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. 2009 Nov;8(11):3117-29.
doi: 10.1158/1535-7163.MCT-09-0448. Epub 2009 Nov 3.

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Zahida Qamri  1 , Anju Preet, Mohd W Nasser, Caroline E Bass, Gustavo Leone, Sanford H Barsky, Ramesh K Ganju
Affiliations

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Zahida Qamri et al. Mol Cancer Ther. 2009 Nov.

Abstract

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.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Primary breast cancer tissues and MDA-MB231, MDA-MB231-luc-D3H2LN, and MDA-MB468 cell lines express CB1 and CB2 receptors. A, representative photomicrographs of immunohistochemical staining of primary breast cancer showing CB1 and CB2 expression. Receptor expression in MDA-MB231, MDA-MB231-luc, and MDA-MB468 cell lines by RT-PCR (B), Western blot (C), and confocal microscopy (D). Human umbilical vein endothelial cells (HUVEC) and Jurkat cells were taken as positive control for CB1 and CB2 receptors, respectively. Data shown are representative of two experiments.
Figure 2
Figure 2
Synthetic cannabinoids inhibit growth and migration of breast cancer cells. MDA-MB231 and MDA-MB468 cells were serum-starved and treated with different concentrations of vehicle, JWH-133, or WIN-55,212-2 (0.1–10 µmol/L) alone, before being subjected to MTT assay (A) and transwell migration (B). C, left, MDA-MB231 cells were grown to 70% confluency and were transfected with 100 pmol CB2 siRNA or nontargeting control using Lipofect AMINE 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the cells were treated overnight with vehicle, JWH-133, or WIN-55,212-2 (10 µmol/L), and subjected to migration assay. The cells that migrated across the filter towards complete medium in 6 h were fixed and stained with 0.1 % crystal violet in PBS for 30 min. The number of migratory cells per membrane was counted in five fields and the percentage of migration determined. Right, the expression of CB2 in siRNA and nontargeted MDA-MB231 cells was evaluated by Western blot using specific anti-CB2 antibody. The experiments were repeated three times and representative values are shown. *, P < 0.05; **, P < 0.01 versus vehicle. Veh, vehicle; Cone, concentration.
Figure 3
Figure 3
Synthetic cannabinoids inhibit breast cancer growth, proliferation, and angiogenesis in vivo. MDA-MB231 cells (3 × 106) were injected s.c. into the right flank of severe combined immunodeficient mice. After tumors were palpable, the mice were treated peritumorally either with 5 mg/kg body wt of vehicle, JWH-133, WIN-55,212-2, or their respective antagonists for 4 wk (n = 6). A and B, tumors were measured every week with external calipers and volume was calculated as volume = length × (width)2/2. C, representative of tumors dissected from different experimental groups. After 4 wk, the tumors were excised from mice and volume was calculated accordingly. D, representative photomicrographs of immunostaining with CD31 (vascularization marker) and Ki67 (proliferation marker) of tumors extracted from treated mice. The results are shown as the percent change in treated animals versus controls. Scale bars, 50 µm. *, P < 0.05; **, P < 0.01, significantly different from vehicle-treated cells. #, P < 0.05; ##, P < 0.01, significantly different from JWH/WIN-treated cells, respectively. Data are representative of two independent experiments.
Figure 4
Figure 4
Synthetic cannabinoids inhibit lung metastasis in vivo. MDA-MB231-luc-D3H2LN cells (1 × 106) were inoculated i.v. into immunodeficient mice. After 24 h of injection, different combinations of 5 mg/kg body wt of vehicle, JWH-1 33, WIN-55,212-2, or their respective antagonists were given i.p. for 4 wk (n = 6). A and B, bioluminescence imaging was done every week and plotted as radiance flux (photons/s). C, representative illustration of imaging of mice. D, after necropsy, lung metastatic lesions were quantified and plotted. *, P < 0.05; **, P < 0.01, significantly different from vehicle-treated cells. #, P < 0.05; ##, P < 0.01, significantly different from JWH/WIN-treated cells, respectively. Data are representative of two independent experiments.
Figure 5
Figure 5
Synthetic cannabinoids delay the growth and progression of mammary tumors in transgenic PyMT mice. Female transgenic mice were treated peritoneally either with 5 mg/kg wt of vehicle or with JWH-133 for 8 wk (n = 6). A, representative illustration of female transgenic PyMT mice with tumors at the time of sacrifice. B, after 8 wk, mammary tumors were excised from mice and weighed. Representative photomicrographs of tumors showing (C) Ki67 (proliferation marker) and (D) CD31 staining (vascularization marker). The results are shown as the percent change in treated animals versus controls. *P < 0.05; **p< 0.01 compared with vehicle-treated.
Figure 6
Figure 6
Synthetic cannabinoids modulate COX-2 signaling in breast cancer cells, arrest cell growth, and induce apoptosis in tumors. A, PGE2 levels in supernatants of MDA-MB231 cells treated with vehicle, JWH-133, or WIN55,212-2 as measured by ELISA assay. B, left, Western blot analysis of COX-2 in MDA-MB231 cells treated with vehicle, JWH-133, or WIN55,212-2. Right, messenger RNA levels for COX-2 determined by real-time PCR in tumors treated with vehicle, JWH-133, or WIN55,212-2. Results are expressed as relative expression compared with control by 2−ΔΔCT. Results are mean ± SD with n = 3 in each group. Inset, Western blot analysis of COX-2 in tumors excised from mice. C, left, Cdc42 activation in MDA-MB231 cells. The densitometric histograms represent relative band intensities normalized to GAPDH. Middle, expression of transcription factors c-Fos and c-Jun in nuclear and cytoplasmic extracts of MDA-MB231 cells treated with either vehicle, JWH-133 (J), or WIN55,212-2 (W). Right, the densitometric histograms represent relative band intensities normalized to Lamin B1. D, top, MDA-MB231 cells were treated with vehicle, JWH-133, or WIN55,21 2-2 for 24 h, stained with propidium iodide, and subjected to flow cytometry. Left, TUNEL staining of tumor tissues. Sections of tumors extracted from mice treated with vehicle, JWH-133, WIN55,212-2, and their antagonists were subjected to TUNEL staining and the positive cells were quantified and plotted. Right, MDA-MB231 cells were treated with 10 µmol/L NS-398 with or without JWH-133, WIN-55,212-2 (10 µmol/L), or vehicle for 24 h, and apoptosis was measured as described in Materials and Methods. *, P < 0.05; * *, P < 0.01, significantly different from vehicle-treated cells. #, P < 0.05; ##, P < 0.01, significantly different from JWH/WIN-treated cells, respectively.
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