Cannabinoid receptors, CB1 and CB2, as novel targets for inhibition of non-small cell lung cancer growth and metastasis

Abstract

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide; however, only limited therapeutic treatments are available. Hence, we investigated the role of cannabinoid receptors, CB1 and CB2, as novel therapeutic targets against NSCLC. We observed expression of CB1 (24%) and CB2 (55%) in NSCLC patients. Furthermore, we have shown that the treatment of NSCLC cell lines (A549 and SW-1573) with CB1/CB2- and CB2-specific agonists Win55,212-2 and JWH-015, respectively, significantly attenuated random as well as growth factor-directed in vitro chemotaxis and chemoinvasion in these cells. We also observed significant reduction in focal adhesion complex, which plays an important role in migration, upon treatment with both JWH-015 and Win55,212-2. In addition, pretreatment with CB1/CB2 selective antagonists, AM251 and AM630, prior to JWH-015 and Win55,212-2 treatments, attenuated the agonist-mediated inhibition of in vitro chemotaxis and chemoinvasion. In addition, both CB1 and CB2 agonists Win55,212-2 and JWH-133, respectively, significantly inhibited in vivo tumor growth and lung metastasis (∼50%). These effects were receptor mediated, as pretreatment with CB1/CB2 antagonists abrogated CB1/CB2 agonist-mediated effects on tumor growth and metastasis. Reduced proliferation and vascularization, along with increased apoptosis, were observed in tumors obtained from animals treated with JWH-133 and Win55,212-2. Upon further elucidation into the molecular mechanism, we observed that both CB1 and CB2 agonists inhibited phosphorylation of AKT, a key signaling molecule controlling cell survival, migration, and apoptosis, and reduced matrix metalloproteinase 9 expression and activity. These results suggest that CB1 and CB2 could be used as novel therapeutic targets against NSCLC.

Figure 1. Expression of cannabinoid receptors in human lung adenocarcinomas and normal lung

Immunohistochemical staining for cannabinoid receptor, CB1 (A) and CB2 (B) expression in human pulmonary adenocarcinoma and normal lungs. Lower panels in both (A) and (B) show controls with the Ab-blocking peptides for CB1 and CB2, respectively.

Figure 2. Inhibition of proliferation and migration in A549 cells with CB1 and CB2 specific cannabinoids

A. A549 cells were incubated in culture medium with 100ng/ml EGF (solid bars) and 10% serum (empty bars) for 72 h in the presence of different concentrations of Win55,212-2 and JWH-015 or vehicle control and then analyzed for proliferation by MTT assay. B. A549 and SW-1573 grown in 6-well culture plates were serum starved for 24 h, pretreated with JWH-015 or Win55,212-2 for 30’ prior to stimulation with EGF (10 ng/ml) and incubated for 48 h. Cell scattering was examined by phase-contrast microscopy and photographed. C. Confluent layers of A549 cells were scratched with sterile tips to form wounds and were cultured in the presence of EGF+vehicle or EGF+cannabinoids in presence or absence of GPCR inhibitor Pertussis toxin (PTX). Quantitative analysis of % wound recolonization shows a significant GPCR-mediated inhibition of the EGF-induced chemotaxis by cannabinoids treatment in the A549 cells. Data represent the mean ± SD, representative experiments (n=3) are shown. (*, P<0.05; **, P<0.001; from EGF or serum stimulated, ^†, P<0.05; ^††, P<0.001; from cannabinoid treated)

Figure 3. CB1 and CB2 receptor mediates inhibition of focal adhesions formation

Confocal microscopic visualization of JWH-015 and Win55,212-2 pretreated A549 cells stimulated with EGF (100ng/ml) for generation of focal adhesions (stained for Vinculin, green) and stress fibers (stained for phalloidin, red). JWH-015 and Win55,212-2 inhibit formation of focal adhesions and stress fibers in NSCLC cells.

Figure 4. CB1 and CB2 receptor mediated inhibition of EGF-induced migration and invasion in NSCLC cell lines

A549 cells were treated with different concentrations of JWH-015 or Win55,212-2 or vehicle alone (ethanol) before being subjected to the EGF-induced (A, C) transwell migration and (C, D) invasion assays. Data represent the mean ± SEM from 3 independent experiments. (*, P<0.05; **, P<0.001; compared to only EGF-stimulated cells, ^†, P<0.05; ^††, P<0.001; to cannabinoid treated cells).

Figure 5. CB1 and CB2 receptor activation inhibits the xenograft growth and metastasis of lung tumors in SCID mice

A549 cells were injected subcutaneously (3 × 10⁶) and intravenously (1 × 10⁶) into immunodeficient SCID mice. Experimental mice were given JWH-133 (1 mg/kg body weight) and Win55,212-2 (0.1 mg/kg body weight) daily for 28 days starting 14 days and 1 day after injection of the cells subcutaneously and intravenously, respectively. At the end of the experiment, the animals were sacrificed; tumors and lungs harvested and weighed before evaluation of other parameters like angiogenesis, proliferation, apoptosis (in tumors) and metastatic lesions (on lungs). Receptor-mediated inhibitory effects of cannabinoids were partially reversed with animals treated with different combinations of agonist and antagonists, AM281 (AM) for CB1 and SR (SR 144528) for CB2. A. Receptor-mediated reduction in tumor growth, B. Changes in the levels of proliferation (% staining for Ki67), vascularization (% staining for CD31) and apoptosis (positive TUNEL staining) with treatment with cannabinoids. (*, P<0.05; **, P<0.001; compared to untreated animals), C. Receptor-mediated inhibition of metastatic lesions and lung weight.

Figure 6. CB1 and CB2 receptor specific ligands inhibit the EGF-induced phosphorylation of AKT and PMA-induced secretion of MMP-9 in NSCLC cells

A549 cells were incubated overnight with different concentrations of JWH-015 and Win55,212-2 (0.1, 0.5 and 1 µM) in serum-free medium supplemented with 0.1% FBS and stimulated for 10 min with 10 ng/ml EGF. The phosphorylation of AKT was analyzed in the cells by Western blot analysis using phospho-specific antibodies against AKT. Total protein levels in each lane are shown in the lower lane. WB: Western blot. Stimulations were repeated three times and representative blots are shown. Analysis for secreted levels of MMP-9 by A549 cells through ELISA shows inhibitory effect of cannabinoids in comparison to the corresponding vehicle-treated cells. A549 cells were treated with different concentrations of JWH-015 and Win55,212-2 for 24 h in serum-free growth medium. Supernatants were collected and levels of MMP-9 were measured by ELISA. (*, P<0.05; **, P<0.001; compared to PMA-treated or untreated cells, ^†, P<0.05; ^††, P<0.001; to cannabinoid treated cells).