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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: Biochem Biophys Res Commun. 2013 Oct 19;441(2):10.1016/j.bbrc.2013.10.057. doi: 10.1016/j.bbrc.2013.10.057

CB1 and CB2 Receptors are Novel Molecular Targets for Tamoxifen and 4OH-Tamoxifen

Paul L Prather a, FeAna FrancisDevaraj b, Centdrika R Dates b, Aleksandra K Greer b, Stacie M Bratton b, Benjamin M Ford a, Lirit N Franks a, Anna Radominska-Pandya b
PMCID: PMC3860589  NIHMSID: NIHMS533744  PMID: 24148245

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.

Keywords: Cannabinoids, Cannabinoid Receptors, G-protein Coupled Receptors, Drug Action, Drug Discovery, Inverse Agonist, Antagonist

INTRODUCTION

Tamoxifen (Tam) is a selective estrogen receptor modulator (SERM) [1] and is one of the most commonly prescribed chemotherapeutic agents for treatment of estrogen receptor (ER)-positive breast cancer [2]. It has been established that the efficacy of Tam against ER-positive breast cancer results primarily from its ability to act as an ER antagonist [3]. However, in addition to efficacy in ER-positive forms of breast cancer, it has been shown that Tam and its P450-generated metabolite, 4-hydroxy-Tam (4OH-Tam), exhibit cytotoxic effects in types of breast cancer that do not express ERs [4,5]. Tam is also effective against several types of cancer derived from tissues not sensitive to estrogen, such as melanoma [6], glioma [7] and pancreatic cancer [8]. Furthermore, Tam appears to be efficacious for treatment of a variety of other diseases not related to cancer, including atherosclerosis [9], osteoporosis [10] and rheumatoid arthritis [11]. These observations suggest that Tam and 4OH-Tam produce therapeutic effects via both ER-dependent and -independent mechanism(s).

The molecular targets responsible for ER-independent effects of Tam and its derivatives are poorly understood; however, several candidates have been proposed [12]. Tam produces cytotoxicity in human prostate cancer cells [13] and astrocytoma [14] via inhibition of protein kinase C (PKC). ER-independent apoptotic effects of Tam in breast cancer cells appear to be mediated via activation of caspases 6, 7, and 9 [15], whereas inhibition of AKT and JNK and sustained activation of MAP kinase and ERK produces cell death in neuronally-derived glioma cells [16]. Additional molecular mechanisms that may underlie ER-independent effects have been reported. Tam has been shown to reduce angiogenesis required for malignant growth and metastasis via inhibition of vascular endothelial growth factor (VEGF) secretion in breast cancer cells [17], and to reverse multi-drug resistance (MDR) in an in vivo animal model of colorectal carcinoma by interaction with mdr1 protein [18].

Cannabinoids are compounds originally isolated from the marijuana plant (Cannabis sativa) that have been shown to reduce tumor growth and progression in many cellular and animal models by acting at two G-protein coupled receptors (GPCRs), cannabinoid receptors type 1 and 2 (CB1R and CB2R) [19]. CB1Rs are expressed in high abundance throughout the CNS, while CB2Rs are expressed predominantly in immune cells and non-neuronal tissues [20]. It is important to note that, like Tam and 4OH-Tam, cannabinoids exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells [21]. Cannabinoids are similarly efficacious against cancer types not derived from estrogen sensitive tissues, including melanoma [22], glioma [23] and pancreatic cancer [24]. These compounds inhibit tumor angiogenesis via modulation of VEGF activity [25], and reverse MDR in a leukemia cell line [26]. In addition, estradiol (E2) regulates expression levels of CB1Rs in human primary tumor colon cancer cell lines making it an estrogen-responsive receptor [27].

Based on the similarity between many of the ER-independent effects of Tam and the actions of cannabinoids, this study investigated whether CBRs are novel molecular targets for Tam and 4OH-Tam. We report that Tam and 4OH-Tam bind to CB1 and CB2Rs and act as inverse agonists, suggesting that many of their ER-independent effects may be mediated via CBRs.

MATERIAL AND METHODS

Materials

Tam, 4OH-Tam, and GDP were purchased from Sigma Aldrich (St. Louis, MO). WIN-55,212-2, CP-55,940, O-2050, and morphine were obtained from Tocris Bioscience (Ellisville, MO). GTPγS was procured from EMD Chemical (Gibbstown, NJ). [3H]CP-55,940 (174.6 Ci/mmol) was purchased from PerkinElmer (Waltham, MA) and [35S]GTPγS (1250 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO).

Mice

The University of Arkansas for Medical Sciences IACUC committee approved the animal use protocol employed in this study. All efforts were made to minimize animal suffering and reduce the number of animals used. B6SJL mice were obtained from an in-house breeding colony. All animals were maintained on a 12 hr light/dark cycle with free access to food and water. Following anesthesia with isoflurane, mice were sacrificed and brains harvested by decapitation and snap frozen using liquid nitrogen for membrane preparation.

Cell Culture

CHO-K1 cells were stably transfected with the human CB2 receptor (CNR2; CHO-hCB2) [28] or the human mu-opioid receptor (MOR, CHO-hMOR) [29]. CHO cells stably expressing hCB1 receptors (CNR1; CHO-hCB1) were purchased from DiscoveRx Corporation (Fremont, CA). CHO-hCB2, CHO-hMOR and CHO-Wild-Type (-WT) cells were cultured in DMEM (Mediatech Inc., Manassas, VA). CHO-hCB1 cells were cultured in F-12K media (ATCC, Manassas, VA). Culture media contained 10% FCS (Gemini Bioproducts, Sacramento, CA), 0.05 IU/mL penicillin, and 50 mg/mL streptomycin (Invitrogen, Carlsbad, CA). For stably transfected cells, 250 μg/mL of Geneticin (G418; Sigma-Aldrich, St. Louis, MO) was added. All cells were maintained in a humidified chamber at 37°C with 5% CO2, harvested weekly, and only cells from passages 5-15 were used in experiments.

Radioligand Binding Assays

Receptor binding experiments were conducted as described previously [28]. Briefly, membranes from mouse brain, CHO-hCB1, or CHO-hCB2 cells were incubated with increasing concentrations of test compound (100 nM to 30 μM) and a fixed concentration of the radiolabeled non-selective CB1/CB2R agonist, [3H]-CP-55,940 (0.2 nM; equivalent to its apparent KD for each receptor subtype and thus predicted to result in ~50% receptor occupancy). After reaching equilibrium binding (90 min), samples were rapidly filtered and the bound radioactivity was assessed by liquid scintillation counting (LSC).

G-protein Activation Assays

[35S]GTPγS binding assays were conducted as described previously [30] in buffer containing 20 mM Hepes (pH 7.4), 100 mM NaCl, 10 mM MgCl2 and 0.1% BSA. Each reaction contained membrane protein (mouse brain, CHO-hCB1, or CHO-hCB2; 50 μg), test compound (10 μ M), [35S]GTPγS (0.1 nM) and GDP (10 μM). Non-specific binding was defined by 10 μM of non-radioactive GTPγS. After 30 min at 30°C, reactions were terminated by filtration and bound radioactivity was determined by LSC. Specificity of drug effect was determined by co-incubation of Tam or 4OH-Tam with a receptor saturating concentration of the CB1R-neutral antagonist O-2050 (1 μM) [31] or by comparison of drug modulation of [35S]GTPγS activity between CBR-transfected and CHO-WT membranes.

Adenylyl Cyclase (AC) Assays

Activity of the intracellular effector AC in intact CHO-hCB1 or -hCB2 cells was measured as reported previously [32]. Cells were seeded into 24 well plates and cultured to confluence. Cells were treated with culture medium containing 0.9% NaCl, 500 μM 3-isobutyl-1-methlyxanthine (IBMX), and 2 μCi/well [3H]adenine. After 2 hrs at 37°C, the [3H]adenine mixture was removed and increasing concentrations of each test compound (0.1 nM to 30 μM) were added for 15 min in a Krebs-Ringer–Hepes buffer containing 500 μM IBMX and 10 μ M forskolin. Reactions were terminated with 50 μl of 2.2 N HCl and [3H]cAMP, separated by alumina column chromatography, and quantified by LSC. Specificity of drug effect was determined by co-incubation of Tam or 4OH-Tam with a receptor saturating concentration of the CB1R-neutral antagonist O-2050 (1 μM) [31] or by comparison of inhibition of AC activity between CBR-expressing and CHO-WT cells.

Statistical Analysis

All data are expressed as the mean ± SEM, and results were obtained from a minimum of three separate experiments, each performed in triplicate. Curve fitting and statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA). Non-linear regression for one-site competition was used to determine the IC50 for competition receptor binding. The Cheng-Prusoff equation [33] was employed to convert the experimental IC50 values obtained from competition receptor binding experiments to Ki values (a quantitative measure of receptor affinity). Curve fitting of concentration-effect curves via non-linear regression was also employed to determine the ED50 (measure of potency) and EMAX (measure of efficacy) for the GTPγS binding and AC-experiments. A one-sample t-test was used to determine whether the modulation of G-protein or AC-activity produced by a 10 μM concentration of test compounds was significantly different than basal levels.

RESULTS AND DISCUSSION

Tam and 4OH-Tam bind to human CB1 and CB2Rs with moderately high affinity

As demonstrated by competition receptor binding studies, Tam and 4OH-Tam produce complete displacement of the non-selective CB1/CB2 agonist [3H]CP-55,940 from human CB1 and CB2Rs, with affinities (Ki values) of 3.2 ± 0.60 or 1.4 ± 0.25 μM, respectively (Figure 1A, N=3 each). Both compounds bind to CB1Rs expressed in mouse brain with similar affinity to that demonstrated for human CB1Rs (data not shown). Tam and 4OH-Tam also bind to human CB2Rs with slightly higher affinities of 1.9 ± 0.31 and 0.9 ± 0.13 μM, respectively (Figure 1B, N=4 each). These results indicate that Tam and 4OH-Tam bind with relatively high affinity to human CB1 and CB2Rs, with a slight selectivity for CB2Rs. Furthermore, 4OH-Tam has slightly higher affinity for both CBRs than does Tam.

Figure 2.

Figure 2

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Tam and 4OH-Tam act as inverse agonists at human CB1 and CB2 receptors to regulate the intracellular effector AC activity in intact cells. A) Modulation of forskolin-stimulated AC-activity was evaluated by incubating intact CHO-hCB1, CHO-hCB2 or CHO-hMOR cells with a receptor saturating concentration of CP-55,950 (1 μM), morphine (1 μM), Tam (10 μM) or 4OH-Tam (10 μM) as described in the Materials and Methods. AC-inhibition produced by the hMOR agonist morphine was evaluated as a positive control in CHO-hMOR cells. The mean ± SEM of basal cAMP levels in the presence of test compounds is presented. B) Increasing concentrations of 4OH-Tam (100 nM - 10 μ M) modulated AC-activity in a concentration-dependent manner in CHO-hCB2 cells. C) Co-incubation with 4OH-Tam produced a parallel shift-to-the-right in the ability of the CB1/CB2 agonist WIN-55,212-2 to inhibit AC-activity in CHO-hCB1 cells, indicative of CB1 receptor antagonism. *Significantly different from basal cAMP levels (P<0.05; One sample t-test).

Interestingly, while this study was in preparation, Kumar and Song published data showing that raloxifene, a benzothiophene-based SERM that is structurally distinct from the triphenylethylene-based Tam [34], binds to CB2Rs [35]. Unfortunately, the ability of raloxifene to bind to CB1Rs was not examined. Although an actual measure of affinity (e.g., Ki or KD) was not reported, a value of approximately 100 nM might be predicted based on the competition receptor binding data reported in that study. These findings suggest that CBR affinity might be a property common to both benzothiophene and triphenylethylene-based SERMs.

Tam and 4OH-Tam act as CB1 and CB2R inverse agonists to regulate G-protein activity in membrane homogenates

CB1 and CB2Rs are members of the Gi/Go-linked class of G-protein coupled receptors [20]. As such, the binding of agonists to these receptors results in activation of G-proteins. Activated G-proteins exchange GTP for GDP and thus when membranes are incubated with [35S]GTPγS (a radiolabeled form of GTP), the degree of G-protein activation can be quantified by measuring increases in the binding of [35S]GTPγBS to G-proteins. The well characterized full CB1/CB2 agonist CP-55,940 (1 μM) activates G-proteins in CHO-hCB1 (55%) and CHO-hCB2 (27%), but not CHO-WT (4%) membranes (Figure 1C). In marked contrast, a receptor saturating concentration (10 μM) of Tam and 4OH-Tam reduces G-protein activity below basal levels in CHO-hCB1 (−33%, −28%) and CHO-hCB2 (−31%, −23%), but not in CHO-WT (−6%, 1%) membranes. Co-incubation with the neutral CB1R antagonist O-2050 (1 μM) [31] also significantly attenuates the reduction in G-protein activity below basal levels produced by Tam and 4OH-Tam in mouse brain membranes (data not shown). These results indicate that Tam and 4OH-Tam act as inverse agonists at CB1 and CB2Rs, blocking activation of Gi-proteins by constitutively active CBRs [36].

Tam and 4OH-Tam act as CB1 and CB2R inverse agonists to regulate AC-activity in intact whole cells

CBRs activate the Gi subclass of G-proteins, which then proceed to inhibit the activity of the downstream intracellular effector AC, resulting in a reduction in intracellular cAMP levels [20]. The CB1/CB2 full agonist CP-55,940 (1 μM) inhibits AC activity in intact CHO-hCB1 (79%) and CHO-hCB2 (86%), but not CHO-hMOR (8%) cells (Figure 2A). The well-characterized mu-opioid agonist morphine (1 μM) inhibits AC activity only in intact CHO-hMOR (66%) cells. A receptor saturating concentration of 4OH-Tam (10 μM) instead increases AC activity (as reflected by increased levels of cAMP) above basal levels in intact CHO-hCB1 (17%) and CHO-hCB2 (175%), but not in CHO-hMOR (1%) cells. Tam (10 μM) appears to be a less efficacious inverse agonist at CBRs than 4OH-Tam, significantly increasing intracellular cAMP levels in CHO-hCB2 (36%), but not CHO-hCB1 (2%) cells. These results indicate that Tam and 4OH-Tam also act as inverse agonists at CB1 and CB2 receptors to regulate activity of the downstream intracellular effector AC, reducing AC inhibition produced by constitutively active CB receptors. Furthermore, 4OH-Tam is a very efficacious inverse agonist at CB2 receptors.

4OH-Tam also produces a concentration-dependent increase in cAMP levels in intact CHO-hCB2 cells with a potency (5.8 ± 0.3 μM) (Figure 2B) that is similar to its affinity for CB2 receptors as determined by competition receptor binding (0.9 μM) (Figure 1B). Demonstration of concentration-dependence and correlation between the potency for effector regulation and receptor affinity, provide additional confirmation that 4OH-Tam indeed acts as a CB2-inverse agonist.

Although 4OH-Tam produces relatively little (though significant) elevations in cAMP levels in CHO-hCB1 cells, co-incubation with this compound produces an 8-fold parallel shift-to-the-right in the potency for the well characterized full CB1/CB2 agonist CP-55,940 to inhibit AC activity (Figure 2C, Kb = 1.6 ± 0.4 μM). This demonstrates that under conditions in which CB1Rs show little constitutive activity, 4OH-Tam acts as an antagonist with a Kb value very similar to its affinity for CB1Rs (Figure 1A; 1.4 μM). Therefore, depending upon the level of CBR constitutive activity, Tam and 4OH-Tam can act as either antagonists or inverse agonists.

Raloxifene was also reported to act as an inverse agonist at CB2Rs to regulate AC-activity in transfected human embryonic kidney cells [35]. This finding, combined with the present work, suggests that these structurally distinct SERMs appear to share the ability to act as inverse agonists at CB2Rs. However, it is interesting to note that although raloxifene (a benzothiophene SERM) appears to have a higher affinity for this receptor, the triphenylethylene SERM, 4OH-Tam, appears to be a much more efficacious CB2R inverse agonist, producing almost 10-fold higher elevations in cAMP levels (193% - Figure 2B versus 20% - [35], respectively). Dissimilar CB2R density between examined cell lines might account for some of the differences observed.

CB1 and CB2Rs are novel molecular targets for Tam and 4OH-Tam and may serve as a mechanism underlying the ER-independent effects often reported for these drugs

Tam and 4OH-Tam bind with very high affinity to ERs (Ki values in the low nM and even sub-nM range) [1] and their ability to act as ER antagonists is clearly responsible for therapeutic effects in ER-positive breast cancer [2]. However, Tam also produces a variety of ER-independent effects, which usually necessitate higher doses than those required to elicit ER-dependent effects [37]. Based on the similarity between many of the ER-independent effects of Tam and the actions of cannabinoids (see Introduction), this study investigated whether CBRs are novel molecular targets for Tam and 4OH-Tam.

As hypothesized, both Tam and 4OH-Tam were found to bind to CB1 and CB2Rs with moderately high affinity. Since the majority of current evidence indicates that the anticancer actions of cannabinoids are due to activation of CB1 and CB2Rs by agonists [38], it might have been predicted that Tam and 4OH-Tam would act as CB1 and/or CB2R agonists; however, our data indicates that they function as inverse agonists. Studies are accumulating that support the alternate view that, in some instances, enhancement of endocannabinoid signaling may serve to promote cancer progression (for review see [39]). In support of this theory, blocking CB1R signaling by inverse agonists has been shown to reduce tumor growth in thyroid cancer [40] and mantle-cell lymphoma [41]. Furthermore, CB1 inverse agonists (including Tam) have been shown slow tumor progression in ER-negative and ER-positive breast cancer [4,42] and colon cancer [43,44], and reduce tumor neoangiogenesis [17,45]. Based on these prior studies and the data presented here, it can be speculated that the novel CBR inverse agonist actions of Tam and 4OH-Tam contribute to many of the high dose and/or ER-independent anti-cancer effects observed for these compounds. In any case, results reported in the present study indicate that both benzothiophene and triphenylethylene SERMs might be used as structural scaffolds for development of a novel class of potent, efficacious and non-toxic anti-cancer drugs acting via CB1 and/or CB2Rs.

Tamoxifen produces cytotoxicity via estrogen-receptor (ER) independent mechanisms.

Tamoxifen binds to CB1 and CB2 cannabinoid receptors and acts as an inverse agonist.

CB1 and CB2 receptors are novel molecular targets for Tamoxifen.

ER-independent effects for Tamoxifen may be mediated via CB1 and/or CB2 receptors.

Figure 1.

Figure 1

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Tam and 4OH-Tam bind to human CB1 and CB2 receptors with moderately high affinity and act as inverse agonists to regulate G-protein activity. Specific binding was determined by incubating 0.2 nM of [3H]CP-55,950 with increasing concentrations of Tam or 4OH-Tam and 100 μg of membranes prepared from A) CHO-hCB1 or B) CHO-hCB2 cells (see Materials and Methods). The Cheng-Prusoff equation [33] was used to convert the experimental IC50 values obtained from competition receptor binding experiments to Ki values, a quantitative measure of receptor affinity. C) Membranes (50 μg) prepared from CHO-hCB1, CHO-hCB2 or CHO-WT cells were incubated in the presence of 0.1 nM [35S]GTPγS with a receptor saturating concentration of CP-55,950 (1 μM), Tam (10 μM) or 4OH-Tam (10 μM) (see Materials and Methods). Modulation of G-protein activity by compounds in non-transfected CHO-WT cells was evaluated to determine if the observed drug effects were mediated specifically via CB1 and CB2 receptors. The mean ± SEM of basal G-protein activity in the presence of test compounds is presented. *Significantly different from basal G-protein activity (P<0.05; One sample t-test).

ACKNOWLEDGMENTS

This work was funded by grants from the NIH [GM075893], DoD [X81XWH-11-1-0795], ABCRB/UAMS TRI CTSA [UL1TR000039] to AR-P.

Non-Standard Abbreviations

4OH-Tam

4OH-tamoxifen

AC

adenylyl cyclase

CB1R

cannabinoid receptor type 1

CB2R

cannabinoid receptor type 2

CHO

Chinese hamster ovary

CP-55,940

5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol

DMEM

Dulbecco’s Modification of Eagle’s Medium

ER

estrogen receptor

GPCR

G-protein coupled receptor

[35S]GTPγS

guanosine 5’-O-(3-[35S]thio)triphosphate

hCB2

human CB2 receptors

hMOR

human mu-opioid receptors

IBMX

isobutyl-methyl-xanthine

SERM

selective estrogen receptor modulator

Tam

tamoxifen

WIN-55,212-2

[(3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone.

Footnotes

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REFERENCES

  • 1.Rochefort H. Mechanism of action of high-affinity antiestrogens. An overview, American Journal of Clinical Oncology. 1991;14(2):S1–S4. doi: 10.1097/00000421-199112002-00003. [DOI] [PubMed] [Google Scholar]
  • 2.Karn A, Jha AK, Shrestha S, Acharya B, Poudel S, Bhandari RB. Tamoxifen for breast cancer. Journal of Nepal Medical Association. 2010;49:62–67. [PubMed] [Google Scholar]
  • 3.Love RR. Tamoxifen therapy in primary breast cancer: biology, efficacy, and side effects. Journal of Clinical Oncology. 1989;7:803–815. doi: 10.1200/JCO.1989.7.6.803. [DOI] [PubMed] [Google Scholar]
  • 4.Perry RR, Kang Y, Greaves B. Effects of tamoxifen on growth and apoptosis of estrogen-dependent and -independent human breast cancer cells. Annals of Surgical Oncology. 1995;2:238–245. doi: 10.1007/BF02307030. [DOI] [PubMed] [Google Scholar]
  • 5.Todorova VK, Kaufmann Y, Luo S, Klimberg VS. Tamoxifen and raloxifene suppress the proliferation of estrogen receptor-negative cells through inhibition of glutamine uptake. Cancer Chemotherapy and Pharmacology. 2011;67:285–291. doi: 10.1007/s00280-010-1316-y. [DOI] [PubMed] [Google Scholar]
  • 6.Beguerie JR, Xingzhong J, Valdez RP. Tamoxifen vs. non-tamoxifen treatment for advanced melanoma: a meta-analysis. International Journal of Dermatology. 2010;49:1194–1202. doi: 10.1111/j.1365-4632.2010.04529.x. [DOI] [PubMed] [Google Scholar]
  • 7.Mastronardi L, Puzzilli F, Ruggeri A. Tamoxifen as a potential treatment of glioma. Anticancer Drugs. 1998;9:581–586. doi: 10.1097/00001813-199808000-00001. [DOI] [PubMed] [Google Scholar]
  • 8.Tomao S, Romiti A, Massidda B, Ionta MT, Farris A, Zullo A, Brescia A, Santuari L, Frati L. A phase II study of gemcitabine and tamoxifen in advanced pancreatic cancer. Anticancer Research. 2002;22:2361–2364. [PubMed] [Google Scholar]
  • 9.Stamatelopoulos KS, Lekakis JP, Poulakaki NA, Papamichael CM, Venetsanou K, Aznaouridis K, Protogerou AD, Papaioannou TG, Kumar S, Stamatelopoulos SF. Tamoxifen improves endothelial function and reduces carotid intima-media thickness in postmenopausal women. American Heart Journal. 2004;147:1093–1099. doi: 10.1016/j.ahj.2003.12.029. [DOI] [PubMed] [Google Scholar]
  • 10.Gennari L, Merlotti D, Valleggi F, Martini G, Nuti R. Selective estrogen receptor modulators for postmenopausal osteoporosis: current state of development. Drugs and Aging. 2007;24:361–379. doi: 10.2165/00002512-200724050-00002. [DOI] [PubMed] [Google Scholar]
  • 11.Williams S, Michael B, Mewar D, Tunn E. Inflammatory osteoarthritis which was precipitated by Arimidex and resolved with tamoxifen. British Medical Journal Case Reports. 2010;2010 doi: 10.1136/bcr.06.2010.3089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Bogush TA, Dudko EA, Bogush EA, Polotskii BE, Tiuliandin SA, Davydov MI. Tamoxifen non-estrogen receptor mediated molecular targets. Oncology Reviews. 2012;6:e15. doi: 10.4081/oncol.2012.e15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rohlff C, Blagosklonny MV, Kyle E, Kesari A, Kim IY, Zelner DJ, Hakim F, Trepel J, Bergan RC. Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21(waf1/cip1) Prostate. 1998;37:51–59. doi: 10.1002/(sici)1097-0045(19980915)37:1<51::aid-pros8>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
  • 14.Sharif TR, Sharif M. A novel approach for examining the anti-proliferative effect of protein kinase C inhibitors against human astrocytoma cells. International Journal of Oncology. 1998;13:685–692. doi: 10.3892/ijo.13.4.685. [DOI] [PubMed] [Google Scholar]
  • 15.Thiantanawat A, Long BJ, Brodie AM. Signaling pathways of apoptosis activated by aromatase inhibitors and antiestrogens. Cancer Research. 2003;63:8037–8050. [PubMed] [Google Scholar]
  • 16.Feng Y, Huang J, Ding Y, Xie F, Shen X. Tamoxifen-induced apoptosis of rat C6 glioma cells via PI3K/Akt, JNK and ERK activation. Oncology Reports. 2010;24:1561–1567. doi: 10.3892/or_00001018. [DOI] [PubMed] [Google Scholar]
  • 17.Garvin S, Dabrosin C. Tamoxifen inhibits secretion of vascular endothelial growth factor in breast cancer in vivo. Cancer Research. 2003;63:8742–8748. [PubMed] [Google Scholar]
  • 18.Shen LZ, Hua YB, Yu XM, Xu Q, Chen T, Wang JH, Wu WX. Tamoxifen can reverse multidrug resistance of colorectal carcinoma in vivo. World Journal of Gastroenterology. 2005;11:1060–1064. doi: 10.3748/wjg.v11.i7.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Velasco G, Sanchez C, Guzman M. Towards the use of cannabinoids as antitumour agents. Nature Reviews of Cancer. 2012;12:436–444. doi: 10.1038/nrc3247. [DOI] [PubMed] [Google Scholar]
  • 20.Howlett AC. Pharmacology of cannabinoid receptors. Annual Review of Pharmacology and Toxicology. 1995;35:607–634. doi: 10.1146/annurev.pa.35.040195.003135. [DOI] [PubMed] [Google Scholar]
  • 21.Caffarel MM, Andradas C, Perez-Gomez E, Guzman M, Sanchez C. Cannabinoids: a new hope for breast cancer therapy? Cancer Treatment Reviews. 2012;38:911–918. doi: 10.1016/j.ctrv.2012.06.005. [DOI] [PubMed] [Google Scholar]
  • 22.Blazquez C, Carracedo A, Barrado L, Real PJ, Fernandez-Luna JL, Velasco G, Malumbres M, Guzman M. Cannabinoid receptors as novel targets for the treatment of melanoma. FASEB Journal. 2006;20:2633–2635. doi: 10.1096/fj.06-6638fje. [DOI] [PubMed] [Google Scholar]
  • 23.Galve-Roperh I, Sanchez C, Cortes ML, Gomez del Pulgar T, Izquierdo M, Guzman M. Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nature Medicine. 2000;6:313–319. doi: 10.1038/73171. [DOI] [PubMed] [Google Scholar]
  • 24.Carracedo A, Gironella M, Lorente M, Garcia S, Guzman M, Velasco G, Iovanna JL. Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes. Cancer Research. 2006;66:6748–6755. doi: 10.1158/0008-5472.CAN-06-0169. [DOI] [PubMed] [Google Scholar]
  • 25.Blazquez C, Casanova ML, Planas A, Gomez Del Pulgar T, Villanueva C, Fernandez-Acenero MJ, Aragones J, Huffman JW, Jorcano JL, Guzman M. Inhibition of tumor angiogenesis by cannabinoids. Faseb J. 2003;17:529–531. doi: 10.1096/fj.02-0795fje. [DOI] [PubMed] [Google Scholar]
  • 26.Holland ML, Panetta JA, Hoskins JM, Bebawy M, Roufogalis BD, Allen JD, Arnold JC. The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells. Biochemical Pharmacology. 2006;71:1146–1154. doi: 10.1016/j.bcp.2005.12.033. [DOI] [PubMed] [Google Scholar]
  • 27.Notarnicola M, Messa C, Orlando A, Bifulco M, Laezza C, Gazzerro P, Caruso MG. Estrogenic induction of cannabinoid CB1 receptor in human colon cancer cell lines. Scandinavian Journal of Gastroenterology. 2008;43:66–72. doi: 10.1080/00365520701559011. [DOI] [PubMed] [Google Scholar]
  • 28.Shoemaker JL, Joseph BK, Ruckle MB, Mayeux PR, Prather PL. The endocannabinoid noladin ether acts as a full agonist at human CB2 cannabinoid receptors. Journal of Pharmacology and Experimental Therapeutics. 2005;314:868–875. doi: 10.1124/jpet.105.085282. [DOI] [PubMed] [Google Scholar]
  • 29.Seely KA, Brents LK, Franks LN, Rajasekaran M, Zimmerman SM, Fantegrossi WE, Prather PL. AM-251 and rimonabant act as direct antagonists at mu-opioid receptors: Implications for opioid/cannabinoid interaction studies. Neuropharmacology. 2012;63:905–915. doi: 10.1016/j.neuropharm.2012.06.046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Seely KA, Brents LK, Radominska-Pandya A, Endres GW, Keyes GS, Moran JH, Prather PL. A Major Glucuronidated Metabolite of JWH-018 Is a Neutral Antagonist at CB1 Receptors. Chem Res Toxicol. 2012;25:825–827. doi: 10.1021/tx3000472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Gardner A, Mallet PE. Suppression of feeding, drinking, and locomotion by a putative cannabinoid receptor 'silent antagonist'. European Journal of Pharmacology. 2006;530:103–106. doi: 10.1016/j.ejphar.2005.11.032. [DOI] [PubMed] [Google Scholar]
  • 32.Rajasekaran M, Brents LK, Franks LN, Moran JH, Prather PL. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors. Toxicology and applied pharmacology. 2013;269:100–108. doi: 10.1016/j.taap.2013.03.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Cheng Y, Prusoff W. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (i50) of an enzymatic reaction. Biochemical Pharmacology. 1973;22:3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  • 34.Goldstein SR, Siddhanti S, Ciaccia AV, Plouffe L., Jr. A pharmacological review of selective oestrogen receptor modulators. Human Reproduction Update. 2000;6:212–224. doi: 10.1093/humupd/6.3.212. [DOI] [PubMed] [Google Scholar]
  • 35.Kumar P, Song ZH. Identification of raloxifene as a novel CB2 inverse agonist. Biochemical and Biophysical Research Communications. 2013;435:76–81. doi: 10.1016/j.bbrc.2013.04.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Aloyo VJ, Berg KA, Clarke WP, Spampinato U, Harvey JA. Inverse agonism at serotonin and cannabinoid receptors. Progress in Molecular and Biological Translational Science. 2010;91:1–40. doi: 10.1016/S1877-1173(10)91001-6. [DOI] [PubMed] [Google Scholar]
  • 37.Saji S, Kuroi K. Application of selective estrogen receptor modulators for breast cancer treatment according to their intrinsic nature. Breast Cancer. 2008;15:262–269. doi: 10.1007/s12282-008-0063-y. [DOI] [PubMed] [Google Scholar]
  • 38.Hermanson DJ, Marnett LJ. Cannabinoids. endocannabinoids, and cancer, Cancer metastasis reviews. 2011;30:599–612. doi: 10.1007/s10555-011-9318-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Pisanti S, Picardi P, D'Alessandro A, Laezza C, Bifulco M. The endocannabinoid signaling system in cancer. Trends in Pharmacological Sciences. 2013;34:273–282. doi: 10.1016/j.tips.2013.03.003. [DOI] [PubMed] [Google Scholar]
  • 40.Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis. FASEB Journal. 2003;17:1771–1773. doi: 10.1096/fj.02-1129fje. [DOI] [PubMed] [Google Scholar]
  • 41.Flygare J, Gustafsson K, Kimby E, Christensson B, Sander B. Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma. FEBS Letters. 2005;579:6885–6889. doi: 10.1016/j.febslet.2005.11.020. [DOI] [PubMed] [Google Scholar]
  • 42.Sarnataro D, Pisanti S, Santoro A, Gazzerro P, Malfitano AM, Laezza C, Bifulco M. The cannabinoid CB1 receptor antagonist rimonabant (SR141716) inhibits human breast cancer cell proliferation through a lipid raft-mediated mechanism. Mol Pharmacol. 2006;70:1298–1306. doi: 10.1124/mol.106.025601. [DOI] [PubMed] [Google Scholar]
  • 43.Motylewska E, Lawnicka H, Melen-Mucha G. Oestradiol and tamoxifen inhibit murine Colon 38 cancer growth and increase the cytotoxic effect of fluorouracil. Endokrynologia Polska. 2007;58:426–434. [PubMed] [Google Scholar]
  • 44.Santoro A, Pisanti S, Grimaldi C, Izzo AA, Borrelli F, Proto MC, Malfitano AM, Gazzerro P, Laezza C, Bifulco M. Rimonabant inhibits human colon cancer cell growth and reduces the formation of precancerous lesions in the mouse colon. International Journal of Cancer. 2009;125:996–1003. doi: 10.1002/ijc.24483. [DOI] [PubMed] [Google Scholar]
  • 45.Pisanti S, Picardi P, Prota L, Proto MC, Laezza C, McGuire PG, Morbidelli L, Gazzerro P, Ziche M, Das A, Bifulco M. Genetic and pharmacologic inactivation of cannabinoid CB1 receptor inhibits angiogenesis. Blood. 2011;117:5541–5550. doi: 10.1182/blood-2010-09-307355. [DOI] [PubMed] [Google Scholar]

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