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. 2011 Jan;162(1):84–93. doi: 10.1111/j.1476-5381.2010.01047.x

Rimonabant reduces keratinocyte viability by induction of apoptosis and exerts topical anti-inflammatory activity in mice

Anna Maria Malfitano 1, Silvio Sosa 2, Chiara Laezza 3, Marco De Bortoli 2, Aurelia Tubaro 2, Maurizio Bifulco 1
PMCID: PMC3012408  PMID: 20880029

Abstract

BACKGROUND AND PURPOSE

There is growing evidence that the cannabinoid CB1 receptor antagonist, rimonabant (SR141716) exerts potential anti-proliferative and anti-inflammatory actions. Here, we have assessed the effects of rimonabant in vitro in murine immortalized keratinocytes and in vivo by assaying the topical anti-inflammatory activity.

EXPERIMENTAL APPROACH

Cell viability and death in a keratinocyte cell line (C5N cells) were measured by Trypan blue exclusion assay and cytotoxicity by sulphorhodamine B test. Cell cycle progression was assessed by flow cytometry and the expression of apoptotic and anti-apoptotic markers, cyclins, pathways of signal transduction and CB1 receptor levels were evaluated by Western blot. The topical anti-inflammatory properties of rimonabant were analysed by inhibition of croton oil-induced ear dermatitis in mice.

KEY RESULTS

Rimonabant reduced cell viability and induced apoptosis as shown by the enhanced number of cells in the subG0 phase of the cell cycle, the expression of Bax and reduced levels of Bcl-2 and X-inhibitor of apoptosis protein. In addition, reduced levels of phosphorylated serine/threonine protein kinase Akt and nuclear factor-kappa B were detected associated with regulation of total nuclear factor-kappa B and inhibitor of kappa B-α, phosphorylated inhibitor of kappa B-α, cyclins D1, E and A. In croton oil-induced ear dermatitis, rimonabant significantly reduced oedema and leukocyte infiltrate.

CONCLUSIONS AND IMPLICATIONS

Rimonabant reduced cell viability, inducing cell death in keratinocytes and decreased croton oil-induced ear dermatitis. Our findings suggest a potential application of rimonabant as a topical anti-inflammatory drug. We did not assess the involvement of CB1 receptors in these effects of rimonabant.

Keywords: cannabinoid receptor CB1, CB1 antagonist, keratinocytes, cell viability, apoptosis, dermatitis, oedema, leukocyte infiltrate

Introduction

Rimonabant (SR141716) [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4methyl pyrazole-3-carboxamide] is a potent and selective cannabinoid CB1 receptor antagonist (receptor nomenclature follows Alexander et al., 2009), inhibiting food intake and exhibiting anti-obesity activity (Rinaldi-Carmona et al., 1994; 1995; Colombo et al., 1998; Simiand et al., 1998; Ravinet Trillou et al., 2003; Carai et al., 2005; Jbilo et al., 2005). It is widely used as a tool to investigate mechanisms by which cannabinoid agonists produce their pharmacological effects and may exert several of its actions by blocking the activation of CB1 receptors, thus modulating the endocannabinoid system, which is tonically activated under certain pathophysiological conditions (Di Marzo and Matias, 2005; Engeli et al., 2005; Matias et al., 2006; Jhaveri et al., 2007;Pertwee, 2009). Anti-tumour properties have been attributed to rimonabant by our group in several cancer cell lines in vitro and in vivo (Sarnataro et al., 2006; Santoro et al., 2008; 2009; Gazzerro et al., 2010). Nonetheless, we very recently provided evidence that rimonabant increases human sperm motility and viability (Aquila et al., 2010). Numerous findings suggested the role of the endocannabinoid system in the control of immunity, reporting the involvement of cannabinoid receptors in immune cell migration (Miller and Stella, 2008) and the efficacy of cannabinoid agonists as antinociceptive and anti-inflammatory agents (Guindon and Hohmann, 2008). We recently demonstrated the immunomodulatory and anti-inflammatory effects of rimonabant on peripheral blood mononuclear cells, showing inhibition of proliferation without induction of apoptosis, block of the G1/S phase of the cell cycle and modulation of the signalling kinases serine/threonine protein kinase Akt (Akt) and the extracellular signal regulated kinase, the transcription factor nuclear factor-kappa B (NF-κB) and its inhibitor (IκB) and the inducible enzymes cyclooxygenase-2 and inducible NO synthase (Malfitano et al., 2008). In rats and mice, rimonabant prevents the rise in tumour necrosis factor-α (TNF-α) serum levels induced by Escherichia coli lipopolysaccharide and relieves neuropathic pain (Smith et al., 2000; Costa et al., 2005).

Other findings suggest a variety of mechanisms of action of rimonabant in the modulation of the inflammatory process. Oral administration of rimonabant dose-dependently prevented indomethacin-induced small intestinal ulcers in rats and inhibited TNF-α levels. However, in CB1 receptor knockout mice, protection against ulcers was lost but TNF-α levels were still inhibited (Croci et al., 2003). Furthermore, rimonabant reduced joint inflammation in a chronic model of adjuvant-induced unilateral arthritis in obese rats and inhibited thermal and mechanical hyperalgesia (Croci and Zarini, 2007). Recent intriguing findings have identified the functional existence of various members of the endocannabinoid system in human and murine skin (Maccarrone et al., 2003; Ibrahim et al., 2005; Ständer et al., 2005). Cannabinoids suppressed in vitro proliferation of cultured epidermal keratinocytes (Ibrahim et al., 2005; Paradisi et al., 2008). In addition, using mice with the double CB1/CB2 receptor knockout, Karsak et al. (2007) elegantly demonstrated that endocannabinoids attenuated allergic contact dermatitis.

The aim of this study was to assess the effects of rimonabant in vitro in murine immortalized keratinocytes and in vivo to evaluate its topical anti-inflammatory activity, using an acute model of inflammation. Our results showed reduced cell viability and induction of apoptosis accompanied by regulation of phosphorylated protein kinase Akt (pAkT), NF-κB (pNF-κB) IκBα (pIκBα) and cyclins D1, E and A. In addition, rimonabant up-regulated the expression of CB1 receptors and in vivo, inhibited croton oil-induced ear dermatitis in mice, reducing oedema and leukocyte infiltrate.

Methods

Cell growth and culture

C5N, a murine immortalized non-tumorigenic keratinocyte cell line (Portella et al., 1998), supplied by Professor G. Portella (Federico II University of Naples) was used in this study. C5N cells were grown at 37°C in a humidified atmosphere of 5% CO2 in Dubelco's Modified Eagle Medium (DMEM) culture medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and glutamine (2 mM). Cells were starved by serum deprivation overnight before each assay.

Cell viability assay

Exponentially proliferating cells (1 × 105) were seeded in 24-well plates. Increasing concentrations of rimonabant (0.3 µM, 1 µM, 3 µM, 10 µM) were added to the culture. After 24 h and 48 h of incubation, cells were harvested, stained with Trypan blue (0.5% solution; Sigma) and counted by haemocytometer.

Cytotoxicity assay

C5N cells (1 × 105) were incubated 24 h in 96-well plates at the concentrations of rimonabant shown above. Cell monolayers were fixed with 10% (wt·vol−1) trichloroacetic acid, stained for 30 min with sulphorhodamine B (0,4% in acetic acid) and then the excess dye was removed by washing repeatedly with 1% (vol vol−1) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density determination at 495 nm using a microplate reader.

Cell cycle analysis

C5N cells (105) were cultured in the presence and in the absence of rimonabant at the concentrations shown above, in DMEM 10% FBS for 24 h and 48 h at 37°C in 24-well plates. To analyse cell cycle progression, cells starved overnight in DMEM medium in the absence of FBS were collected, washed twice with phosphate-buffered saline (PBS) and re-suspended in 300 µL of PBS; 700 µL of 70% ethanol were added slowly to the cells on a vortex mixer and kept at −20°C for 1 h. Propidium iodide (PI; 10 µg·mL−1) in PBS containing 100 U·mL−1 DNase-free RNase was added to the cells; after 15 min at room temperature, cells were subjected to flow cytometric analysis using Summit v4.3 program. Each sample was analysed using 10 000 events corrected for debris and aggregate populations.

Electrophoresis and immunoblots

Cell extracts were prepared from C5N cells. Cells were treated with rimonabant at 0.3 µM, 1 µM, 3 µM and 10 µM. After 24 h and 48 h of incubation, cells were washed twice with PBS, re-suspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 50 mM EDTA, 100 mM NaF, 2 mM Na orthovanadate, glycerol, 10 mM Na4P2O7 and 10% Triton at pH 7.5) and passed through a 23-gauge needle, 10 times before centrifugation at 12 000 g at 4°C. Supernatants were collected and the protein concentration evaluated by the Bradford method (Bradford, 1976). Equal amounts of protein extracts (50 µg) were boiled in sample buffer and analysed by electrophoresis in 12% sodium dodecylsulphate polyacrylamide gel, separated proteins were transferred to nitrocellulose membranes (Amersham Hybond ECL, Amersham GE Healthcare, Buckinghamshire, UK) (8 V for 1 h). The blots were blocked in PBS containing 0.1% Tween-20 and 5% non-fat dry milk for 1 h at room temperature. The membrane was then probed overnight with primary antibodies (1:1000) (host species : rabbit) specific for NF-κB p65 (Cell Signaling Technology Inc., Danvers, MA, USA) and its phosphorylated form, pNF-κB p65 (Cell signalling), IκBα (Cell signalling), pIκBα (Cell signalling), Bax (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), Bcl-2 (Santa Cruz Biotechnology Inc.), the X-inhibitor of apoptosis protein (XIAP) (BD Transduction Laboratories, Franklin Lakes, NJ, USA), pAkt (Cell signalling), Akt (Cell signalling), cyclin (Cyc) D1 (Santa Cruz Biotechnology Inc.), Cyc E (Santa Cruz Biotechnology Inc.), Cyc A (Santa Cruz Biotechnology Inc.) and CB1 receptors (Santa Cruz Biotechnology Inc.). Immunodetection of specific proteins was carried out with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad, Life Science Research, Hercules, CA, USA), using the enhanced chemiluminescence system (Amersham GE Healthcare). Actin (Santa Cruz Biotechnology Inc., anti-rabbit) as control was used to normalize.

Topical anti-inflammatory activity in vivo

Croton oil induced ear dermatitis

All animal care and experimental procedures complied with the Italian D.L. no. 116 of January 27, 1992 and associated guidelines in the European Communities Council Directive of November 24, 1986 (86/609 ECC). Male CD-1 mice (Harlan Italy; S. Pietro al Natisone, Italy) were anaesthetized by intraperitoneal injection of ketamine hydrochloride (145 mg·kg−1). Skin inflammation was induced on their right ear (surface: about 1 cm2) applying 80 µg of croton oil dissolved in 15 µL of acetone. The left ear remained untreated as preliminary experiments showed that the vehicle did not affect the inflammatory response or induce irritation. Control mice received only the irritant while other mice received the irritant and rimonabant or the non-steroidal anti-inflammatory drug (NSAID) indomethacin, used as reference. Mice were killed by cervical dislocation 6 h or 24 h after the induction of inflammation, at the times of maximum oedema response and leukocyte infiltrate in control mice respectively. Disks (6 mm diameter) were punched out of the ear to quantify the oedematous response and leukocyte infiltrate (Tubaro et al., 1985). A total of 10 animals were used for each group of treatment.

Evaluation of the oedematous response

Oedema was quantified by the difference in weight between the samples taken from the right treated and left untreated ears. The anti-oedema activity was expressed as percent inhibition of oedema in mice treated with the test substances with respect to oedema of animals treated with the irritant alone (Tubaro et al., 1985).

Evaluation of the granulocyte infiltrate

The cellular infiltrate was quantified in the treated ears measuring the myeloperoxidase activity, as index of the presence of neutrophilic granulocytes, in the same ear samples as used to determine oedema (Tubaro et al., 1985). Myeloperoxidase was extracted by hexadecyltrimehylammonium bromide (HTAB; Sigma-Aldrich, Milan, Italy), according to Bradley et al. (1982), and the enzyme activity was measured by a colorimetric assay using tetramethylbenzidine (TMB; Sigma-Aldrich, Milan, Italy) as chromogen (Andrews and Krinsky, 1981). Each ear sample, suspended in 1 mL buffered saline (0.1 M sodium acetate buffer at pH 4.2), containing 0.1% HTAB (wt v−1), was homogenized by Ultra-Turrax (Ika-Werk, Staufen, Germany) for 5 s at 20 000 r.p.m. The homogenate was centrifuged at 15 000 g for 5 min, and the supernatant was used for the colorimetric assay, because preliminary experiments revealed that the pellet contained less than 5% of total myeloperoxidase activity. In each well of a 96-well microplate, 25 µL of the supernatant was mixed with 50 µL of the chromogen solution [2.83 mM TMB dissolved in 0.1 M sodium acetate buffer at pH 4.2, containing 0.1% (wt v−1) HTAB]. The enzyme reaction was started by adding 75 µL of 0.7 mM hydrogen peroxide. After 5 min of incubation at 25°C, the reaction was blocked by 50 µL of 4 M acetic acid, containing 10 nM sodium azide. The absorbance was read at 620 nm using an automated microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Myeloperoxidase activity was expressed as enzyme units in 1 mL of supernatant. One unit of peroxidase activity was defined as the amount of enzyme oxidizing 1 nmol of TMB min−1. The enzyme activity of each sample was determined in duplicate.

Statistical analysis

Results are expressed as means ± SD and were analysed by Student's t-test; P values less than 0.05 were considered to be statistically significant. In vivo data were analysed by one-way analysis of variance followed by Dunnett's test for multiple comparisons of unpaired data, and a probability level lower than 0.05 was considered as significant. The dose giving 50% inhibition of the oedematous response (ID50) was calculated by graphic interpolation of the logarithmic dose–effect curves.

Materials

Rimonabant [N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-pyrazole-3-carboxamide] was kindly provided by Sanofi-Aventis (Montpellier, France) and was dissolved in dimethylsulfoxide. The vehicle did not induce any positive result in any assay. Croton oil and indomethacin were purchased from Sigma Aldrich (Milan, Italy), while ketamine hydrochloride (Inoketam100) was from Virbac (Milan, Italy).

Results

Rimonabant reduces C5N cell viability

C5N cells were cultured in the presence and in the absence of rimonabant at increasing concentrations ranging from 0.3 µM to 10 µM. Cell survival was determined after 24 h and 48 h of treatments by Trypan blue staining and cells were counted by haemocytometer. Results obtained showed that rimonabant caused decrease of cell viability after 24 h treatment (Figure 1A). The inhibitory effect was maintained after 48 h of treatment only at the highest concentration used (Figure 1B).

Figure 1.

Figure 1

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Rimonabant reduces C5N cell viability. C5N cells cultured in the presence and in the absence of rimonabant (SR), at the concentrations shown, were collected after 24 h (A) and 48 h (B) of treatment, stained by Trypan blue and counted by haemocytometer. Data for control (CTR) represent the effects of the vehicle. The histograms shown representative of four experiments. Data are means ± SD; *P < 0.05, significantly different from control values.

Cytotoxic effects of rimonabant in C5N cells

In order to assess cell cytotoxicity following rimonabant treatment, we performed sulphorhodamine B staining. Cells were cultured in triplicates at increasing concentrations ranging from 0.3 µM to 10 µM of rimonabant and collected after 24 h of incubation, as at this time point the reduced cell viability reached its maximum. We found decreased cell survival at the highest concentrations used (Figure 2).

Figure 2.

Figure 2

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Cytoxicity of rimonabant. C5N cells incubated with rimonabant, at the concentrations shown, were collected after 24 h treatment and stained by sulphorhodamine B. The optical density (OD) at 495 nm was determined by a microplate reader. The histogram shown is representative of three independent experiments. Data are means ± SD; *P < 0.05, significantly different from control values.

Rimonabant effects on cell cycle progression

To better characterize the inhibition of cell viability by rimonabant and correlate this effect with cell cycle progression, we analysed cell cycle distribution by flow cytometry. C5N cells were cultured in DMEM + 10%FBS and treated with rimonabant at increasing concentrations ranging from 0.3 µM to 10 µM. Rimonabant enhanced the number of cells in the subG0 phase of the cell cycle in a concentration-dependent manner after 24 h of treatment (Figure 3).

Figure 3.

Figure 3

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Rimonabant increased the proportion of cells in the subG0 phase of the cell cycle. C5N cells cultured in the presence and in absence of rimonabant (SR) at the concentration showed in the histograms, were collected after 24 h of incubation, stained with propidium iodide and analysed by flow cytometry. The cell cycle profile of a single experiment representative of three is reported in the figure. The per cent of cells in each phase of the cell cycle is shown for each histogram and represent the mean of three independent experiments.

Rimonabant effects on the expression levels of NF-κBp65, pNF-κBp65, IκBα, pIκBα, Bax, Bcl-2, XIAP, pAkt, Akt, Cyc D1, Cyc E, Cyc A and CB1 receptors

In order to assess potential pathways involved in the effects of rimonabant, we analysed in cell extracts, the expression levels of NF-κBp65, pNF-κBp65, IκBα, pIκBα, pAkt and Akt. We found a reduction of the expression levels of pAkt and pNF-κBp65 following rimonabant treatment, together with no changes of total Akt, enhanced expression of both total NF-κBp65 and IκBα and pIκBα after 24 h treatment and their reduced expression after 48 h at the highest concentrations (Figure 4; Table 1).

Figure 4.

Figure 4

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Effects of rimonabant on the expression levels of NF-kBp65, pNF-kBp65, IκBα, pIκBα, Bax, Bcl-2, xIAP, pAkt, Akt, Cyc D1, Cyc E, Cyc A and CB1 receptors in C5N cells. Cell extracts obtained from cells cultured in the presence and in absence of rimonabant at 24 h and 48 h treatment, were analysed by Western blotting. The concentrations of rimonabant used are shown above the experimental records. A representative blot for NF-kBp65, pNF-kBp65, IκBα, pIκBα, Bax, Bcl-2, XIAP, pAkt, Akt, cyclins D1, E and A (Cyc D1, E and A) and CB1 receptors (CB1) is shown in the figure. Summary data from these experiments are shown in Table 1. IκBα, inhibitor of nuclear factor kappa B alpha; NF-κB, nuclear factor kappa B, pNF-κB, phosphorylated NF-κB; pIκBα, phosphorylated IκBα; XIAP, X-inhibitor of apoptosis protein.

Table 1.

Effects of rimonabant (SR; 0.3–10 µM) on NF-κB, components of apoptosis, cyclins (Cyc) and CB1 receptor (CB1) proteins in C5N keratinocytes

24 h 48 h
ctr SR 0.3 SR 1 SR 3 SR 10 ctr SR 0.3 SR 1 SR 3 SR 10
NF-κB 1.2 1 2* 2* 4* 4 4 1* 0.5* 0*
pNF-κB 1 0.8* 0.8* 0.5* 0.5* 0.8 0.4 0.5* 0.25* 0*
IκBα 0.5 1 1.5* 2* 2* 3 2.8* 3.3* 3* 0.01*
pIκBα 0.5 0.5 0.6* 1* 1* 0.5 0.5 0.3* 0* 0*
Bax 1 2 2* 4* 4* 0.7 4 4* 4* 4*
Bcl-2 0.95 1 1* 1* 0.1* 1.5 1.3 0.5* 0.25* 0.1*
XIAP 1 1 1* 1.2* 0.95* 0.85 0.8 0.3* 0.35* 0.2*
pAkt 2 1.5 1* 0.5* 0.57* 2 0.8 1* 0.4* 0.01*
Akt 2 2 2* 2* 2* 2 2 2* 2* 2*
Cyc D1 1 2 3* 3* 3* 2 1 1* 0.5* 0*
Cyc E 0 0.5 0.5* 0.6* 1* 0.9 1.5 1.2* 0.5* 0.2*
Cyc A 0.5 0.5 0.6* 0.75* 0.75* 0.8 1 0.9* 0* 0*
CB1 0.5 1 1.5* 2* 1* 0.5 0.95 2* 1.5* 0.6*
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Cell extracts were analysed by Western blotting (as illustrated in Figure 4) after 24 h and also after 48 h incubation with rimonabant. Immunoreactive bands were quantified with the Quantity One programme. The table shows quantification of the intensity of bands (as means; n= 3), normalized to the intensity of the actin bands.

*

P < 0.05,

significantly different from corresponding control (ctr); Student's t-test.

IκBα, inhibitor of nuclear factor kappa B alpha; NF-κB, nuclear factor kappa B, pNF-κB, phosphorylated NF-κB; pIκBα, phosphorylated IκBα; XIAP, X-inhibitor of apoptosis protein.

To better characterize the effects of rimonabant on cell death we investigated the expression levels of the apoptotic cell marker, Bax and anti-apoptotic Bcl-2 and XIAP. We found enhanced expression of Bax after 24 h and 48 h treatment and reduced expression of Bcl-2 after 24 h at 10 µM more pronounced after 48 h at the highest concentrations. Reduced levels of XIAP were detected after 48 h (Figure 4; Table 1). As rimonabant modified cell cycle progression, we investigated the expression levels of various cell cycle regulatory molecules, including cyclin D1, cyclin E and cyclin A. As shown in Figure 4, treatment of cells with rimonabant increased the expression of these cyclins after 24 h in comparison to untreated cells (Table 1) but after 48 h of treatment the protein expression of cyclin D1, cyclin E and cyclin A was reduced in a dose-dependent manner.

Furthermore, we investigated potential modification of the CB1 receptor, that is expressed in C5N cells as detected in the control (Figure 4), following treatment with the CB1 receptor antagonist. Interestingly, we found an induction of the CB1 receptor at all time points and concentrations of rimonabant used with respect to the untreated control cells. This effect was not maintained at 10 µM after 48 h treatment (Figure 4; Table 1).

Topical anti-inflammatory actions of rimonabant in vivo

The topical anti-inflammatory activity of rimonabant was evaluated as inhibition of the croton oil-induced ear dermatitis in mice in comparison with that of the NSAID indomethacin. After 6 h, when oedema formation in control mice is maximum (Tubaro et al., 1985), rimonabant induced a dose-dependent reduction of oedema (Table 2). At the same doses, indomethacin also reduced the oedematous response. The anti-oedema potency of the two compounds was assessed from the dose–response curves, calculating the relevant ID50 values. The ID50 of rimonabant was 0.34 µmol·cm−2, showing a potency comparable to that of the reference NSAID indomethacin (ID50= 0.26 µmol·cm−2) (Table 2). The anti-inflammatory activity of an equimolar dose of rimonabant and indomethacin (0.3 µmol·cm−2) was further evaluated on both oedema and leukocyte infiltrate, at the times of their maximum in control mice (6 h and 24 h respectively) (Tubaro et al., 1985). Six hours and 24 h after induction of inflammation, rimonabant reduced oedema and indomethacin exerted a similar effect after 6 h but no effect after 24 h (Table 3). Rimonabant also significantly reduced also the recruitment of neutrophilic granulocytes in the ear tissue: at 6 h and 24 h (Table 4). The reference compound, indomethacin, exerted a comparable effect at 6 h and 24 h after the dermatitis induction (Table 4).

Table 2.

Dose-dependent anti-oedema activity of rimonabant and indomethacin in croton oil-induced ear dermatitis after 6 h

Substance Number of mice Dose (µmol·cm−2) Oedema (mg) Mean ± SD % reduction ID50 (µmol·cm−2)
Controls 10 – 7.0 ± 1.0 – –
Rimonabant 10 0.1 5.3 ± 0.6* 24 0.34
10 0.3 3.4 ± 0.6** 51
10 1.0 2.1 ± 0.7** 70
Indomethacin 10 0.1 5.2 ± 0.4* 26 0.26
10 0.3 3.0 ± 0.4** 57
10 1.0 1.4 ± 0.3** 80
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*

P < 0.005,

**

P < 0.001, significantly different from control values; one-way anova.

Table 3.

Effect of rimonabant and indomethacin on the oedematous response in croton oil-induced ear dermatitis after 6 h and 24 h

6 h 24 h
Substance Number of mice Dose (µmol cm2-1) Oedema (mg) mean ± SD % reduction Oedema (mg) mean ± SD % reduction
Controls 10 – 7.0 ± 0.8 – 2.3 ± 0.6 –
Rimonabant 10 0.3 3.0 ± 0.6** 57 1.2 ± 0.4* 48
Indomethacin 10 0.3 2.7 ± 0.9** 61 2.2 ± 0.6 4
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*

P < 0.005,

**

P < 0.001, significantly different from control values; one-way anova.

Table 4.

Effect of rimonabant and indomethacin on the leukocyte infiltrate in croton oil-induced ear dermatitis after 6 h and 24 h

6 h 24 h
Substance Number of mice Dose (µmol·cm−2) MPO (EU) mean ± SD % reduction MPO (EU) mean ± SD % reduction
Controls 10 – 18.6 ± 1.9 – 30.5 ± 4.5 –
Rimonabant 10 0.3 13.0 ± 1.8* 30 22.2 ± 2.1* 27
Indomethacin 10 0.3 11.7 ± 1.4* 37 20.6 ± 4.7* 32
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*

P < 0.005,

significantly different from control values; one-way anova.

MPO, myeloperoxidase; EU, enzyme units (nmol tetramethylbenzidine hydrolysed min−1).

Discussion and conclusions

In this study we assessed the effects of the CB1 receptor antagonist, rimonabant (Rinaldi-Carmona et al., 1994) in vitro, in a murine-immortalized keratinocyte cell line and in vivo, in a model of skin inflammation to evaluate potential anti-inflammatory properties of this drug related to epidermal dysfunction. We observed that rimonabant reduced C5N cell viability and induced apoptosis as showed by the enhanced level of Bax, reduction of Bcl-2 and XIAP and the increased number of cells in the subG0 phase of the cell cycle. Previous reports had demonstrated that cannabinoids inhibited proliferation of epidermal keratinocytes (Ibrahim et al., 2005; Wilkinson and Williamson, 2007; Paradisi et al., 2008), hair matrix keratinocytes (Telek et al., 2007; Dobrosi et al., 2008) and induced intraepithelial apoptosis and premature regression of hair follicles. A fundamental role of NF-κB has been described in apoptosis and inhibition of the NF-κB action in TNFα-stimulated keratinocytes showed significant apoptosis (Banno et al., 2005). In addition, NF-κB blocked apoptosis by induction of anti-apoptotic genes, such as the Bcl-2 homologs and IAPs (Hu et al., 1998; Zong et al., 1999; Chen et al., 2000). In agreement with this finding, we detected down-regulation of XIAP, reduction of pNF-κBp65, associated with enhanced expression of pIκBα, total NF-κB and IκB at early time points and reduced levels at late time points likely due to degradation effects at the highest concentration of rimonabant. Evidence showing inhibition of pAkt related to induction of apoptotic pathways (Kumar et al., 2009), support our finding demonstrating that rimonabant down-regulates pAkt while no effect was observed on total Akt. Cyclin D1 has been described as a direct NF-κB target gene in several cell types (Hinz et al., 1999), TNF-α increased the binding of NF-κB proteins to the κB site on the cyclin D1 promoter (Zhang et al., 2007), indeed NF-κB activation was essential for induction of cyclin D1 (Schmidt-Ullrich et al., 2006). We detected concomitant accumulation of NF-κB and induction of cyclin D1, after 48 h at the highest concentration of rimonabant. The decreased levels of NF-κB were accompanied by reduced expression of cyclin D1, similar effects we observed in the levels of cyclin A and E. Interestingly, we found that rimonabant enhanced considerably the levels of CB1 receptors and that this effect was lost at the highest concentration after 48 h of treatment, probably due to the longer time of incubation of the antagonist and sequestration of its receptor. However, even if we found up-regulation of CB1 receptors, the effects of rimonabant that we observed might not be mediated by these receptors. Further studies are needed to determine if the action of rimonabant is due to CB1 receptor blockade. Several studies demonstrated antinociceptive properties of locally administered cannabinoids in animal models (Hohmann, 2002; Walker and Huang, 2002; Mbvudula et al., 2004) and these effects were dependent on CB1 receptors (Agarwal et al., 2007). CB1 receptor-mediated antinociception has been attributed to activation of CB1 receptors (Ahluwalia et al., 2000; Ständer et al., 2005; Amaya et al., 2006), indeed stimulation of cannabinoid receptors reduced contact allergic inflammation (Karsak et al., 2007). Rimonabant has been proved to be a strong anti-inflammatory agent (Costa, 2007), attenuating weight gain in Zucker rats, lowering neutrophil and monocyte counts, platelet activation and aggregation and circulating pro-inflammatory cytokine levels (Di Marzo and Szallasi 2008). However, it is still not clear if the anti-inflammatory actions of rimonabant are mediated by CB1 receptors and due to antagonist, or more likely, to inverse agonist effects (Karsak et al., 2007).

Recently, the vanilloid receptor (TRPV1) was also proposed to be involved in contact allergic dermatitis, as there was up-regulation of TRPV1 receptors and palmitoyl ethanolamide levels in ear keratinocytes. Also, palmitoyl ethanolamide inhibited ear inflammation in mice in vivo, and this inhibition was attenuated by antagonism at TRPV1 receptors (Petrosino et al., 2010). In the model of acute inflammation used here, croton oil-induced ear dermatitis in mice (Tubaro et al., 1985), we found that rimonabant exhibited a dose-dependent anti-oedema activity comparable to that of the NSAID indomethacin at 6 h after dermatitis induction. Rimonabant was still active after 24 h, when indomethacin had lost its anti-oedema effect, as previously reported (Tubaro et al., 1985; Giangaspero et al., 2009). Moreover, rimonabant significantly reduced the leukocyte infiltrate, as did the equimolar dose of indomethacin.

Our findings showed that rimonabant reduced cell viability, was cytotoxic, induced apoptosis in C5N keratinocytes by regulation of Akt, NF-κB, IκBα pathways, cyclins D1, E and A and up-regulated CB1 receptors. We have provided evidence that the apoptotic effect in vitro is related to anti-inflammatory properties in vivo. The finding that rimonabant decreased croton oil-induced ear dermatitis in mice suggests a potential therapeutic application of the CB1 receptor antagonist as a topical anti-inflammatory drug.

Acknowledgments

This work was supported by Sanofi-Aventis (M.B.) and Associazione Educazione e Ricerca Medica Salernitana (ERMES).

Glossary

Abbreviations

FBS

fetal bovine serum

NSAID

non-steroidal anti-inflammatory drug

TMB

tetramethylbenzidine

TRPV1

vanilloid receptor

Conflicts of interest

None.

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