CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen

Abstract

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.

Tamoxifen Isomers and Metabolites Exhibit Distinct Affinity and Activity at Cannabinoid Receptors: Potential Scaffold for Drug Development

“…g ., >10 times K i ; Table 1) that would be predicted to produce a maximal response. As anticipated [16, 29, 34], incubation with the known CB1R agonist CP-55,940 or CB1R inverse agonist AM-281 significantly increased or decreased [ 35 S]GTPγS binding, respectively (Fig 3A). Consistent with actions as inverse agonists, all SERMs examined (except E-4OHT) reduced basal [ 35 S]GTPγS binding to levels similar to that produced by the full CB1R inverse agonist AM-281 [36].…”

“…However, due to the adverse effects of currently available drugs acting at CBRs, FDA approval of therapeutic cannabinoids unfortunately remains elusive. Recent studies by our group [16] and others [17, 18] have shown that several clinically available, FDA-approved drugs in the selective estrogen receptor modular (SERM) class (e.g. Z-Tamoxifen, Z-4-hydroxytamoxifen, and Raloxifen) also bind and modulate activity of CB1 and CB2Rs.…”

Selective Estrogen Receptor Modulators: Cannabinoid Receptor Inverse Agonists with Differential CB1 and CB2 Selectivity

“…Competition receptor binding was conducted by using 0.2 nM of the radioligand [ 3 H]CP-55,940, a high-affinity and non-selective cannabinoid agonist as reported previously (Prather et al, 2013). The final volume of each sample was 1 ml, containing 50 mM Tris-HCl buffer (pH 7.4), 0.05% bovine serum albumin, 5 mM MgCl 2 , increasing concentrations of non-radioactive competing SERM ligands, and either 100 μg of CHO-hCB1 or 50 μg of CHO-hCB2 membrane homogenates.…”

“…However, many of these SERMs at higher doses have surprisingly been shown to also produce cytotoxicity in cancers devoid of the ERs (Nagahara et al, 2013). Our laboratory and others (Kumar and Song, 2013; Prather et al, 2013) have suggested that cannabinoids receptors (CBRs) might represent one potential ER-independent mechanism responsible for SERM cytotoxicity in some forms of cancer. For example, SERMs bind to the CBRs with moderate to high affinity (Kumar and Song, 2013; Prather et al, 2013), and SERMs and cannabinoids exhibit overlapping anti-proliferative, anti-angiogenic and pro-apoptotic actions.…”

“…Tamoxifen has been used safely for decades and binds non-selectively to CB1 and CB2 receptors with affinity in the low μM range (Kumar and Song, 2013; Prather et al, 2013). To identify cannabinoids with improved pharmacological characteristics relative to tamoxifen, and investigate the usefulness of SERM scaffolds for future cannabinoid drug development, the purpose of this study was to characterize the affinity and activity of SERMs in newer structural classes at CBRs.…”