Cannabinoid receptor agonists signal through the inhibition of the AC and PKA pathways (26). This is also one of the main signaling pathways of estrogens (27–29), activated by the binding of 17β-estradiol to ERs (Fig. 1) and partly by non-genomic mechanisms of estrogen action (30).

Endocannabinoids decrease the expression of EGF receptors (31) and significantly inhibit the EGF-induced proliferation, migration and invasion of non-small cell lung cancer cell lines (32). The EGF cytoplasmic signaling pathways influence ER activity. The activation of EGF receptors leads to the MAPK-mediated phosphorylation of ERα; in addition, EGF receptors activate p38/MAPKs that activate ERα/β (33) (Fig. 1).

After binding to ERs, estrogens can promote the activation of activating transcription factor (ATF)-2/CREB to induce the expression of cyclin D1 and can promote the activation of ATF-1/CREB to induce the expression of B-cell lymphoma 2 protein (Bcl-2). Cyclin D1 and Bcl-2 are important for their proliferative and anti-apoptotic effects (34). The β-estradiol through the MAPK pathway, independently from the PKA pathway (35). Yet again, cannabinoids appear to have an opposite effect. It has been shown that cannabinoid agonists inhibit cAMP response elements (CRE) (36), the binding deoxyribonucleic acid (DNA) sequences for CREB (Fig. 1).

Endocannabinoids inhibit the mitogenic action of prolactin (37), which is among its others functions also an important inducer of carcinogenesis in breast cancer. Both estrogens and endocannabinoids regulate the expression of prolactin receptors (37,38).

Cannabinoids cause a reduction in VEGF expression and inhibit angiogenesis through CB1 receptors. In mouse thyroid carcinoma, the reported anticancer effects of the CB1 receptor agonist, Met-F-anandamide, may be due to the inhibition of angiogenesis, as a consequence of VEGF signal blocking, the overexpression of cyclin-dependent kinase inhibitor 1 (p21) (39) and interference with VEGF receptor type 2 activation (40). By binding to EREs, 17β-estradiol directly regulates VEGF gene transcription in endometrial cells and in Ishikawa adenocarcinoma cells. This mechanism may also be important in the estrogenic regulation of VEGF production and angiogenesis in estrogen target tissues, i.e., breast, bone, heart and skin (41,42).

Cannabinoids signal apoptosis via a pathway involving CB receptors. This pathway is sustained by ceramide accumulation and extracellular signal-regulated Raf kinase activation (43). The Raf kinase is activated by ER through non-genomic mechanisms. It has been demonstrated that in Chinese hamster ovary cells, serine 522 in the ligand binding domain of ERα interacts with caveolin-1. Caveolin-1 is a structural protein in caveolae that binds Raf, proto-oncogene tyrosine-protein kinase (Src), growth factor receptor-bound protein 7, rat sarcoma protein (Ras), mitogen-activated protein kinase kinase (MEK), EGF receptor and ERα at the plasma membrane, forming a 'signalsome' for the rapid activation of intracellular signaling (44, and refs therein). The protein Raf is important in the activation of MAPK and other kinases that are activated by estrogens (35).

The MAPK pathway is generally important in gene expression, cell proliferation and apoptosis (45). Moreover, the activation of ERα or ERβ differentially affects proliferation and apoptosis. The 17β-estradiol-ERα complex activates multiple signaling pathways, including p38/MAPK, extracellular-signal-regulated kinase (ERK)/MAPK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) which are involved in cell cycle progression and apoptotic cascade prevention. The 17β-estradiol-ERβ complex activates only the p38/MAPK pathway, which in turn leads to cell apoptosis (46). The ER-17β-estradiol complex combined with the insulin-like growth factor-1 (IGF-1) receptor is also a MAPK signaling pathway activator (47). Many apoptotic effects are also linked to CB receptors and the activation of MAPK pathways (48); e.g., ceramide synthesis is induced by cannabinoids and leads to the activation of the p38/MAPK pathway (49) (Fig. 2).

Cannabinoids cause the downregulation of PI3K-Akt and ERK1/2 kinase signaling, which in turn inhibits proliferation and induces apoptosis (51). By contrast, 17β-estradiol activates PI3K-mediated signaling, which causes rapid endothelial nitric-oxide synthesis (52) and promotes cell cycle progression (Fig. 3), also involving increased cyclin D1 expression (53).

C-Jun N-terminal kinase may be a potential target of ceramide action in the induction of apoptosis in a number of cell types (43). Estrogens also influence c-Jun N-terminal kinase. Estrogen receptors can associate with promoters/enhancers of the transcription factors ATF-2 and c-Jun. The activator protein-1 (AP-1) complex, consisting also of c-Jun, plays an important role in cell proliferation (34). Estrogen receptors enhance the transcription of genes that contain AP-1 (54). The activation of ERα activates AP-1-dependent transcription, whereas the activation of ERβ inhibits AP-1-dependent transcription (55). Cannabinoid agonists inhibit AP-1-mediated transcriptional activities, the latter being induced in several types of tumors (34) (Fig. 3).

Current evidence indicates that a small population of ERα and ERβ localized at the plasma membrane exists within caveolar lipid rafts. It is at the plasma membrane that 17β-estradiol-ER associates with the scaffolding protein, caveolin-1, and a variety of signal transduction cascades activations occur, including ERK and other enzymes [phospholipase C (PLC), protein kinase C (PKC), PI3K, nitric oxide synthase] (35). In bones, ERα is present in the caveolae of bone-forming osteoblasts. The ERα transmits survival signals through the activation of the Src/adapter protein Shc/ERK pathway and prolongs the lifespan of osteoblasts (56). Cannabinoids activate ERK1/2 kinases, leading to G1 cell cycle arrest (57) (Fig. 3). The activation of either CB1 or CB2 receptors in colon cancer cells induces the Raf-MEK-ERK pathway to promote apoptosis and triggers the synthesis of ceramide (58).

There is evidence to indicate that certain enzymes involved in the synthesis or degradation of endocannabinoids are also regulated by estrogens. Estrogens and progesterone downregulate uterine enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression via their nuclear receptors (59). Protein NAPE-PLD is an important enzyme in the synthesis of anandamide (60). Two endocannabinoids, anandamide and 2-AG, are oxidized by cyclooxygenase-2 (COX-2) (61). The 17β-estradiol exhibits a tendency to increase COX-2 expression and prostaglandin E2 synthesis in primary human uterine microvascular endothelial cells (62). As mentioned above, FAAH activity is inhibited by 17β-estradiol, which probably leads to higher concentrations of endocannabinoids after rising plasma estrogen levels (19). Accordingly, a homeostatic association between cannabinoids and estrogens can be proposed; increased levels of estrogens result in higher concentrations of endocannabinoids, which in turn inhibits the hypothalamic-pituitary-gonadal axis and leads to a decrease in estrogen levels (21). It also appears that endocannabinoids are released as a response to estrogen action and that they act via several molecular pathways, generally contradicting the effects of estrogens.

There is strong epidemiological, biological and clinical data that connects sex hormones, particularly estrogens, to breast cancer. The ERα is most directly implicated in the pathophysiology of the disease and its presence in tumor tissue is one of the most important disease prognostic factors. The role of other sex hormone receptors in the pathophysiology of breast cancer, i.e., ERβ and androgen receptors (ARs), has been less clearly investigated (63).

A recent study suggested that tamoxifen can act as a CB1 and CB2 inverse agonist, thus producing cytotoxicity via an ER-independent mechanism of action (25).

It has been shown that the activation of CB2 receptor by THC reduces the progression of the cell cycle and promotes the apoptosis of human breast cancer cells (64). Cannabidiol and most potently its analogue, 01663, induce breast cancer cell death through apoptosis and autophagy in vitro (65), and they inhibit breast cancer cell proliferation and invasion in vivo (66); other synthetic cannabinoid receptor agonists also inhibit tumor growth and breast cancer metastasis (67). On the other hand, a previous study found that THC stimulates breast cancer growth and metastasis in vivo by the suppression of the body's antitumor immune response (68).

Endometrial cancer is the most common gynecological malignancy. Prolonged unopposed estrogen exposure is associated with the majority of type I endometrial cancers. Estrogen replacement therapy, prescribed to control menopausal symptoms, increases the risk of developing endometrial cancer by 2–20-fold (69). Endometrial cancers are thought to arise from estrogen exposure, not balanced by the differentiating effects of progesterone (70). Currently, estrogen antagonists and progesterone analogues are used in endometrial cancer treatment (70).

A previous study demonstrated that anandamide may be a possible risk factor in endometrial cancer. Anandamide was shown to decrease both CB1 and CB2 receptor transcript levels in endometrial cancer tissues. In addition, plasma anandamide concentrations were significantly higher in patients with endometrial cancer than in the control group. This suggests that increased tissue and plasma anandamide concentrations may be in some way be linked to the pathophysiology of endome-trial cancer (71).

In another study, the immunohistochemical analysis of endometrial biopsies revealed that CB2 receptors were selectively expressed in cancer cells, with a very weak expression in healthy cells in the same biopsies. Mass spectrometry analysis of endometrial carcinoma lipid extracts also revealed a significant increase in 2-AG levels in comparison with samples obtained from healthy subjects. No significant increase in the levels of anandamide was detected. The elevation of 2-AG is possibly due to the decrease in the expression of monoacylglycerol (MAGL), an important enzyme necessary for 2-AG breakdown (72).

The bone remodeling process is influenced by many factors, including estrogens and endocannabinoids. Imbalances in bone remodeling mechanisms cause one of the most common degenerative diseases in developed countries, osteoporosis (73).

Osteoblasts and osteoclasts are influenced by estrogens at both the cellular and molecular level. Estrogens increase collagen I and osteoprotegerin expression (74,75), there is evidence to suggests that estrogens have inhibitory effects on osteoblast apoptosis (76).

Cannabinoids also appear to modulate bone structure. Compared to CB1 receptors, CB2 receptors have been reported to have a significantly higher expression in osteoblasts, osteoclasts and osteocytes (77). Selective CB2 receptor agonists/antagonists may therefore successfully regulate bone remodeling. Importantly, selective CB2 receptor ligands are not generally psychoactive, making them more suitable for potential clinical use (78).

There is evidence of estrogen and cannabinoid interactions in bone cells. Exposure to 17β-estradiol has been shown to lead to the increased expression of CB2 receptors in osteoclasts (21). In our recent study on primary human osteoblasts, we reported a possible synergistic interaction between 17β-estradiol and a selective CB2 antagonist/inverse agonist (79).

The pathophysiology underlying atherosclerosis is a combination of endothelial cell dysfunction and vascular inflammation, accompanied by a build-up of lipids, cholesterol and calcium within the tunica intima. In combination, these can result in plaque formation, thrombosis and cardiovascular insufficiency (80).

An increase in cardiovascular incidents in post-menopausal women suggests that estrogens play an essential protective role against cardiovascular disease. Menopause creates unhealthy changes in plasma lipoprotein levels that can be reversed by post-menopausal estrogen replacement therapy (81). Studies have demonstrated that estrogens are important for normal cell proliferation in blood vessels. When physiological angiogenesis is lacking or insufficient, a setting is created for various cardiovascular diseases (82). Estrogens regulate lipid and cholesterol levels and may provide protection by increasing plasma high-density lipoprotein levels (83). Furthermore, estrogens may modulate inflammatory responses within vascular cells, may cause stem cell death and may also be involved in the development of hypertrophy (84).

The high expression of CB1 and CB2 receptors in atherosclerotic plaques indicates an important role of the endocannabinoid system in atherosclerosis (80). A higher expression of CB1 receptors is also associated with cardiovascular risk factors, such as obesity and dyslipidemia and CB1 agonists have been shown to increase the amount of reactive oxygen species, and thus to induce the apoptosis of endothelial cells in coronary arteries (85,86). In an animal model of atherosclerosis, CB1 antagonists have proven useful; they reduce the accumulation of oxygenated low-density lipoproteins in macrophages, reduce inflammatory reactions in small blood vessels, decrease the proliferation of smooth muscle cells in vessel walls and, consequently, delay disease progression (80). The CB2 receptors have also been proven to play a significant role in the pathogenesis of atherosclerosis. In a previous study, the progression of atherosclerotic plaques in a mouse model was shown to be attenuated by the administration of THC. This effect was nullified by the subsequent administration of a selective CB2 antagonist (87). On the other hand, CB2 agonists reduce the accumulation of lipids in human foam cells (88). Cannabinoids also lower the expression of CD36 receptor, which promotes the release of pro-inflammatory cytokines and increases its own expression (89).

The induction of prostatic cancer by androgens (specifically by testosterone replacement therapy) is still a controversial issue (105), but there is little doubt that the activation of AR by testosterone and dihydroxytestosterone contributes significantly to the progression of metastatic prostate cancer (106). Cannabinoids, on the other hand, have some protective functions. Synthetic cannabinoid quinoines have anti-proliferative effects in vitro and prostate antitumor activity in vivo (107). Another study found that cannabidiol inhibited prostate carcinoma growth in vitro and in vivo via the stimulation of intrinsic pathways of apoptosis. The pro-apoptotic effects of cannabidiol were due to transient receptor potential cation channel subfamily M member 8 (TRPM8) antagonism, the downregulation of AR, p53 activation and the elevation of reactive oxygen species (108).