoptosis‐related decrease of Bcl‐2 and increase of Bcl‐2‐associated X protein as a response to treatment with JZL184. However, these studies did not evaluate the contribution of cannabinoid‐activated receptors to the observed antiproliferative effects. In another study, lentivirus‐mediated MAGL knockdown in HT29 colon cancer cells and MDA‐MB‐231 breast cancer cells was associated with increased Akt phosphorylation, thereby exhibiting growth inhibition (Sun et al., 2013). Again, in this study, phosphorylation of Akt was constitutively suppressed by MAGL, but the contribution of cannabinoid receptors to this phenomenon was not addressed.