Cannabinoid Receptor Type 1 (CB1) Activation Inhibits Small GTPase RhoA Activity and Regulates Motility of Prostate Carcinoma Cells

Abstract

The cannabinoid receptor type 1 (CB1) is a G protein-coupled receptor that is activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). The CB1 and its endogenous and synthetic agonists are emerging as therapeutic targets in several cancers due to their ability to suppress carcinoma cell invasion and migration. However, the mechanisms that the CB1 regulates cell motility are not well understood. In this study, we examined the molecular mechanisms that diminish cell migration upon the CB1 activation in prostate carcinoma cells. The CB1 activation with the agonist WIN55212 significantly diminishes the small GTPase RhoA activity but modestly increases the Rac1 and Cdc42 activity. The diminished RhoA activity is accompanied by the loss of actin/myosin microfilaments, cell spreading, and cell migration. Interestingly, the CB1 inactivation with the selective CB1 antagonist AM251 significantly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes cell migration. This finding suggests that endogenously produced EC activate the CB1, resulting in chronic repression of RhoA activity and cell migration. Consistent with this possibility, RhoA activity is significantly diminished by the exogenous application of AEA but not by 2-AG in PC-3 cells (cells with very low AEA hydrolysis). Pretreatment of cells with a monoacylglycerol lipase inhibitor, JZL184, which blocks 2-AG hydrolysis, decreases the RhoA activity. These results indicate the unique CB1 signaling and support the model that EC, through their autocrine activation of CB1 and subsequent repression of RhoA activity, suppress migration in prostate carcinoma cells.

The cannabinoid receptors, type 1 (CB1) and type 2 (CB2), are G protein-coupled receptors (GPCR) that are emerging as important regulators of the malignant phenotype in a wide variety of cancers (1–6). These receptors can be activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), which are synthesized in cells (7). Autocrine signaling by these EC is regulated by their biosynthesis and hydrolysis by specific enzymes. Fatty acid amide hydrolase (FAAH) hydrolyzes both AEA and 2-AG, whereas monoacylglycerol lipase (MGL) hydrolyzes 2-AG but not AEA (8). Enzymatic hydrolysis of EC is the major mechanism to terminate the EC actions. This autocrine signaling system offers multiple cancer therapeutic targets, including the cannabinoid receptors and the EC as well as the EC-hydrolyzing enzymes FAAH and MGL.

The CB1 has been demonstrated as a suppressor of cell migration and invasion in many types of cancer, including colon, breast, cervical, and lung cancer (1). We previously demonstrated that EC and synthetic cannabinoids, through the CB1, inhibit invasion and migration of prostate carcinoma cells (9–12). The ability of CB1 to suppress cancer cell migration and invasion defines it as a potential inhibitor of cancer metastasis. Despite this important function of the CB1 in cancer, the molecular mechanisms that diminish cell migration upon the CB1 activation have remained elusive.

Small GTPases in the Rho family are well-known regulators of cell migration (13–17), making them intriguing candidates as potential participants in CB1-dependent signaling pathways that suppress cell motility. Rho family GTPases regulate cell migration by regulating the organization of the actin/myosin cytoskeleton, cell adhesion, spreading, and polarity (13–17). Multiple Rho family members, including RhoA, Rac1, and Cdc42, coordinate these dynamic changes in the cytoskeleton (15–17). Activated, GTP-bound RhoA promotes cell migration by activating Rho-associated kinase, which enhances myosin phosphorylation and the formation of actin/myosin microfilaments (13). The activated, GTP-bound forms of Rac1 and Cdc42 promote the formation of lamellopodia and filopodia, respectively, which defines cell polarity and promotes directional movement (13–16). Loss of cell migration can occur when the activities of these small GTPases are diminished. For example, inactivation of RhoA can diminish the Rho-associated kinase activity, diminish the formation of actin/myosin microfilaments, and diminish cell-substrate adhesion, leading to cell rounding and loss of cell motility (13,17). A recent study of human breast carcinoma cell line MDA-MB-231 demonstrated that 2-methyl-2′-F-anandamide, a synthetic stable analog of AEA,. inhibits RhoA activity, induces RhoA translocation from membrane to cytosol, and decreases actin stress fibers and inhibits cell migration (18).

Among small GTPases in prostate carcinoma cells, RhoA is an important promoter of invasion and migration (19–25). Increased RhoA activity, which is often accompanied by increased cell migration and invasion, can be induced by stimulating multiple GPCR expressed by prostate carcinoma cells, including specific GPCR for thrombin, thromboxane A2, lysophosphatidic acid (LPA), and bombesin (20, 23–27). In striking contrast, there are very few GPCR such as the angiotensin type II receptor (28–30) that has been demonstrated to inactivate RhoA. We hypothesized that the CB1 might represent this type of inhibitory GPCR and uniquely suppress RhoA activity to diminish the migration of prostate cancer cells.

To test the role of the CB1 as a potential inhibitory regulator of RhoA in prostate cancer, we examined the effects of CB1 agonists and antagonists on the activities of Rho family members RhoA, Rac1 and Cdc42, in prostate carcinoma cells. RhoA is posttranslationally modified by the geranylgeranyl isoprenoid moieties by enzyme geranylgeranyl transferase (31, 32). This prenylation is one of critical steps for the translocation of RhoA to the cellular membrane as an early event to induce actin fiber stress and focal adhesion formation (33). Thus, we examined the CB1 activation on changes in RhoA in the cellular membrane, actin/myosin microfilaments, and cell migration. We also examined the effects of the endocannabinoid system (EC and enzymes hydrolyzing EC) on the activity of RhoA. This study defines the CB1 as a GPCR that uniquely represses RhoA activity in prostate cancer cells, providing a novel mechanism of the CB1 activation to diminish the migration of aggressive prostate carcinoma cells.

Materials and Methods

Materials

Human hormone-independent prostate carcinoma PC-3 and DU-145 cells and hormone-dependent prostate carcinoma LNCaP cells were obtained from the American Type Culture Collection (Manassas, VA). Eagle's minimal essential medium (RPMI 1640), goat antimouse IgG-horseradish peroxidase (HRP) antibody, and goat antirabbit IgG-HRP antibody were obtained from Invitrogen (Carlsbad, CA). l-Glutamine-penicillin-streptomycin solution, (R)-(+)-WIN55,212-2, ADP-ribosyltransferase C3 (C3 exoenzyme), mitomycin C, and LPA were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was obtained from Hyclone (Logan, UT). 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM251; a selective CB1 antagonist) was obtained from Tocris Bioscience (Ellisville, MO). [N-(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]hepta-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4methylbenzyl)-pyrazole-3-carboxamide (SR144528, a selective CB2 antagonist), was obtained from Research Triangle Institute (Research Triangle Park, NC). Arachidonic acid (AA), AEA 2-AG, 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-l)(hydroxy)methyl]piperidine-1-carboxylate (JZL184; a monoacylglycerol lipase inhibitor), and 2-arachidonylglyceryl ether (noladin ether, NE, a CB1 agonist); were obtained from Cayman Chemical (Ann Arbor, MI). Y-27632 was obtained from Calbiochem (Gibbstown, NJ). Enzyme-linked immunosorbent assay (G-LISA) RhoA activation assay kit, and pull-down activation assay kits for RhoA, Rac1, and Cdc42 were obtained from Cytoskeleton, Inc. (Denver, CO). EZ-Detect Rho activation kit (pull-down), enhanced chemiluminescence Western blotting kit, and bicinchoninic acid protein assay kit were obtained from Pierce Biotechnology, Inc. (Rockford, IL). Primary antibody to phosphorylated RhoA (Ser 188) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), mouse monoclonal antibody to lactate dehydrogenase (LDH) was from Abnova (Walnut, CA) and mouse monoclonal antibody to sodium potassium-ATP (Na,K-ATPase; sodium-potassium pump) was from Cell Signaling Technology (Danvers, MA). SA-2 human IgM antibody to the myosin heavy chain and fluorescein isothiocyanate (FITC)-labeled antihuman IgM were provided by C.L.W. All other chemicals and solvents used were of the analytical or highest purity grades. Distilled and deionized water was used in all pertinent experiments.

Cell culture

Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, l-glutamine (2 mm), streptomycin (100 μg/ml), and penicillin (100 U/ml) at 37 C in 5% CO₂. Cells were cultured as described in specific assays.

Immunofluorescence imaging of microfilaments in prostate carcinoma cells

To examine actin-myosin organization, cells were cultured on glass coverslips in 24-well plates in complete medium for 48 h. Then the complete medium was replaced with serum-free medium for 24 h. Cells were treated with either vehicle or WIN55,212-2 (a CB agonist, 500 nm), NE (an enzymatically stable putative EC for CB1, 10 μm), AM251 (a selective CB1 antagonist, 500 nm), AM251+NE, AM251+WIN55,212-2, and C3 exoenzyme (a RhoA inhibitor, 1 μg/ml) at 37 C for 90 min. The concentrations of pharmacological agents were chosen in the range of their EC₅₀ and dissociation constant values and the previous and current studies that inhibit cell motility but do not cause cell death. Cells were fixed and incubated with SA-2 human IgM antibody to the myosin heavy chain and FITC-labeled antihuman IgM antibody as described (34, 35). The images were captured using a Nikon Eclipse E600 fluorescence microscope (Nikon Instruments, Inc., Melville, NY).

G-LISA of small GTPase RhoA activity

Cells were grown in 100-mm dishes in complete media to about 80% confluency, and the media were replaced with serum-free media overnight. Then cells were treated at 37 C with pharmacological agents. Group 1 was LPA (a RhoA activator, 10 μm) for 1, 5, 10, 15, and 30 min; group 2 was AM251 (500 nm) for 1, 5, 10, 15, 30, 45, and 60 min; group 3 was WIN55,212-2 (500 nm) for 1, 5, 15, and 30 min; and group 4 was AM251 (500 nm)+WIN55,212-2 (100, 500, and 1000 nm) for 5 min. Then cells were lysed in cold lysis buffer, and the samples were prepared as described in the G-LISA RhoA activation assay.

The GTP-bound RhoA was determined after incubation with anti-RhoA primary antibody (1:250 dilution) and shaken on a microplate shaker for 45 min followed by a 45-min incubation with the secondary HRP-labeled antibody (1:62.5 dilution). Detection was made by the incubation with the HRP detection reagent at 37 C for 15 min, followed by the HRP stop buffer. The absorbance, corresponding to the level of RhoA activity (GTP bound RhoA), was measured on a microplate reader at 490 nm.

Measurement of activity of small GTPases by pull-down assay

Cells were treated with AM251 (500 nm), WIN55,212-2 (500 nm), and AM251+WIN55,212-2 at 37 C for 5 min. Cells were lysed in lysis buffer (25 mm Tris-HCl; 150 mm NaCl; 5 mm MgCl₂; 5% glycerol; 1% Nonidet P-40; 1 mm dithiothreitol, pH 7.5) supplemented with protease inhibitors. Activated RhoA was determined using pull-down assay kits from two suppliers (Pierce and Cytoskeleton). RhoA was separated on 10% SDS-PAGE Ready Gels and transferred to a nitrocellulose membrane. Blots of GTP-bound RhoA and total RhoA were incubated at 4 C overnight with anti-RhoA primary antibody (1:1000 dilution) followed by incubation with goat antimouse IgG (H+L) at room temperature for 1 h. Detection was made by enhanced chemiluminescence Western blotting kit and captured by Fuji film x-ray (Tokyo, Japan). Band intensities were analyzed using Image J software from the National Institutes of Health (Bethesda, MD) and normalized to the band intensities of the total RhoA.

To determine the effects of EC on RhoA activity, PC-3 cells were treated at 37 C with AEA (1 μm) and 2-AG (1 μm) for 5 min, JZL184 (an inhibitor of MGL, 10 μm) for 40 min to block the hydrolysis of endogenous 2-AG, and JZL184 (10 μm) for 40 min and then followed by 2-AG (1 μm) for 5 min. Activated RhoA was determined by pull-down assay and G-LISA as described above.

Activated Rac1 and activated Cdc42 were determined using pull-down activation assay kits similar to the protocol for the RhoA activity as described above.

Measurement of RhoA membrane translocation

PC-3 cells were treated with AM251 (500 nm) at 37 C at various times. In another set of experiments, cells were treated with vehicle control, (R)-()-[2,3-Dihydro-5-methyl-3-(4-mortpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (a CB agonist) (WIN55212-2; 500 nm), AM251 (500 nm), and a WIN55212-2 (500 nm) and AM251 (500 nm) at 37 C for 30 min. Then cells were added with ice-cold HEPES containing protease inhibitors for 5 min, scraped, pulled through a 27-gauge needle three times, and sonicated at a 20-sec burst for five times. The samples were centrifuged at 800 × g at 4 C for 15 min to remove particulates or debris. Then the supernatant was centrifuged at 150,000 × g at 4 C for 45 min, and the supernatant was separated as the cytosolic fraction. The pellets were dissolved in HEPES buffer and broken down by a glass rod and repeated pipetting and centrifuged at 800 × g at 4 C for 15 min to remove particulates from the membrane fractions. RhoA was separated on 10% SDS-PAGE Ready Gels and transferred to a nitrocellulose membrane, and total RhoA was incubated at 4 C overnight with anti-RhoA primary antibody (1:1000 dilution) as described above. The nitrocellulose membrane for the membrane fractions was then reprobed with Na,K-ATPase antibody (1:1000 dilution), and the nitrocellulose membrane for the cytosolic fractions was reprobed with LDH antibody (1:1000 dilution) for loading control.

Measurement of p-RhoA (Ser 188)

PC-3 cells were treated with WIN55,212-2 (500 nm) or AM251 (500 nm) at 37 C for different times. Cells were lysed in lysis buffer (25 mm Tris-HCl; 150 mm NaCl; 5 mm MgCl₂; 5% glycerol; 1% Nonidet P-40; 1 mm dithiothreitol, pH 7.5) supplemented with phosphatase inhibitors and protease inhibitors. The samples were centrifuged at 800 × g at 4 C for 15 min to remove particulates. Then the supernatant was used for Western blot analysis for p-RhoA (Ser 188) on 10% SDS-PAGE Ready Gels and transferred to a nitrocellulose membrane. The p-RhoA was incubated at 4 C overnight with anti-p-RhoA (Ser 188) primary antibody (1:2000 dilution), followed by the secondary antibody, and detected as described above. The nitrocellulose membrane was then reprobed with total RhoA antibody (1:1000 dilution) for loading control and normalization.

Cell motility measurements

Cell migration was determined by in vitro scratch assay as we previously described (12). In addition, a single cell migration assay (34) and migration through filter pores of the modified Boyden Chamber using X-celligence system (Roche Applied Science, Indianapolis, IN) to determine the effects of CB1 agonists and antagonists. For in vitro scratch assay, PC-3 and DU-145 cells (2 × 10⁶ cells) were plated in 35-mm dishes and cultured to 100% confluent monolayer. Cells were scraped to form a track. Cells were washed five times with serum-free media and treated with vehicle or pharmacological agents for 5 or 24 h. the images of the tracks were taken at 0 and 5 h or 24 h incubation at 37 C. Each treatment was performed in four dishes and repeated in three or four separate experiments. Migration was determined by the difference between the initial track widths (0 h) and the final track widths (5 or 24 h). Cell migration was reported as the percentage of the migration of the control cells.

Pharmacological agents were divided into two main study groups. Group 1 was synthetic CB1 agonists and antagonists: WIN55,212-2 (500 nm), NE (10 μm), AM251 (500 nm), NE+AM251, WIN55,212-2+AM251, and SR144528 (a selective CB2 antagonist, 500 nm). Group 2 was inhibitors of RhoA and Rho-associated kinase: AM251 (500 nm), C3 exoenzyme (1 μg/ml), Y-27632 (a Rho associated kinase inhibitor, 10 μm), C3 exoenzyme+AM251, and Y-27632+AM251.

For single-cell migration measurement, cells were transfected with green fluorescence protein (GFP) and allowed to migrate on gold colloids as previously described (34). For cell migration measurement by X-celligence system, cells (30,000 cells/well) were plated in the upper chamber (with 8.0 μm filter pores without Matrigel) of the Migration CIM-Plate of the RTCA DP Analyzer (Roche Applied Science) in the serum-free media. Media containing 10% fetal bovine serum was placed in the bottom chamber. Pharmacological agents were added in the upper chamber and the system was equilibrated for 30 min in the incubator at 37 C. Then cell migration was monitored and recorded every 15 min for up to 72 h. Four wells were used and averaged for each treatment.

Each treatment of migration measurements by the scratch assay was repeated with cells in the presence of mitomycin C (10 μm) to block the contribution of cell growth during the cell migration assay, or cells were stained with trypan blue for cell death visualization. These experiments were to determine that the observed change in migration was not from the cell growth or death.

Determination of 2-AG

Endogenous 2-AG concentrations in PC-3 cells in the absence and presence of JZL184 (10 μm) for 40 min was determined by liquid chromatography-electrospray ionization-mass spectrometry as previously described (9).

Statistical analysis

The means of the measured values of each treatment group were compared using a Student's t test (two tailed). Means were considered statistically different at P < 0.05.

Results

Effects of CB1 agonists and antagonists on cell motility

We previously reported that invasion of prostate carcinoma PC-3 and DU-145 cells is significantly diminished by activating the CB1 with different agonists, including the putative and enzymatically stable EC NE (a CB1 agonist) and the synthetic CB agonist WIN55212-2 (9). The cell invasion is also diminished by the inhibitors of enzymes hydrolyzing EC (9–12). In contrast, PC-3 cell invasion is increased by the selective CB1 antagonist AM251 (9). These results prompted us to investigate the ability of the CB1 to regulate cell migration in prostate carcinoma cells. Cells treated with the CB1 agonists NE and WIN55212-2 migrated into the scratched area significantly slower than the control cells. Interestingly, cells treated with the CB1 antagonist AM251 migrated into the scratched area significantly faster than the control cells (Fig. 1, A and D). Treatment of the cells with the selective CB2 antagonist SR144528 did not significantly affect the cell migration (Fig. 1D), indicating the greater role of CB1 than CB2 in these responses. At 5 h of treatment, the relative migration of PC-3 cells that were treated with CB1 agonists was lower than the control, whereas cells treated with a CB1 antagonist was higher (Fig. 1E). The results at 5 h were similar to the relative cell migration at 24 h of treatment (Fig. 1D). Treatment of cells with C3 exoenzyme to inhibit RhoA and Y27632 to inhibit Rho-associated kinases significantly inhibited cell migration (Fig. 1F). Experiments using DU-145 cells produced results similar to those of PC-3 cells (Fig. 1G).

Fig. 1.

Migration of PC-3 and DU-145 cells. A, Examples of migration of PC-3 cells by scratch assay of cells treated with vehicle, WIN55212-2 (500 nm), NE (an enzymatically stable putative EC for CB1,10 μm), AM251 (500 nm), WIN55212-2 (500 nm)+AM251 (500 nm), and NE (10 μm)+AM251 (500 nm) at 37 C. Images were taken at 0 and 24 h of treatment. B, Examples of single-cell migration of PC-3 cells-expressing GFP. PC-3 cells were treated with vehicle (upper panels) and NE (10 μm) (lower panels) at 37 C for 4 h. Left panels (i) are white-light images, and right panels (ii) are fluorescence images. Arrows indicate live GFP-expressing cells. C, Examples of migration of PC-3 cells determined by X-celligence at 37 C in an incubator. Cells in the upper chamber were treated with vehicle (d), WIN55212-2 (500 nm) (f), NE (10 μm) (e), AM251 (500 nm) (a), WIN55212-2 (500 nm)+AM251 (500 nm) (c), and NE (10 μm)+AM251 (500 nm) (d) during the detection of cells in the bottom chamber of the modified Boyden chamber (as described in the system protocol). Data are the average of four wells per treatment. D, Average migration of PC-3 cells treated with vehicle, NE (10 μm), WIN55212-2 (500 nm), NE (10 μm)+AM251 (500 nm), WIN55212-2 (500 nm)+AM251 (500 nm), AM251 (500 nm), and SR144528 (500 nm) at 37 C for 24 h. E, Average migration of PC-3 cells treated with vehicle, NE (10 μm), WIN55212-2 (500 nm), NE (10 μm)+AM251 (500 nm), WIN55212-2 (500 nm)+AM251 (500 nm), and AM251 (500 nm) at 37 C for 5 h. F, Average migration of PC-3 cells treated with vehicle, AM251 (500 nm), C3 exoenzyme (1 μg/ml), C3 exoenzyme (1 μg/ml)+AM251 (500 nm), Y-27632 (10 μm), and Y-27632 (10 μmol/liter)+AM251 (500 nm) at 37 C for 24 h. G, Average migration of DU-145 cells treated with vehicle, NE (10 μm), WIN55212-2 (500 nm), NE (10 μm)+AM251 (500 nm), WIN55212-2 (500 nm)+AM251 (500 nm), AM251 (500 nm), C3 exoenzyme (1 μg/ml), C3 exoenzyme (1 μg/ml)+AM251 (500 nm), Y-27632 (10 μm), and Y-27632 (10 um)+AM251 (500 nm) at 37 C for 24 h (n = 12–18). *, Significantly lower than control with P < 0.05; #, significantly higher than control with P < 0.05; %, significantly lower than AM251 treatment with P < 0.05.

The assay was repeated in the presence of mitomycin C to block cell proliferation. The relative responses of the cells to the CB1 agonists and antagonists were similar in the presence and absence of mitomycin C (data not shown). A single PC-3 cell migration was also examined as they migrated over colloidal gold coated with fibronectin (34). This analysis indicated that PC-3 cells treated with the CB1 agonist NE clearly migrate slower than the control cells (Fig. 1B). PC-3 cells treated with NE and WIN55212-2 slowed the migration of cells through the filter pores of the Boyden chamber by X-celligence, whereas AM251 promoted the cell migration as compared with the control cells (Fig. 1C). Taken together, these results indicate that CB1 activation by agonists diminishes, whereas the CB1 deactivation by antagonist enhances, the migration of PC-3 and DU-145 cells.

Direct inhibition of RhoA by C3 exoenzyme, as well as direct inhibition of Rho-associated kinases by Y27632, inhibits the migration of PC-3 cells (Fig. 1F). Interestingly, C3 exoenzyme or Y27632 also blocked the AM251-induced migration (Fig. 1F). These results indicate that the disruption of RhoA signaling pathways by either C3 exoenzyme or Y27632 diminishes the ability of PC-3 cells to migrate and diminishes the migratory response by the selective CB1 antagonist AM251. Again, the results for DU-145 cells (Fig. 1G) were similar to the results for PC-3 cells.

Cell morphology and actin-myosin microfilament organization

To determine the CB1 ability to regulate the organization of the actin/myosin cytoskeleton, the effects of CB agonists and antagonists on the myosin-containing microfilaments were examined (Fig. 2). Nontreated PC-3 cells exhibit a moderately spread morphology and detectable myosin-containing microfilaments, indicating an actin-myosin association in these cells (Fig. 2A). Activating the CB1 with the agonists WIN55,212-2 (Fig. 2B) or NE (Fig. 2C) causes the cells to become rounded and smaller, with diminished myosin-containing microfilaments, indicating diminished actin-myosin interactions. In contrast, inactivating the CB1 with the selective CB1 antagonist AM251 induces cell spreading and the formation of myosin-containing microfilaments (Fig. 2D), indicating increased actin-myosin interactions. AM251 also abolishes the inhibitory effects of WIN55212-2 on cell spreading and microfilament formation (Fig. 2, F and G). Treatment with the selective CB2 antagonist SR144528 did not induce significant changes in cell shape or microfilament formation (Fig. 2E), indicating that the CB2 is not a major participant in these responses. Direct inhibition of RhoA by the C3 exoenzyme also causes cell rounding and loss of myosin-containing microfilaments (Fig. 2H), similar to the responses of the cells treated with WIN55212-2 and NE (Fig. 2, B and C).

Fig. 2.

Cell morphology and actin-myosin microfilaments in PC-3 cells. Cells were grown on coverslips in complete media for 48 h and then in serum-free media for 24 h and treated with vehicle (A), WIN55212-2 (500 nm) (B), NE (10 μm) (C), AM251 (500 nm) (D), SR144528 (a selective CB2 antagonist, 500 nm) (E), WIN55212-2 (500 nm)+AM251 (500 nm) (F), NE (10 μm)+AM251 (500 nm) (G), and C3 exoenzyme (1 μg/ml) (H) at 37 C for 90 min. Cells were fixed and incubated with SA-2 human IgM antibody to the myosin heavy chain and FITC-labeled antihuman IgM antibody. Cell images were captured using a Nikon Eclipse E600 fluorescence microscope. Shown were examples of identical results from three separate experiments. Identical results were obtained in DU-145 cells.

Effects of CB1 agonists and antagonists on small GTPase RhoA activity

Our observation that activating the CB1 with either WIN55212-2 or NE induces the same morphological changes as inhibiting RhoA with C3 exoenzyme (Fig. 2, B, C, and H) is consistent with the hypothesis that CB1 activation inhibits RhoA activity. To directly test this possibility, changes in RhoA activity induced by CB1 activation and inactivation were first examined using the G-LISA, which provides a faster assay for a large number of samples.

As a control experiment, the LPA receptor, a known GPCR that regulates RhoA activity and promotes the migration of PC-3 cells (20), was examined. Consistent with previous reports, activating the LPA receptor by LPA in PC-3 cells significantly increases RhoA activity, with the maximal activation occurring within about 5 min and diminishing thereafter (Fig. 3A).

Fig. 3.

Activated RhoA (RhoA-GTP) in PC-3 cells as determined by G-LISA. A, PC-3 cells were incubated with LPA (10 μm) at 37 C from 0 to 30 min. B, PC-3 cells were incubated with AM251 (500 nm) at 37 C from 0 to 60 min. C, PC-3 cells were incubated with WIN55212-2 (500 nm) at 37 C from 0 to 30 min. D, PC-3 cells were incubated with AM251 (500 nm) and WIN55212-2 at various concentrations at 37 C for 5 min (n = 6–12). #, Significantly higher than control with P < 0.05; %, significantly lower than AM251 treatment with P < 0.05.

Activating the CB1 with the agonist WIN55212-2 significantly decreases RhoA activity, with the maximal inhibition occurring within about 5 min and being sustained for 30 min (Fig. 3C). In contrast, inactivating the CB1 with the antagonist AM251 significantly increases RhoA activity, with the maximal activation occurring within about 5 min and diminishing to the control level (Fig. 3B). This AM251-mediated increase in RhoA activity is diminished in the presence of increasing concentrations of WIN55,212 (Fig. 3D).

To confirm the results obtained using the G-LISA, a pull-down assay was used to examine the ability of the CB1 to regulate RhoA activity. Consistent with the results of the G-LISA, the pull-down assay indicated that activating CB1 with the agonist WIN55212-2 decreases RhoA activity, whereas inactivating CB1 with the antagonist AM251 increases RhoA activity (Fig. 4A). Furthermore, WIN55212-2 abolishes the increase in RhoA activity due to AM251 (Fig. 4A). Similar RhoA activity in response to WIN55212-2 and AM251 treatment occurred in DU-145 cells (Fig. 4B) and a lesser extent in LNCaP cells (Fig. 4C).

Fig. 4.

Activity of RhoA (RhoA-GTP) by CB1 agonists and antagonists. A, PC-3 cells were treated with vehicle (lane 1), WIN55212-2 (500 nm) (lane 2), WIN55212-2 (500 nm)+AM251 (500 nm) (lane 3), and (AM251 (500 nm) lane 4) at 37 C for 5 min. Example of immunoreactive bands of RhoA-GTP and total RhoA (above) and the average relative RhoA activity (the ratio of RhoA-GTP to total RhoA) from five separate experiments (below). B, DU-145 cells were treated with vehicle (lane 1), WIN55212-2 (500 nm) (lane 2), WIN55212-2 (500 nm)+AM251 (500 nm) (lane 3), and AM251 (500 nm) (lane 4) at 37 C for 5 min. An example of immunoreactive bands of RhoA-GTP and total RhoA (above) and the average relative RhoA activity (the ratio of RhoA-GTP to total RhoA) from four separate experiments (below) is shown. C, LNCaP cells were treated with vehicle (lane 1), WIN55212-2 (500 nm) (lane 2), WIN55212-2 (500 nm)+AM251 (500 nm) (lane 3), and AM251 (500 nm) (lane 4) at 37 C for 5 min. An example of immunoreactive bands of RhoA-GTP and total RhoA (above) and the average relative RhoA activity (the ratio of RhoA-GTP to total RhoA) from three separate experiments (below) is shown. *, Significantly lower than control with P < 0.05; #, significantly higher than control with P < 0.05; %, significantly lower than AM251 treatment with P < 0.05. D, Effects of EC AEA and 2-AG and inhibition of 2-AG hydrolysis by JZL184 on RhoA activity. Top panels are examples of immunoreactive bands of RhoA-GTP and total RhoA from PC-3 cells treated with vehicle (lane 1), AEA (1 μm, 5 min) (lane 2), JZL184 (10 μm, at 40 min pretreatment) (lane 3), 2-AG (1 μm, 5 min) (lane 4), and JZL184 for 40 min followed by 2-AG for 5 min (lane 5). Bottom panels are the relative RhoA activity for the same treatments as determined by G-LISA (n = 6). *, Significantly lower than control with P < 0.05; **, significantly lower than JZL184 treatment with P < 0.05.

Effects of endocannabinoids on small GTPase RhoA activity

To determine the effects of EC on RhoA activity, the ability of AEA and 2-AG to regulate RhoA activity was examined in PC-3 cells. PC-3 cells have undetectable concentrations of endogenous AEA (9) and extremely low expression and activity of fatty acid amide hydrolase (FAAH), which hydrolyzes AEA (12, 36). Exogenous AEA application inhibits RhoA activity (Fig. 4D) similarly to the synthetic agonist WIN55212-2 (Fig. 4A). On the other hand, the exogenous 2-AG application does not inhibit RhoA activity (Fig. 4D).

We previously demonstrated that PC-3 cells do not have hydrolysis activity for AEA, but they have high hydrolysis activity for 2-AG to AA and glycerol, which both arachidonic acid and glycerol cannot activate CB1 (11, 12). Inhibiting 2-AG hydrolysis using specific inhibitors increases endogenous 2-AG concentrations and diminishes invasion in PC-3 cells (10, 11). In this study, the application of 2-AG to PC-3 cells for 5 min does not significantly alter RhoA activity; however, JZL184, a specific inhibitor for MGL, the major enzyme hydrolyzing 2-AG, significantly diminishes RhoA activity (Fig. 4D). These results are consistent with the increase of 2-AG concentrations from 26.47 ± 3.74 ng/mg protein in control PC-3 cells to 66.21 ± 10.43 ng/mg protein in JZL184-treated cells. Treatment of PC-3 cells with both JZL184 and 2-AG produces the greatest loss of RhoA activity (Fig. 4D), which most likely occurs due to the protected 2-AG to enhance CB1 activation.

Effects of CB1 agonists and antagonists on small GTPase RhoA membrane translocation

PC-3 cells were treated with AM251 at various times and RhoA in membrane fractions was determined by Western blot analysis. Na,K-ATPase was used as a loading control. RhoA in membrane fractions increased at 15 min through 30 min and slightly decreased at 60 min (Fig. 5A). PC-3 cells were then treated with WIN55212-2 and AM251 for 15 min and RhoA in cytosolic fractions and membrane fractions were determined. LDH and Na,K-ATPase were used as loading control in each cellular fraction, respectively. WIN55212-2 blocked whereas AM251 enhanced the RhoA translocation to the membrane (Fig. 5B). These results indicate that the CB1 activation reduces and the CB1 inactivation enhances the RhoA translocation from the cytosol to the membrane.

Fig. 5.

Effects of CB1 agonists and antagonists on the RhoA membrane translocation. A, RhoA in membrane fractions of PC-3 cells treated with AM251 (500 nm) at 37 C for various times (an example of three separate experiments). Na,K-ATPase was used as a loading control. B, RhoA in cytosolic fractions and membrane fractions of PC-3 cells. Top panel, Examples of immunoreactive bands of RhoA in cytosolic and membrane fractions of PC-3 cells treated with vehicle control (lane 1), (WIN55212-2 (500 nm) (lane 2), AM251 (500 nm) (lane 3), and WIN55212-2 (500 nm)+AM251 (500 nm) (lane 4) at 37 C for 15 min. Also shown are bands for LDH and Na,K-ATPase in each fraction, respectively. Bottom panel, Average of RhoA band intensity in membrane fractions at 15 min treatment normalized to the Na,K-ATPase band intensity (n = 3).

Effects of CB1 agonist and antagonist on p-RhoA (Ser 188)

PC-3 cells were treated with WIN55212-2 (500 nm) or AM251 (500 nm) at 37 C for various times and p-RhoA (Ser 188) was determined by Western blot analysis. WIN55212-2 induced a rapid increase of p-RhoA (Ser 188) that reached a plateau after 15 min (Fig. 6A). On the other hand, AM251 reduced p-RhoA (Ser 188) as a function of time (Fig. 6B). These results indicate that the activation of CB1 increased Ser 188 phosphorylation of RhoA and the inactivation of CB1 diminished Ser 188 phosphorylation of RhoA.

Fig. 6.

Effects of CB1 agonist and antagonist on phosphorylated RhoA (Ser 188) (p-RhoA) and total RhoA (t-RhoA). A, PC-3 cells were treated with WIN55212-2 (500 nm) at 37 C for 0, 3, 5, 15, 30, and 60 min. An example of immunoreactive bands of p-RhoA and t-RhoA (left panel) is shown. Average of fold change of p-RhoA to t-RhoA ratio as normalized to the control (right panel). B, PC-3 cells were treated with AM251 (500 nm) at 37 C for 0, 3, 5, 15, 30, and 60 min. An example of immunoreactive bands of p-RhoA and t-RhoA (left panel) is shown. Average of fold change of p-RhoA to t-RhoA ratio as normalized to the control (right panel) is shown (n = 3).

Effects of CB1 agonists and antagonists on Rac1 and Cdc42 activity

Because CB1 activation diminishes RhoA activity (Figs. 3 and 4), CB1 activation might also diminish the activities of other Rho family members, such as Rac1 and Cdc42. Contrary to this expectation, activating the CB1 with the agonist WIN55212-2 modestly increases Rac1 and Cdc42 activity, whereas inactivating the CB1 with the antagonist AM251 decreases Rac1 and Cdc42 activity (Fig. 7, A and B). AM521 also diminishes the stimulatory effects of WIN55212-2 on Rac1 and Cdc42 activity (Fig. 7, A and B). Taken together, these results indicate that CB1 activation diminishes RhoA activity but modestly increases Rac1 and Cdc42 activity. Intriguingly, these different changes in RhoA, Rac1, and Cdc42 occur at about 5 min of CB1 activation, suggesting that the signals that induce these different changes in the small GTPases occur at relatively short and similar time after CB1 activation.

Fig. 7.

Activated Rac1 (Rac1-GTP) and activated Cdc42 (Cdc42-GTP) as determined by pull-down assay. A, PC-3 cells were treated with vehicle (lane 1), WIN55212-2 (500 nm) (lane 2), WIN55212-2 (500 nm)+AM251 (500 nm) (lane 3), and AM251 (500 nm) (lane 4) at 37 C for 5 min. An example of immunoreactive bands of Rac1-GTP and total Rac1 (above) and the average relative Rac1 activity (the ratio of Rac1-GTP to total Rac1) from three separate experiments (below) is shown. B, PC-3 cells were treated with vehicle (lane 1), WIN55212-2 (500 nm) (lane 2), WIN55212-2 (500 nm)+AM251 (500 nm) (lane 3), and AM251 (500 nm) (lane 4) at 37 C for 5 min. An example of immunoreactive bands of Cdc42-GTP and total Cdc42 (above) and the average relative Cdc42 activity (the ratio of Cdc42-GTP/total Cdc42) from three separate experiments (below) is shown. *, Significantly lower than control with P < 0.05; #, significantly higher than control with P < 0.05; %, significantly lower than WIN55212-2 treatment with P < 0.05. Identical results were obtained in DU-145 cells with identical treatments.

Discussion

This study identifies the CB1 and its endogenous agonists (EC) AEA and 2-AG as unique suppressors of RhoA activity in prostate carcinoma cells, which contributes to the endocannabinoid system-dependent suppression of migration by these cells. This conclusion is supported by our finding that the CB1 activation in these cells diminishes RhoA membrane translocation, diminishes RhoA activity, disrupts microfilament organization, induces cell rounding, and ultimately slows cell migration. Direct inhibition of RhoA with C3 exoenzyme in the prostate carcinoma cells mimics several of these effects of the CB1 activation. Taken together, these results indicate that the loss of RhoA activity on the CB1 activation provides a new and unique mechanism for the inhibitory effects of CB1 agonists on the migration and invasion of prostate carcinoma cells.

Interestingly, the effects of WIN55212-2 and AM251 on changes in RhoA activity in LNCaP cells were less than in PC-3 and DU-145 cells. These relative changes in RhoA activity are in agreement with previous observations that the effects of CB1 agonists and antagonists on cell migration and invasion were less in LNCaP than PC-3 and DU-145 cells (9, 12). Again, the higher expression and activity of enzymes hydrolyzing EC such as FAAH in LNCaP cells could contribute to the hydrolysis of EC such as 2-AG and the lower CB1 activation in these cells (12).

EC produced by prostate carcinoma cells may keep the CB1 chronically activated, resulting in chronic repression of RhoA activity and controlled cell migration. This possibility is supported by our finding that competing with EC for the CB1 by the selective CB1 antagonist AM251 rapidly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes the cell migration. It is currently unclear whether AEA or 2-AG plays a greater role in this process. PC-3 cells express undetectable levels of endogenous AEA and very low FAAH expression and activity of AEA hydrolysis (9, 12); thus, exogenous AEA applied to PC-3 cells inhibits RhoA activity (Fig. 4D). Due to the very low endogenously produced AEA concentration, this EC might not act as the major autocrine suppressor of RhoA activity in PC-3 cells. In contrast, 2-AG is abundantly produced in PC-3 cells (9, 10); however, 2-AG is hydrolyzed to AA and glycerol, resulting in reduced CB1 activation (11, 12). Exogenously added 2-AG does not significantly inhibit RhoA activity (Fig. 4D). Strong evidence suggests that the binding sites of CB1 are located in the transmembrane domains (37–41), and our studies indicated that PC-3 cells have high 2-AG hydrolysis activity, even in the membrane fractions (11, 12). It is likely that exogenously added 2-AG has less opportunity to activate CB1 due to its rapid hydrolysis in the cell membranes (11). The endogenously produced 2-AG probably has to go through this similar event to compete with the binding to the CB1. The possibility that endogenous 2-AG can activate the CB1 is supported by our finding that inhibiting the hydrolysis of endogenous 2-AG with JZL184 increases 2-AG concentration and diminishes RhoA activity, similar to the effects of activating the CB1 with other agonists such as WIN22512–2 (Figs. 3 and 4). These results support the model that EC activate CB1 in an autocrine fashion to repress RhoA activity and suppress cell migration in prostate carcinoma cells (Fig. 8). The results also indicate the importance and complexity of the endocannabinoid system (including the CB receptors, EC, and enzymes that affect the balance of endogenous concentrations of EC) as the endogenous regulators of carcinoma cells.

Fig. 8.

A simplified diagram depicting the proposed signaling pathway of the CB1 activation that inhibits the activity of small GTPase RhoA to inhibit motility of PC-3 cells. PC-3 cells have undetectable endogenous AEA and very low expression of FAAH, a membrane enzyme that hydrolyzes AEA and 2-AG (12, 36). However, PC-3 cells have high specific hydrolysis activity for 2-AG (11, 12) in the membrane fractions (12). The binding sites of CB1 have been suggested to be in the transmembrane domains (37–41). Thus, the inhibition of enzymes hydrolyzing EC is important for an increase of EC concentrations, the CB1 activation and the activity of CB1 signaling pathway(s) to inhibit cell motility.

Our findings identify the CB1 as a unique inhibitory GPCR that diminishes RhoA activity on the receptor activation. This inhibitory property distinguishes the CB1 from other GPCR. Most known GPCR generally increase RhoA activity on receptor activation (20, 23–25). The CB1 activation inhibits the activity of RhoA but modestly stimulates the activities of Rac1 and Cdc42. These results support previous studies indicating that RhoA is the major promoter of motility of prostate carcinoma cells. These results are also similar to previous reports indicating that diminished RhoA activity is often accompanied by increased Rac1 activity in a variety of cell types (42–44). For example, inactivation of RhoA or Rho-associated kinase in rat mammary carcinoma cells stimulates Rac1 activity and enhances cell protrusion but diminishes cell motility (45). The mechanisms that are responsible for this cross talk between RhoA and Rac1 currently have not been clearly defined.

There are multiple kinases and regulatory proteins that could participate in the specific inhibition of RhoA on CB1 activation. For example, Rho GDP-dissociation inhibitor, which is a regulatory protein that sequesters RhoA, Rac1, and Cdc42 in the inactive GDP-bound forms (46), is one of the candidates that might participate in CB1-dependent signaling pathways. Phosphorylation of Ser 188 in the C terminus of RhoA promotes the interaction of RhoA with Rho GDP-dissociation inhibitor (46) and diminishes RhoA activity (29, 47–50). We found that WIN55212-2 increases p-RhoA at Ser 188 (a putative inhibitory site), whereas AM251 decreases p-RhoA (Ser 188). These data suggest that CB1 activation induces phosphorylation of RhoA and represents a pathway for the CB1 regulation of RhoA activity. However, the kinases and/or other signaling events that cause CB1-mediated changes in the activity of the Rho family GTPases are currently not known and warrant further investigation.

In conclusion, these findings indicate that CB1 activation diminishes the activity of RhoA, but not Rac1 or Cdc42, as a signaling pathway that regulates motility in prostate carcinoma cells. The signaling events that cause CB1-mediated changes in the activity of Rho family GTPases warrant further investigation. This loss of RhoA activity after CB1 activation is accompanied by the loss of actin/myosin microfilaments, diminished cell adhesion, and reduced cell migration. This study defines the CB1 as a unique GPCR that represses RhoA activity, providing a mechanism to diminish the motility of prostate carcinoma cells. These findings also suggest therapeutic implications of a combination of multiple therapeutic targets of the endocannabinoid system and RhoA signaling pathway in prostate cancer.