CXCR4/CXCL12 signaling axis has been shown to promote migration and invasion of breast cancer cells to distant sites of metastasis such as the lung, brain, bone, lymph nodes, and liver [19], [31], [38]. Hence, to evaluate the potential of CB2 as a possible therapeutic target, the effect of CB2 agonist JWH-015 on CXCL12-induced cell migration was investigated. As shown in Fig. 2, JWH-015 (20 µM) showed significant inhibition of CXCL12-induced migration of MCF-7/CXCR4 and SCP2 cells. We further analyzed the effect of JWH-015 on CXCL12-induced invasion using wound healing assays. We observed a significant inhibition of CXCL12-induced wound healing in MCF-7/CXCR4, SCP2, and NT 2.5 after overnight pretreatment with JWH-015 (20 µM) compared to the vehicle-treated cells (Fig. 3). Thus these results suggest that CB2 receptor specific agonists significantly reduced CXCL12/CXCR4-induced migration and invasion of breast cancer cells.

Breast cancer is the second leading cause of cancer death in women in the United States. Despite recent advances in hormonal therapies, mortality still remains high due to breast cancer metastasis to other organs. Synthetic cannabinoids that bind to cannabinoid receptors CB1 and CB2 have been shown to inhibit migration, metastasis, and invasion of various cell types including breast cancer cells [4], [5], [6], [37], [41]. However, not much is known about the mechanisms by which CB1 and CB2 mediate their inhibitory effects. The majority of breast cancers have been shown to overexpress chemokine receptor CXCR4, which has been correlated with poor prognosis [34]. Furthermore, high CXCR4 expression was also correlated to poor clinical outcome in triple negative breast cancers, which are difficult to treat [39], [42]. Here, we report for the first time that CB2 specific synthetic agonist inhibits CXCL12-induced migration and invasion of breast cancer cell lines in vitro. Furthermore, we have shown that this compound inhibits tumor growth in vivo and downmodulates CXCR4 phosphorylation and downstream signaling.   Marijuana has been used in medicine for millennia, but it was not until 1964 that delta9-tetrahydrocannabinol (delta9-THC), its major psychoactive component, was isolated in pure form and its structure was elucidated. Shortly thereafter it was synthesized and became readily available. However, it took another decade until the first report on its antineoplastic activity appeared. In 1975, Munson discovered that cannabinoids suppress Lewis lung carcinoma cell growth. The mechanism of this action was shown to be inhibition of DNA synthesis. Antiproliferative action on some other cancer cells was also found. In spite of the promising results from these early studies, further investigations in this area were not reported until a few years ago, when almost simultaneously two groups initiated research on the antiproliferative effects of cannabinoids on cancer cells: Di Marzo's group found that cannabinoids inhibit breast cancer cell proliferation, and Guzman's group found that cannabinoids inhibit the growth of C6 glioma cell. Other groups also started work in this field, and today, a wide array of cancer cell lines that are affected is known, and some mechanisms involved have been elucidated.  ​Anandamide was the first brain metabolite shown to act as a ligand of "central" CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 microM and 83-92% maximal inhibition at 5-10 microM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 microM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55, 940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1-0.5 microM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor. (THC) exhibits antitumor effects on various cancer cell types, but its use in chemotherapy is limited by its psychotropic activity. We investigated the antitumor activities of other plant cannabinoids, i.e., cannabidiol, cannabigerol, cannabichromene, cannabidiol acid and THC acid, and assessed whether there is any advantage in using Cannabis extracts (enriched in either cannabidiol or THC) over pure cannabinoids. Results obtained in a panel of tumor cell lines clearly indicate that, of the five natural compounds tested, cannabidiol is the most potent inhibitor of cancer cell growth (IC(50) between 6.0 and 10.6 microM), with significantly lower potency in noncancer cells. The cannabidiol-rich extract was equipotent to cannabidiol, whereas cannabigerol and cannabichromene followed in the rank of potency. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of xenograft tumors obtained by s.c. injection into athymic mice of human MDA-MB-231 breast carcinoma or rat v-K-ras-transformed thyroid epithelial cells and reduced lung metastases deriving from intrapaw injection of MDA-MB-231 cells. Judging from several experiments on its possible cellular and molecular mechanisms of action, we propose that cannabidiol lacks a unique mode of action in the cell lines investigated. At least for MDA-MB-231 cells, however, our experiments indicate that cannabidiol effect is due to its capability of inducing apoptosis via: direct or indirect activation of cannabinoid CB(2) and vanilloid transient receptor potential vanilloid type-1 receptors and cannabinoid/vanilloid receptor-independent elevation of intracellular Ca(2+) and reactive oxygen species. Our data support the further testing of cannabidiol and cannabidiol-rich extracts for the potential treatment of cancer. Cannabinoids, the active components of the hemp plant Cannabis sativa, along with their endogenous counterparts and synthetic derivatives, have elicited anti-cancer effects in many different in vitro and in vivo models of cancer. While the various cannabinoids have been examined in a variety of cancer models, recent studies have focused on the role of cannabinoid receptor agonists (both CB(1) and CB(2)) in the treatment of estrogen receptor-negative breast cancer. This review will summarize the anti-cancer properties of the cannabinoids, discuss their potential mechanisms of action, as well as explore controversies surrounding the results. It has been recently shown that cannabinoids, the active components of marijuana and their derivatives, inhibit cell cycle progression of human breast cancer cells. Here we studied the mechanism of Delta(9)-tetrahydrocannabinol (THC) antiproliferative action in these cells, and show that it involves the modulation of JunD, a member of the AP-1 transcription factor family. THC activates JunD both by upregulating gene expression and by translocating the protein to the nuclear compartment, and these events are accompanied by a decrease in cell proliferation. Of interest, neither JunD activation nor proliferation inhibition was observed in human non-tumour mammary epithelial cells exposed to THC. We confirmed the importance of JunD in THC action by RNA interference and genetic ablation. Thus, in both JunD-silenced human breast cancer cells and JunD knockout mice-derived immortalized fibroblasts, the antiproliferative effect exerted by THC was significantly diminished. Gene array and siRNA experiments support that the cyclin-dependent kinase inhibitor p27 and the tumour suppressor gene testin are candidate JunD targets in cannabinoid action. In addition, our data suggest that the stress-regulated protein p8 participates in THC antiproliferative action in a JunD-independent manner. In summary, this is the first report showing not only that cannabinoids regulate JunD but, more generally, that JunD activation reduces the proliferation of cancer cells, which points to a new target to inhibit breast cancer progression., the active components of Cannabis sativa (marijuana), and their derivatives produce a wide spectrum of central and peripheral effects, some of which may have clinical application. The discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has attracted much attention to cannabinoids in recent years. One of the most exciting and promising areas of current cannabinoid research is the ability of these compounds to control the cell survival/death decision. Thus cannabinoids may induce proliferation, growth arrest, or apoptosis in a number of cells, including neurons, lymphocytes, and various transformed neural and nonneural cells. The variation in drug effects may depend on experimental factors such as drug concentration, timing of drug delivery, and type of cell examined. Regarding the central nervous system, most of the experimental evidence indicates that cannabinoids may protect neurons from toxic insults such as glutamaergic overstimulation, ischemia and oxidative damage. In contrast, cannabinoids induce apoptosis of glioma cells in culture and regression of malignant gliomas in vivo. Breast and prostate cancer cells are also sensitive to cannabinoid-induced antiproliferation. Regarding the immune system, low doses of cannabinoids may enhance cell proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis. The neuroprotective effect of cannabinoids may have potential clinical relevance for the treatment of neurodegenerative disorders such as multiple sclerosis, Parkinson's disease, and ischemia/stroke, whereas their growth-inhibiting action on transformed cells might be useful for the management of malignant brain tumors. Ongoing investigation is in search for cannabinoid-based therapeutic strategies devoid of nondesired psychotropic effects. It has been proposed that cannabinoids are involved in the control of cell fate. Thus, these compounds can modulate proliferation, differentiation, and survival in different manners depending on the cell type and its physiopathologic context. However, little is known about the effect of cannabinoids on the cell cycle, the main process controlling cell fate. Here, we show that Delta(9)-tetrahydrocannabinol (THC), through activation of CB(2) cannabinoid receptors, reduces human breast cancer cell proliferation by blocking the progression of the cell cycle and by inducing apoptosis. In particular, THC arrests cells in G(2)-M via down-regulation of Cdc2, as suggested by the decreased sensitivity to THC acquired by Cdc2-overexpressing cells. Of interest, the proliferation pattern of normal human mammary epithelial cells was much less affected by THC. We also analyzed by real-time quantitative PCR the expression of CB(1) and CB(2) cannabinoid receptors in a series of human breast tumor and nontumor samples. We found a correlation between CB(2) expression and histologic grade of the tumors. There was also an association between CB(2) expression and other markers of prognostic and predictive value, such as estrogen receptor, progesterone receptor, and ERBB2/HER-2 oncogene. Importantly, no significant CB(2) expression was detected in nontumor breast tissue. Taken together, these data might set the bases for a cannabinoid therapy for the management of breast cancer. expression profiling has revealed that the gene coding for cannabinoid receptor 1 (CB1) is highly up-regulated in rhabdomyosarcoma biopsies bearing the typical chromosomal translocations PAX3/FKHR or PAX7/FKHR. Because cannabinoid receptor agonists are capable of reducing proliferation and inducing apoptosis in diverse cancer cells such as glioma, breast cancer, and melanoma, we evaluated whether CB1 is a potential drug target in rhabdomyosarcoma. Our study shows that treatment with the cannabinoid receptor agonists HU210 and Delta(9)-tetrahydrocannabinol lowers the viability of translocation-positive rhabdomyosarcoma cells through the induction of apoptosis. This effect relies on inhibition of AKT signaling and induction of the stress-associated transcription factor p8 because small interfering RNA-mediated down-regulation of p8 rescued cell viability upon cannabinoid treatment. Finally, treatment of xenografts with HU210 led to a significant suppression of tumor growth in vivo. These results support the notion that cannabinoid receptor agonists could represent a novel targeted approach for treatment of translocation-positive rhabdomyosarcoma. and metastasis of aggressive breast cancer cells is the final and fatal step during cancer progression, and is the least understood genetically. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Clearly, effective and nontoxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. Using a mouse model, we previously determined that metastatic breast cancer cells became significantly less invasive in vitro and less metastatic in vivo when Id-1 was down-regulated by stable transduction with antisense Id-1. It is not possible at this point, however, to use antisense technology to reduce Id-1 expression in patients with metastatic breast cancer. Here, we report that cannabidiol (CBD), a cannabinoid with a low-toxicity profile, could down-regulate Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive phenotype of breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. These effects seemed to occur as the result of an inhibition of the Id-1 gene at the promoter level. Importantly, CBD did not inhibit invasiveness in cells that ectopically expressed Id-1. In conclusion, CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the down-regulation of tumor aggressiveness. Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis.  We found that CBD inhibited the survival of both estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines and induced apoptosis in a concentration-dependent manner. Moreover, at these concentrations, CBD had little effect on MCF-10A cells, nontumorigenic, mammary cells (19). These data enhance the desirability of CBD as an anticancer agent, because they suggest that CBD preferentially kills breast cancer cells, while minimizing damage to normal breast tissue.    In this study we showed that CBD induced both apoptosis and autophagy-induced death in breast cancer cells. PCD by apoptosis is well-documented, whereas autophagy-mediated cell death is a relatively recent discovery, and there is much to be learned about the factors that prejudice autophagy toward self-protection or self-destruction. Recent studies support growing evidence that ER stress can trigger autophagic cell death (39, 40). We observed the induction of ER stress in MDA-MB-231 cells within 2 hours of CBD treatment, followed by LC3-II accumulation, leading us to conclude that CBD-induced autophagy in breast cancer cells does not maintain homeostasis. Rather, in this context, autophagy is cytodestructive and leads to PCD. modulate the growth of hormone sensitive breast cancer cells as shown in Fig.​Fig.33 and ​and4.4. JWH-O15 inhibits hormone sensitive breast cancer metastasis by modulating CXCL12/CXCR4 signaling axis [27, 54]. Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Breast tumor cells express FAAH abundantly. Inhibition of FAAH (siRNA-FAAH or FAAH inhibitor URB597) induced cell death by activating nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway and heme oxygenase-1 (HO-1) induction and transcription [55]. Anandamide inhibits basal and nerve growth factor (NGF) induced proliferation of MCF-7 and EFM-19 cells in culture through CB1 receptor and Δ9-THC inhibits 17beta-estradiol-induced proliferation of MCF7 and MCF7-AR1 cells [45, 56-58]. Δ9-THC also inhibits cell proliferation of ER−/PR+ breast cancer cells. The effects of anandamide and Δ9-THC were mediated by blocking transition from one phase of cell cycle to another, G1-S and G2-M respectively [49, 56, 59-60]. Cell cycle arrest is responsible for apoptotic cell death. The analog of anandamide, Met-F-AEA reduces MDA-MB-231 proliferation by arresting cells in the S phase of the cell cycle [60]. Anandamide inhibits adenylyl cyclase (AC) and thus activating the Raf-1/ERK/MAP pathway in ER+/PR+ breast cancer cells whilst THC activates the transcription factor JunD to finally execute action towards apoptosis in ER−/PR+ breast cancer cells [49, 56, 59]. One study shows that anandamide inhibits proliferation of MDA-MB31 cells by modulating Wnt/β-catenin signaling pathway [61]. This effect is occurred by inhibition of the cyclin-dependent kinase CDK2 [50, 60].   Non-THC cannabinoids have also been tested in cancer (Izzo et al., 2009; Gertsch et al., 2010; Russo, 2011). Cannabidiol (CBD), which is very abundant in certain strains of Cannabis, has very low affinity for CB1 and CB2 receptors, and activates TRPV1 channels (Bisogno et al., 2001). This compound induces apoptosis in a triple-negative breast carcinoma cell line and inhibits tumour cell growth and metastasis (Ligresti et al., 2006; Ramer et al., 2010; McAllister et al., 2011; Aviello et al., 2012). CBD and other non-THC cannabinoids [i.e. cannabigerol (CBG), cannabichromene (CBC), cannabidiolic acid (CBDA) and Δ9-tetrahydrocannabidiolic acid (THCA)] have been assessed against a number of tumour cell lines distinct in origin and typology. These compounds have been compared with extracts [known as ‘botanical drug substances’, (BDS)] from corresponding Cannabis strains (Ligresti et al., 2006). Indeed, the testing of BDS enriched in a certain cannabinoid might demonstrate potentially important synergistic effects between cannabinoid and non-cannabinoid cannabis components, which, in turn might be useful therapeutically. The results obtained indicated that, of these five pure compounds and BDS tested, CBD and CBD–BDS were usually the more effective inhibitors of cancer cell growth, with little or no activity on non-cancer cells (Ligresti et al., 2006). CBD inhibits also glioblastoma growth and potentiates the action of THC on this type of tumour (Torres et al., 2011). However, these effects are only marginally dependent upon interaction with cannabinoid and TRPV1 receptors (Massi et al., 2004; Vaccani et al., 2005; Torres et al., 2011). and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression. cancer is a very common disease that affects approximately 1 in 10 women at some point in their lives. Importantly, breast cancer cannot be considered a single disease as it is characterized by distinct pathological and molecular subtypes that are treated with different therapies and have diverse clinical outcomes. Although some highly successful treatments have been developed, certain breast tumors are resistant to conventional therapies and a considerable number of them relapse. Therefore, new strategies are urgently needed, and the challenge for the future will most likely be the development of individualized therapies that specifically target each patient's tumor. Experimental evidence accumulated during the last decade supports that cannabinoids, the active components of Cannabis sativa and their derivatives, possess anticancer activity. Thus, these compounds exert anti-proliferative, pro-apoptotic, anti-migratory and anti-invasive actions in a wide spectrum of cancer cells in culture. Moreover, tumor growth, angiogenesis and metastasis are hampered by cannabinoids in xenograft-based and genetically-engineered mouse models of cancer. This review summarizes our current knowledge on the anti-tumor potential of cannabinoids in breast cancer, which suggests that cannabinoid-based medicines may be useful for the treatment of most breast tumor subtypes.


Cannabis -vs- Breast Cancer