Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms
Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than Δ9-tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival.
We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells.
Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h. Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1–10 µM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and intracellular Ca2+). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis.
CONCLUSIONS AND IMPLICATIONS
These data support the clinical testing of CBD against prostate carcinoma.
The rapid progress of the research on cannabinoids has contributed to the understanding of the biological actions of these molecules and of their medical significance, which encompass a broad spectrum of physiological and pathological mechanisms in diverse cell types (Bab, 2011). Cannabinoids can be used for treatment of the nausea and vomiting associated with chemotherapy in cancer patients (Robson, 2005; Galal et al., 2009). There is also evidence that Δ9-tetrahydrocannabinol (THC) and synthetic agonists of cannabinoid CB1 and CB2 receptors, as well as endocannabinoids, are promising regulators of malignant cell growth (receptor and channel nomenclature follows Alexander et al., 2011). In most cases, these actions have been attributed to the ability of these compounds to activate the cannabinoid receptors or, (as in the case of anandamide) the transient receptor potential (TRP) vanilloid type-1 (TRPV1) channel (Munson et al., 1975; De Petrocellis et al., 1998; Maccarrone et al., 2000; Bifulco et al., 2001; Jacobsson et al., 2001; Sanchez et al., 2001; Casanova et al., 2003; Ligresti et al., 2003; Mimeault et al., 2003; Contassot et al., 2004; Caffarel et al., 2010; Guindon and Hohmann, 2011). Cannabinoid receptor agonists, apart from their pro-apoptotic and anti-proliferative anticancer activities, may also affect tumour cell angiogenesis, migration, invasion, adhesion and metastasis (Blázquez et al., 2003; Portella et al., 2003; Preet et al., 2008).
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).
Prostate carcinoma is a major life-threatening disease in men and WHO predicts that deaths from this type of cancer will double over the next 30 years (Bahnson, 2007; Jemal et al., 2009). Hence, novel therapeutic approaches are urgently required. Endocannabinoids, through interaction with CB1 receptors and synthetic endocannabinoid-vanilloid hybrids via stimulation of TRPV1 channels have been shown to inhibit nerve growth factor (NGF)-induced proliferation of human prostate PC-3 cells (Melck et al., 2000). However, THC can induce apoptosis of these cells via a receptor-independent mechanism (Ruiz et al., 1999), but also increase the production of the pro-proliferative factor, NGF (Velasco et al., 2001). A role for CB2 receptors in the induction of prostate carcinoma cell (PCC) apoptosis has been described (Sarfaraz et al., 2005; Olea-Herrero et al., 2009). On the other hand, the prototype TRPV1 agonist, capsaicin, produces both pro-proliferative and pro-apoptotic effects on PCCs (Sanchez et al., 2005; 2006; Czifra et al., 2009; Ziglioli et al., 2009; Malagarie-Cazenave et al., 2009; 2011) and not necessarily via TRPV1 activation, but depending on the sensitivity of the cells to androgen. Moreover, it has been suggested that other TRP channels play a role in PCC survival. TRP channels of melastatin-type 8 (TRPM8) are over-expressed in androgen-dependent PCC lines in a manner dependent on androgen receptor (AR) activation (Horoszewicz et al., 1983; Tsavaler et al., 2001; Henshall et al., 2003; Zhang and Barritt, 2004; Bidaux et al., 2005; 2007). In contrast, TRP channel of vanilloid type-2 (TRPV2) are down-regulated by AR, and their activation stimulates PCC migration (Monet et al., 2010). These findings are relevant to current investigations of the anti-tumour activity of non-THC cannabinoids, as many such compounds and the corresponding BDS antagonize TRPM8 channels and activate and subsequently desensitize TRPV2 and TRPV1 channels (Qin et al., 2008; De Petrocellis et al., 2008; 2011). Furthermore, most of these compounds are also able to inhibit endocannabinoid inactivation (De Petrocellis et al., 2011). Therefore, they might act as ‘indirect’ cannabinoid receptor agonists, similar to synthetic compounds previously found to inhibit PCC growth (Nomura et al., 2011).
In the current study we tested 12 pure cannabinoids and nearly all the corresponding BDS on PCC growth in vitro and in vivo. We investigated the cellular and molecular mechanisms of the putative effects of these compounds using both AR-positive and -negative PCC lines, under different culturing conditions, in the presence or absence of currently used chemotherapeutic agents, and after differentiation into a more malignant phenotype. By employing pharmacological, molecular biology, cell biology and immunofluorescence techniques in vitro, as well as xenograft tumour and survival studies in athymic mice, we suggest that non-THC cannabinoids, and CBD in particular, (much like THC, but without the typical psychotropic effects of this compound) might provide the bases for the development of novel therapeutic strategies for the treatment of prostate carcinoma.
Read full Study at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570006/
In conclusion, the in vitro data presented here allow us to suggest that non-THC cannabinoids, and CBD in particular, retard proliferation and cause apoptosis of PCC via a combination of cannabinoid receptor-independent, cellular and molecular mechanisms. Our data, however, do not argue against the previously suggested role of CB1 and CB2 receptors in prostate carcinoma (Sarfaraz et al., 2005; Olea-Herrero et al., 2009), although they do exclude the participation of these receptors in the effects of non-THC cannabinoids. Indeed, the effects reported here, together with previously reported cannabinoid receptor-mediated effects of THC on PCCs, might encourage clinical studies on cannabinoids and Cannabis extracts as a therapy for human prostate carcinoma, either as single agent or in combination with existing compounds. Our additional observation that differentiation of an ‘androgen-dependent’ cell into a more malignant and ‘androgen-unresponsive’ phenotype increases its sensitivity to the pro-apoptotic effect of CBD might provide a new strategy to deal with the frequent loss of efficacy of AR antagonists against prostate carcinoma growth seen after only a few years of treatment.
Read full Study at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570006/