1.6.1. Cannabinoids and Ovarian Cancer

Among gynaecological cancers, those of the ovary have the highest morbidity and mortality rates [140]. In an attempt to establish if there is a possible role for the ECS in ovarian pathophysiology, we [45] studied the expression levels of different components of the ECS [50], and demonstrated expression of CB₁, CB₂, and the NAE-modulating enzymes NAPE-PLD and FAAH in normal human ovaries using immunohistochemistry [45]. Additionally, AEA concentrations in follicular fluid after ovarian stimulation by hormones (following an in vitro fertilisation protocol that caused an increase in follicle size) were directly correlated with follicle size, suggesting that AEA is indeed involved in the hormonal maturation of follicles and oocytes [45,50]. Furthermore, data exist to indicate that AEA, OEA, and PEA are all elevated in follicular fluids of ovarian cancer patients and women with ovarian cysts [59].

Bagavandoss and colleagues demonstrated CB₁ and FAAH expression in ovarian surface epithelium, the site from which some ovarian cancers often arise, providing another clue for a possible involvement of the ECS in ovarian cancer [96]. Regarding the expression of CB₁ in ovarian cancer, Messalli and coworkers [141] showed that CB₁ expression was moderate in benign and borderline epithelial rat ovarian tumours, but was increased in invasive ovarian tumours, suggesting a correlation between the extent of expression of the ECS components and the prognosis for patients with more aggressive ovarian cancer [141]. The levels of lysophospholipids such as lysophosphatidylinositol (an endogenous GPR55 agonist) in blood and ascitic fluids were also found to be elevated in ovarian cancer patients compared to healthy controls, a finding associated with proliferation and the metastatic potential of ovarian cancer cells [142]. Hofman and colleagues [143] more recently found that elevated lysophosphatidylinositol levels in the ovarian cancer cell lines OVCAR-3, OVCAR-5, and COV-362 resulted in GPR55-dependent angiogenesis. Their conclusion was based on experiments where pharmacological inhibition and genetic deletion of GPR55 reduced the pro-angiogenic potential of lysophosphatidylinositol in these cell lines. Additionally, they demonstrated that the mitogen-activated protein kinase pathway triggered via GPR55 by phosphorylation of ERK1/2 and p38, which are signalling molecules known to be involved in proliferative and migratory responses, could be curtailed by chemical interventions [143]. This observation suggests that some ovarian cancers might be amenable to pharmaceutical intercession. In addition, other components of the endocannabinoid system are important here. For example, the 2-AG degrading enzyme MAGL has been shown to be upregulated in aggressive human ovary cancer cells [65], and it is also thought to be involved in oncogenic signalling and, hence, in increased migration, invasion, and survival of many other cancer cell types [144]. These data suggest that identification of an effective drug that targets the ECS to treat ovarian cancer may have applications in the treatment of other cancers too. The application of such therapies would need to be timely, because MAGL overexpression in non-aggressive cancer cells often results in tumours that subsequently exhibit an increased pathogenic phenotype [65]. Moreover, the application of an MAGL inhibitor led to a reversion of the enhanced pathogenicity [65]. Thus, the involvement of the ECS, and especially the 2-AG signalling pathways in ovarian cancer, may fuel expectations on new therapeutics to combat this and other types of cancer. Some preliminary evidence suggests that OEA and its structural analogues may also have a beneficial effect on inhibiting ovarian cancer growth, but these data need to be confirmed in vivo [60]. There is little evidence that plant-derived (phyto)cannabinoids have any effect on ovarian biology or ovarian cancer development or progression, a concept that came from a study where SKOV-3-derived tumours were grown on the chorioallantoic membrane of fertilised chicken eggs [145], and then were treated with CBD-containing nanoparticles. The data indicated that CBD caused a 1.35- to 1.50-fold reduction in tumour size depending on the type of CBD formulation used [145]. The authors indicated that these nanoparticle preparations might be useful in the treatment of peritoneal metastases of ovarian cancer, possibly with lower adverse drug effects [145]. Furthermore, the preparations also reduced SKOV-3 ovarian cancer cell numbers in vitro, to almost zero within 48 h, possibly making this a good candidate for a randomised clinical trial. Of course, many additional studies are required before any candidate CBD formulation can be used in such clinical trials.

1.6.2. Cannabinoids and Fallopian Tube Cancer

Fallopian tube cancer is a relatively rare gynaecological cancer (Figure 2). It is often categorised as being part of ovarian cancer (especially as there is emerging evidence that most surface epithelial ovarian cancers maybe of fimbrial origin), but it is important to study it as a separate entity. Just like other parts of the female reproductive tract, the oviduct (Fallopian tube) expresses all the components of the ECS, with CB₁ and FAAH expression intimately associated with proper oviductal function [49,98,99]. When dysfunctional, the risk of ectopic pregnancy is markedly increased [46,49,99,100,146]. There is little evidence on the effect of cannabinoids on human oviductal function, but in the murine oviduct [100], THC reduces fertility because of the increased number of ectopically implanting embryos. In the bovine oviduct, there is gradation of AEA, OEA, and PEA concentrations in the oviductal epithelial cells with low levels in the isthmus and significantly higher levels of OEA and PEA (but not AEA) in the ampulla at the same point of the oestrous cycle [67]. These levels significantly fluctuated during the oestrous cycle [67], as they do in the human oviduct (Fallopian tube) during the menstrual cycle and along its length [49], with OEA causing a reduction in epithelial cell cilia beat frequencies [146], an effect that is likely to prevent timely movement of fertilised oocytes and precipitate ectopic pregnancy [49]. Although possible relationships between the ECS, cannabinoids, and oviductal cancer currently do not exist (Table 1), the fact that dysregulation of the ECS in the fallopian tube is related to the development of ectopic pregnancy makes us speculate that there could be a role for the ECS in oviductal cancer, and that such a possibility deserves to be investigated.

1.6.3. Cannabinoids and Endometrial Cancer

Endometrial cancer, which is classified into type 1 and 2 [147], is the fourth most common cancer in women [148] and the most common gynaecological cancer. Various therapies exist depending on the disease grade and stage. Prognosis is poor, especially in those women with late presentation/detection [147]. Guida and coworkers [74] reported an upregulation of CB₂ expression in endometrial cancer, whereby immunostaining was only successful in transformed malignant cells, while being completely absent in normal endometrial tissue. Furthermore, 2-AG levels were increased, but MAGL expression was decreased in comparison to controls, while AEA levels and FAAH expression were unaffected [74]. Similarly, Jove and colleagues [117] demonstrated that CB₁ and CB₂ were expressed at higher levels in stage III and IV endometrial carcinoma that has a poor prognosis. Unlike Guida and coworkers, the latter researchers found, by immunohistochemistry, an increase in CB₁ expression, but no change in CB₂ expression in stage 1 endometrial carcinoma tissue compared to normal endometrial tissue [117]. These observations were at odds with those of Risinger and coworkers, who found a decrease in CB₁ receptor at the transcriptional level in stage 1 tissue [75]. These contradictory observations prompted us to investigate the ECS in endometrial cancer, using more than a single technique to interrogate CB₁ and CB₂ expression in endometrial cancer [70,72,73]. Our data indicated that CB₁ and CB₂ expression are decreased not only at the transcript level, but also at the protein level in both types 1 and 2 (stage 1) endometrial cancers (Table 1; Figure 3). We concluded that the discrepancy between these and previous studies was due to technical issues in the different methodologies used, including tissue sampling [72,73]. Furthermore, we examined the concentrations of plasma and tumour levels of AEA, OEA, and PEA in women with and without endometrial cancer, and showed that although the levels of all three N-acylethanolamines were increased in the tumours and in blood, only AEA and PEA were significantly higher in the plasma of such patients [29,68]. These data suggest that the differential catabolism of these three N-acylethanolamines might explain the different patterns of expression in endometrial cancer and plasma. We subsequently discovered that the apparent discrepancy between the tissue levels and plasma concentrations of OEA in the sample patient cohort was due to a decrease in the expression of FAAH in the tumour [78], without any change in the expression of NAPE-PLD (Table 1; Figure 3). The latter study also allowed us to define cut-off values for plasma AEA, OEA, and PEA concentrations (>1.36, >4.97, and 27.5 nM, respectively) that could be used in the prediction of endometrial cancer in symptomatic women [39], an observation that awaits confirmation in a larger, multicentre trial.

The effects of phytocannabinoids, such as THC, on the progression of endometrial cancer were recently evaluated by Zhang and collaborators [149]. They found that THC inhibited endometrial cancer cell proliferation and migration through decreased expression of matrix metalloproteinase-9, an effect mimicked by matrix metalloproteinase-9 gene silencing [149]. More recently, the effect of THC and CBD on endometrial cancer cell survival was investigated on Ishikawa and Hec50co cells [76], which are models of type 1 and type 2 endometrial cancer, respectively. The expression of all components of the ECS, including TRPV1, was demonstrated in these cells, supporting our in vivo observations (Figure 3). Additionally, treatment of the cells with AEA or CBD (>5 μM) reduced cell viability and was linked to an increase in reactive oxygen species production and caspase-3/-7 activity, which are markers of apoptosis [76]. Interestingly, in both endometrial cancer cell lines, THC had no effect on tumour cell survival, suggesting that in vitro findings in cancer cell lines cannot directly be translated to the in vivo situation. It is also interesting to note that the doses of CBD used in the in vitro study [76] greatly exceed those which are possible to achieve through recreational use of C. sativa or C. indica; this observation possibly explains the lack of anecdotal reports [136,138,139] that support anti-tumour benefits of marijuana use in patients with endometrial cancer [150]. The findings regarding the pro-apoptotic action of AEA in endometrial cancer cell lines are in keeping with the observations by Contassot and colleagues [84], who described AEA-driven cervical cancer cell apoptosis via TRPV1 activation. The danger from such studies, however, is that the data can be misinterpreted by agencies on the internet who wish to sell their Cannabis products, without an appreciation of the dangers they are potentially placing their customers in [138,139,150], and as summarised in [136], “…As always, the results must be met with scepticism and caution. The concentrations of cannabinoids used in these tests are quite high; it may not be safe to administer the amounts necessary to reach these concentrations. Furthermore, cells growing in a dish are very different from cancer cells in the body. They generate their own signals that cause them to grow out of control, which may counteract the effects of cannabinoids. In addition, individual genetic differences may influence how any particular patient will respond to medication. Nevertheless, these results point to CBD and other cannabinoids as a potential treatment for this common type of cancer…” [136]. This level of scepticism is appropriate because the drugs being promoted as anticancer therapies have not undergone the rigorous pre-clinical studies and randomised clinical trials for the treatment they are being advertised for [136,138,139,150]. The danger here is that the multiple pleiotropic effects that cannabinoids (especially THC and CBD) exert on the female body have not been discovered, and adverse side-effects of self-treatment by patients with gynaecological cancers, either by topical or oral routes, could result in serious morbidities or mortality, especially as the effects of these phytocannabinoids go well beyond the effect of apoptosis [151]. Indeed, interactions between (endo)cannabinoids and the stromal cells of the endometrium are often ignored in cancer studies, and these cells of the endometrium are also affected by (endo)cannabinoids [55,71]. It is thus essential that more laboratory-based and clinical studies in this area are performed.

1.6.4. Cannabinoids and Cervical Cancer

Cervical cancer is the second leading cause of malignancy-related deaths in women worldwide due to the lack of customisable and effective treatments (especially in low- and middle-income countries), with more than 250,000 deaths being reported annually [152]. A possible role of the ECS in the development of cervical cancer has been elucidated in recent years. Contassot and coworkers [84] reported a strong expression pattern of CB₁ and CB₂, as well as of TRPV1, in cervical carcinoma cell lines and biopsies. In addition, it was shown that AEA had a pro-apoptotic effect on cervical carcinoma cell lines (HeLa and Caski) [84], which were not inhibited, but were instead enhanced by CB₁ and CB₂ antagonists. On the other hand, the TRPV1 selective antagonist capsazepine protected the cell lines from AEA-induced apoptosis, indicating an important role of the TRPV1 channel in the pro-apoptotic action of AEA [92]. Additionally, it was demonstrated by Ramer and collaborators [153] that CBD decreased the invasiveness of cancer cells in a concentration-dependent manner. This effect was observed in the cervical cancer cell lines HeLa and C33A, as well as in the lung cancer cell line A549, and seemed to be mediated by the upregulation of TIMP-1 via CB₁/CB₂ and TRPV1. TIMP-1 is an inhibitor of matrix metalloproteinases, and as such, it prevents the movement of cells out of the tissue and, hence, a metastatic disease, as has been observed in a patient with ovarian cancer treated with CBD [154].

The activation of p38 and p42/44 mitogen-activated protein kinases was identified as an upstream event in TIMP-1 upregulation [153]. In agreement with these findings, it was reported that treatment of different cervical cancer cell lines (HeLa, SiHa, ME-180) with CBD led to a decrease in cell proliferation [155]. Furthermore, CBD induced cell death by the accumulation of cells in the sub-G0 phase (cell death phase) of the cell cycle, a finding that was most likely caspase-dependent because caspase-9 as well as caspase-3 were upregulated upon CBD treatment [155]. Hence, CBD may be an additional therapeutic tool for the treatment of cervical cancer, yet additional in vivo studies, similar to that performed on a single ovarian cancer patient [154], will be needed to clarify the impact of CBD on cervical cancer.

1.6.5. Cannabinoids and Vaginal Cancer

Vaginal cancer is uncommon, and the American Cancer Society estimated that >6000 women will be diagnosed with it in 2020. The estimated lifetime risk is 1 in 1100 (i.e., less than 0.1%). Of the 6000 USA women expected to be diagnosed with vaginal cancer in 2020, 1450 will die because they have this disease [156]. The role of the ECS in vaginal cancer has not been fully examined. We [80] have demonstrated that CB₁ and FAAH are expressed in the normal vagina; however, there are no data on the expression of other components of the ECS (Table 1), nor on what their normal function might be. What happens to the expression of these factors or what effects cannabinoid and eCB ligands might have on the vagina or on cells of vaginal tumours is uncertain/unclear (Table 1). The internet is one source of information, and for the vagina, it is reported that some women experience a “vaginal high” when using cannabinoids, especially as a topical application [135]. The problem with these data is that only 40% of women experience this “psychological” effect [135]. Nevertheless, these statements have led some internet sites to suggest that different cannabis-containing preparations might be useful for the treatment of some of the symptoms associated with vaginal cancer [134,157]. Obviously, a lot more information is needed on the role of cannabinoids and eCBs in the human vagina, and especially in vaginal cancer.

1.6.6. Cannabinoids and Vulvar Cancer

Vulvar cancer is a less common gynaecological cancer [158]. The vulva is very similar to normal thin skin and is known to express CB₁ and FAAH [80], but it is not known if it contains all the main components of the ECS (Table 1). The only existing evidence that cannabinoids have an effect on the vulva comes from a less-than-reliable internet source [137]. A C. sativa ethanolic extract and a purified CBD preparation had anti-inflammatory effects on keratinocytes and skin fibroblasts in vitro, suggesting that CBD was the main active ingredient that would be effective in wound injury [159].

This seems important because women with vulvar cancer often undergo radical surgery to remove their malignancy, which causes disfiguration of the female external genitalia, and causes significant long-term emotional and physical instability [160]. Indeed, the use of the CBD derivative VCE-004.3 on skin fibrosis and inflammation [161] demonstrated a CB₂/PPARγ-dependent effect, and suggested that similar compounds might be beneficial for patients with vulvar cancer who have undergone surgery and need topical treatment for the pruritus; the latter is associated with skin fibrosis and inflammation, especially as VCE-004.3 appears to inhibit mast cell degranulation [161]. The toxicity profile of such topical administrations remains to be determined; however, ethanolic extracts of THC, CBD, and other cannabinoids appear in the blood shortly after administration; thus, some caution is advised, also in the light of the pleiotropic effects of these compounds [162]. Obviously, more detailed analysis of the role of the ECS and of plant-derived cannabinoids in the treatment of vulvar cancer is warranted.