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Introduction

Recent bodies of evidence implicate that the endocannabinoid system (ECS) (Pacher et al., 2006; Di Marzo, 2008; Pertwee et al., 2010), as one of the new players in cutaneous neuroendocrinology (Slominski and Wortsman, 2000; Slominski et al., 2008), may play a significant role in controlling skin homeostasis (Bíró et al., 2009). Endocannabinoids such as anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) (Calignano et al., 1998; Karsak et al., 2007), enzymes involved in the synthesis and metabolism of these lipid mediators (Berdyshev et al., 2000; Maccarrone et al., 2003), as well as cannabinoid receptors (CB), were all identified on various skin cell populations (Casanova et al., 2003; Ibrahim et al., 2005; Stander et al., 2005; Blazquez et al., 2006; Karsak et al., 2007; Telek et al., 2007; Dobrosi et al., 2008; Tóth et al., 2011).

Of further importance, the cutaneous ECS was shown to regulate the well-balanced growth and differentiation program of skin cells. Namely, AEA was reported to inhibit the differentiation of cultured human epidermal keratinocytes (Maccarrone et al., 2003; Paradisi et al., 2008), whereas we have recently shown that AEA suppressed growth and induced apoptosis of these cells (Tóth et al., 2011). Likewise, we have also presented that the locally produced AEA inhibited in vitrohair shaft elongation and induced apoptosis-driven premature catagen regression (Telek et al., 2007). Of further importance, using a human sebaceous gland–derived cultured sebocyte model, we have demonstrated that endocannabinoids (AEA and 2-AG), produced by these cells, constitutively enhanced lipid production and induced chiefly apoptosis-driven cell death (hallmarks of sebocyte differentiation and hence a model of holocrine sebum production) (Dobrosi et al., 2008).

Interestingly, although the in situ expressions of both CB₁ and CB₂ have been described on epithelial cells of human eccrine sweat glands (Stander et al., 2005), we lack functional data on the role of endocannabinoids in the regulation of biology of the smallest appendage of the mammalian skin. Therefore, in this study, we aimed at defining the effects of the most extensively studied endocannabinoids (i.e., AEA and 2-AG) on growth and survival of human eccrine sweat gland cells. By using cultured NCL-SG3 eccrine sweat gland cell culture model and by employing combined pharmacological and molecular approaches, we provide the first evidence that endocannabinoids markedly suppress cellular proliferation, induce chiefly apoptosis-driven cell death, and alter expression/production of various intracellular proteins (e.g., cytokeratins (CKs)) and lipids in human sweat gland epithelial cells.

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Results

Endocannabinoids inhibit growth and induce chiefly apoptosis-driven cellular death in NCL-SG3 cells

Using colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and fluorimetric CyQuant assays, we found that both AEA and 2-AG dose-dependently suppressed the viable cell number and proliferation of NCL-SG3 cells (Figure 1a and b). To define whether this effect was due to the induction of cell death (apoptosis and/or necrosis), a series of functional assays was performed. As measured by quantitative fluorimetric determinations, both endocannabinoids significantly decreased mitochondrial membrane potential (reflecting mitochondrial disturbance) (Figure 1c) and induced the activation of pro-apoptotic caspases (Figure 1d), hallmarks of apoptosis (Green and Reed, 1998; Susin et al., 1998; Tóth et al., 2011). In addition, higher concentrations of 2-AG significantly increased the release of glucose-6-phosphate-dehydrogenase (Figure 1e) and Sytox Green accumulation to the cells (Figure 1f), two complementary indicators of necrosis/cytotoxicity. (Intriguingly, AEA did not induce necrosis.) These findings suggested that the endocannabinoids suppressed cellular growth and induced chiefly apoptosis-driven cell death of human sweat gland cells.

 

Endocannabinoids markedly alter expressions of certain CKs and other cytoskeleton proteins involved in cutaneous differentiation

In most epithelial cells, the cessation of proliferation and the induction of apoptosis are accompanied by the onset of the differentiation program of the cells (Candi et al., 2005; Proksch et al., 2008). Expressions of various intermediate filament CKs as well as of other cytoskeleton proteins (e.g., involucrin, filaggrin, loricrin), which were shown to be involved in differentiation of certain skin cells (e.g., epidermal and hair follicle keratinocytes), have previously been described on human eccrine sweat gland epithelium (Ohnishi and Watanabe, 1999; Langbein et al., 2005; Tharakan et al., 2010). Yet, the exact details of the differentiation process have not been fully elucidated in cultured sweat gland epithelial cells.

Therefore, we first analyzed the expression of various epithelial “differentiation markers” such as CKs (CK1, 7, 8, 10, 14, 18, and 19) (Candi et al., 2005; Moll et al., 2008), as well as of involucrin, filaggrin, and loricrin, in NCL-SG3 cells. We could not detect the expression of the epidermal differentiation markers CK1 and 10 (data not shown), whereas all other molecules were identified (Figure 2). Of great importance, however, we found that expressions of these markers differentially altered in parallel with the age of the cultures. Namely, while levels of CK7, 14, 18, and 19 were highest in the pre-confluent, highly proliferating cultures (Figure 2a), expressions of CK8, involucrin, filaggrin, and loricrin were predominant in the post-confluent, and hence growth-arrested (and presumably more differentiated) ones (Figure 2b). It appears, therefore, that expression profiles of these molecules in NCL-SG3 sweat gland cells, in part similar to those described, e.g., in cultured epidermal keratinocytes (Papp et al., 2003, 2004; Candi et al., 2005; Langbein et al., 2005; Moll et al., 2008), are strongly affected by the growth rate (i.e., proliferation vs. growth arrest) of the cells.

 

We then investigated the effects of endocannabinoids. Pre-confluent (30–40%) NCL-SG3 cells were treated with AEA and 2-AG (10 μm each) for 48 hours and the levels of the above markers were analyzed by quantitative “real-time” PCR (Q-PCR). As seen in Figure 2c and d, both endocannabinoids significantly (yet differentially) elevated the expressions of the “post-confluent” markers, i.e., CK8, involucrin, filaggrin, and loricrin. In addition, levels of some of the markers of the highly proliferating pre-confluent cells, i.e., CK7 by 2-AG and CK14 by both AEA and 2-AG, were markedly suppressed by endocannabinoid treatment.

Endocannabinoids stimulate lipid synthesis in NCL-SG3 cells

Another key function of sweat gland cells is to secrete various substances. As we have previously shown that endocannabinoids markedly induced lipid synthesis in cultured human sebocytes (Dobrosi et al., 2008), and furthermore, as sweat gland epithelial cells were shown to synthesize a wide array of lipids (Takemura et al., 1989), we also assessed whether endocannabinoids modulate the lipid synthesis of NCL-SG3 cells. As measured by semiquantitative Oil Red-O histochemistry and by quantitative Nile Red-based fluorimetry, both endocannabinoids (as early as after 24 hours treatment) markedly and dose-dependently elevated neutral (but not polar) lipid synthesis of the cells (Figure 2e and f). As neutral lipids reflect intracellularly accumulated “de novo” synthesized ones, our data argue that endocannabinoids (besides modulating cell growth, survival, and expressions of various cytoskeleton proteins) may exert a profound role in the regulation of secretory activity of human eccrine sweat gland cells by modifying the composition of the produced sweat.

Cellular effects of endocannabinoids are not mediated by CB₁ or CB₂expressed on NCL-SG3 cells

We then investigated the putative involvement of certain receptor-mediated signal-transduction systems in mediating the cellular actions of endocannabinoids. Conforming previous data (Stander et al., 2005), we first identified the expressions of both metabotropic CB subtypes (CB₁, CB₂) on sweat gland–derived cells using western blot (Figure 3a) and immunocytochemistry (data not shown) techniques. In addition, transcription of genes encoding the above proteins was demonstrated by reverse-transcriptase–PCR (data not shown) and by Q-PCR (Figure 3b). During the above analysis, we also observed that the expression of CB₁ and CB₂ markedly altered in parallel with the culturing of the cells (Figure 3a and b). Specifically, the level of CB₁ monotonously increased during culturing and reached its maximum in the post-confluent states. In contrast, the expression of CB₂ was the highest in the pre-confluent cultures, whereas its level markedly decreased with reaching confluence of the cells. These data further suggested that the cannabinoids might play a role in the regulation of growth (and most probably of differentiation) of the human sweat gland–derived cells.

 

To assess the roles of the CB receptors, we have employed various antagonists and the RNA interference (RNAi) techniques. In these experiments, to equally assess changes in cellular growth, survival, and secretory activities, we determined cellular viability/proliferation (MTT assay) and lipid production (Nile Red fluorimetry) of NCL-SG3 cells.

First, various inhibitors of CB subtypes (AM251 for CB₁, AM630 for CB₂) were employed. These inhibitors could not modify the actions of the endocannabinoids, namely both AEA and 2-AG were still able to suppress cellular viability and stimulate lipid synthesis in the presence of either antagonist (Figure 4a and b).

 

As the pharmacological natures of AM251 and AM630 (i.e., also acting as inverse agonists) (Pertwee et al., 2010) may compromise their specificities on the CBs, a series of RNAi experiments against the CBs were carried out in accordance with the optimized techniques developed in our previous studies (Dobrosi et al., 2008; Tóth et al., 2009, 2011). Western blot analysis (Supplementary Figure S1 online) as well as Q-PCR determination (data not shown) revealed that expressions of both CB₁and CB₂ were significantly and specifically “knocked down” at day 3 after transfection by two out of three RNAi probes employed. However, this phenomenon was reversible as we observed a “return” of their expression at day 4. Scrambled RNAi probes had no effect on the expression of either CB₁ or CB₂.

We then investigated the effects of endocannabinoids on NCL-SG3 cells with “silenced” CB₁ or CB₂. Of great importance, in perfect agreement with the above pharmacological data, the molecular “knockdown” of CB₁ or CB₂ could not prevent the growth-inhibitory and differentiation-promoting cellular actions of AEA and 2-AG, namely both endocannabinoids were able to suppress cellular viability and induce lipid production (Figure 4c and d). Therefore, these results, in line with the above data obtained with the antagonists, collectively argue for the fact that CB₁– or CB₂-coupled intracellular signaling mechanisms most probably do not participate in mediating the effects of endocannabinoids on human sweat gland epithelial cells.

Cellular effects of endocannabinoids are not mediated by TRP channels expressed on NCL-SG3 cells

Recent intriguing data from our laboratory and from others, however, suggest that cannabinoid compounds, besides CB₁ or CB₂, may also target other molecules (Pertwee et al., 2010). Among these, certain transient receptor potential (TRP) ion channels were implicated as “ionotropic CB receptors” (Zygmunt et al., 1999; Di Marzo et al., 1998, 2001; Akopian et al., 2009). These molecules, which function as highly Ca²⁺-permeable ion channels, are the heat-sensitive TRPV1, TRPV2, TRPV3, and TRPV4, as well as the cold-activated TRPM8 and TRPA1 (Akopian et al., 2009; Vriens et al., 2009; Di Marzo and De Petrocellis, 2010; Pertwee et al., 2010). Hence, in the next phase of our experiments, we investigated the possible involvement of these molecules.

We first defined the expression profile of TRP channels on NCL-SG3 cells. As revealed by Q-PCR analysis, human sweat gland epithelial cells express all thermosensitive TRPV channels (Supplementary Figure S2a online). However, we could not detect the expression of cold-sensitive TRPM8 and TRPA1 (data not shown).

We then investigated whether the TRP channel–mediated Ca²⁺ influx to NCL-SG3 cells is involved in the action of cannabinoids. Using fluorimetric image plate reader (FLIPR)-based Ca²⁺ imaging, we found that endocannabinoids (tested up to 50 μmconcentration) did not modify the intracellular Ca²⁺ concentration in NCL-SG3 cells (data not shown). In addition, we also observed that neither the “universal” TRP channel antagonists Ruthenium Red nor the suppression of extracellular Ca²⁺concentration affected the cellular effects of endocannabinoids to suppress cellular growth and to induce lipid synthesis (Supplementary Figure S2b and c online). These findings collectively suggest that TRP channels are most probably not involved in mediating the actions of cannabinoids in human sweat gland epithelial cells.

Endocannabinoids selectively stimulate the MAPK pathway in NCL-SG3 cells

Although we failed to identify surface membrane receptor/channel coupled mechanisms, we also aimed at identifying putative endocannabinoid-activated intracellular signaling pathways. Cannabinoids were shown to induce various signal-transduction mechanisms, such as e.g., the mitogen-activated protein kinase (MAPK), protein kinase C isoenzymes, phosphatidylinositide 3-kinase (Howlett, 2005; Pacher et al. 2006); hence, we investigated the involvement of these mechanisms.

Using various pharmacological tools, we found that the general protein kinase C inhibitor GF109203X and the phosphatidylinositide 3-kinase inhibitor Wortmannin did not affect the growth-inhibitory and lipid synthesis-promoting action of the endocannabinoids (Figure 5a and b). However, of great importance, the MAPK inhibitor PD098059 markedly (almost completely) prevented the effects of both AEA and 2-AG. Furthermore, the endocannabinoids also induced the transient phosphorylation of the MAPK Erk1/2 (p42/44) (Figure 5c), which effect was also abrogated by the application of the aforementioned antagonist (Figure 5d). These findings collectively argued for the crucial involvement of the MAPK pathway in mediating the actions of endocannabinoids in human sweat gland epithelial cells.

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Discussion

In this study, we present early evidence that prototypic endocannabinoids inhibit proliferation, induce cell death, and upregulate secretory activity (lipid synthesis) in cultured human eccrine sweat gland epithelial cells. These data support the concept that human sweat glands may also function as novel targets for endocannabinoids, as emerging members of the skin neuroendocrine regulatory circuits (Slominski and Wortsman, 2000; Slominski et al., 2008).

In the course of our experiments, a key goal was to identify how endocannabinoids affect the differentiation process of the sweat gland cells. However, as we have detailed above, the exact “differentiation marker pattern” of cultured sweat gland epithelial cells is not fully known. Therefore, we have investigated the expressions of those cytoskeleton proteins (e.g., various CKs, involucrin, filaggrin, loricrin), which, on the one hand, were shown to be expressed in human sweat glands, and, on the other hand, were found to be involved in differentiation of certain skin cells such as, e.g., epidermal and hair follicle keratinocytes (Candi et al., 2005; Langbein et al., 2005). Our experiments resulted in several findings, which to our knowledge is previously unreported: (i) This is the first demonstration that, to our best knowledge, shows that levels of these molecules in cultured human sweat gland epithelial cells are strongly affected by the growth rate (i.e., proliferation vs. high cell density–induced growth arrest) of the cells. (ii) Moreover, we have also found that expression patterns of some of these markers were very similar to those described (by us and others) in human epidermal keratinocytes (Papp et al., 2003, 2004; Candi et al., 2005; Langbein et al., 2005; Moll et al., 2008). Namely, in both cell types, highest levels of the “differentiation markers” involucrin, filaggrin, and loricrin were detected in the post-confluent (hence more differentiated) cultures. Nevertheless, it should also be noted that the cytoskeleton protein profile of sweat gland cells, by far, was not identical to those of epidermal keratinocytes as, e.g., expressions of the “classical” epidermal markers CK1 and 10 were not detected in cultured sweat gland cells. (iii) Of further importance, we have also shown that endocannabinoids upregulated the expression of the above “post-confluent” markers (i.e., involucrin, filaggrin, and loricrin), whereas suppressed expressions of some of those cytoskeleton proteins that were expressed at high levels in the pre-confluent (hence greatly proliferating) cultures. Taken together, these findings collectively argue that endocannabinoids not only inhibit cell growth and induce cell death, but may also promote the differentiation process in the human sweat gland epithelium.

We also wished to uncover the signaling pathways that were involved in mediating the cellular actions of endocannabinoids. Quite unexpectedly, we were unable to find evidence for the involvement of the “classical” metabotropic CB₁ and CB₂receptors (lack of effects of CB₁ or CB₂ antagonists or RNAi-mediated silencing of these receptors), or the “ionotropic CB receptor” TRP channels (lack of elevation of intracellular Ca²⁺ concentration upon endocannabinoid challenge) expressed by these cells. Although these observations suggested a CB₁/CB₂/TRP-independent cellular action of the endocannabinoids—similar to those seen on various cell types, including e.g., leukocytes, endothelial cells, etc. (Rockwell et al., 2006; McCollum et al., 2007)—we have intriguingly found that both AEA and 2-AG selectively stimulated the MAPK pathway (but not the phosphatidylinositide 3-kinase and protein kinase C signal-transduction mechanisms). As the MAPKs are most often activated by receptor-coupled processes, further studies are now invited to define the exact intracellular signaling of endocannabinoids in human sweat gland cells.

Comparison of the above findings with our previously published data obtained on other human skin adnexal structures revealed another exciting feature of the cutaneous effects of cannabinoids. Namely, our presentations that endocannabinoids (i) inhibit human hair growth and induce apoptosis by activating CB₁ (Telek et al., 2007); (ii) promote lipid synthesis and apoptosis in human sebaceous gland–derived sebocytes via CB₂-mediated signaling (Dobrosi et al., 2008); (iii) inhibit proliferation, induce cell death, and stimulate lipid synthesis and differentiation of human sweat gland epithelial cells by activating non-CB₁/CB₂-coupled signal-transduction pathways; and (iv) highlight the existence of cell type-specific and (most probably) receptor-selective regulatory endocannabinoid mechanisms in the human skin appendages.

Of further importance, sweat gland epithelial cells apparently are not only targets but sources of endocannabinoids. Indeed, mass spectrometry analysis revealed that NCL-SG3 sweat gland cells produce the prototypic endocannabinoids AEA and 2-AG. However, the concentrations of the endocannabinoids determined in NCL-SG3 cells (AEA, 15 fmol per 10⁶ cells; 2-AG, 0.2 pmol per 10⁶ cells) were much less than those found in other human cultures skin cells (e.g., AEA, 160 fmol per 10⁶ cells; 2-AG, 4.2 pmol per 10⁶ cells in human SZ95 sebocytes) (Telek et al., 2007; Dobrosi et al., 2008). In addition, we were also able to identify the expression of those enzymes that are involved in the synthesis (NAPE-PLD, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D; DAGL, diacylglycerol lipase-α and -β) and degradation (FAAH, fatty acid amid hydrolase; MAGL, monoacylglycerol lipase) of the endocannabinoids (Supplementary Figure S3 online). Interestingly, similar to CB₁ and CB₂ receptors (Figure 3), we also detected marked fluctuations in the expressions of the elements of the ECS further suggesting the role of the ECS in the regulation of growth (and most probably of differentiation) of the human sweat gland–derived cells. Finally, by performing immunohistochemical labeling on human skin sections, we were able to identify the elements of the ECS on eccrine sweat glands in situ (Supplementary Figure S4 online), which findings perfectly complement the above PCR data obtained on NCL-SG3 cells.

Evidently, further explorative research efforts are needed to define whether alterations in the activities of the above enzymes can modify the endocannabinoid production (hence the endocannabinoid “tone”) in the human sweat glands in situ. Nevertheless, our findings now warrant proof-of-principle clinical studies to test the therapeutic value of cutaneous ECS-targeted approaches in the clinical management of multitude of human skin diseases. Specifically, based on this preclinical data in human sweat gland model cell cultures, it is envisaged that agents increasing the cutaneous endocannabinoid “tone” (such as employing endocannabinoids or molecules that upregulate the production of endocannabinoids by, e.g., stimulating their synthesis of inhibiting their degradation) (Di Marzo, 2008; Bíró et al., 2009) may be successfully applied in certain sweat gland disorders (e.g., benign or malignant tumors) characterized by unwanted cell growth—similar to those we have previously suggested for the management of various growth and inflammatory conditions of the human pilosebaceous unit (e.g., hair growth problems, acne vulgaris) (reviewed in Bíró et al., 2009).

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Materials and Methods

Materials

Throughout the experiments, the following agents were used: AEA, 2-AG (Cayman, Ann Arbor, MI); AM251, GF109203X, Ruthenium Red (Sigma-Aldrich, St Louis, MO); AM630 (Tocris, Ellisville, MO); and PD098059, Wortmannin (Calbiochem, Nottingham, UK).

Cell culturing

Human eccrine sweat gland–derived NCL-SG3 epithelial cells were cultured in William’s Medium E medium (Invitrogen, Paisley, UK) supplemented with 5% fetal bovine serum, 10 μg ml⁻¹ insulin–transferrin–selenium mixture, 20 ng ml⁻¹epidermal growth factor (all from Invitrogen), and 2 mm l-glutamine, 10 ng ml⁻¹hydrocortisone, and antibiotic mixture (all from Sigma-Aldrich).

Western blotting

Cell lysates were subjected to SDS-PAGE (8% gels were loaded with 30 μg protein per lane), transferred to BioBond nitrocellulose membranes (Whatman, Maidstone, UK), and then probed with the rabbit primary antibodies against CB₁ and CB₂ (both 1:200). A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1,000; Bio-Rad, Hercules, CA) was used as a secondary antibody, and the immunoreactive bands were visualized by a SuperSignal West Pico Chemiluminescent Substrate-enhanced chemiluminescence kit (Pierce, Rockford, IL) using LAS-3000 Intelligent Dark Box (Fuji, Tokyo, Japan). To assess equal loading, membranes were re-probed with an anti-cytochrome C antibody (1:50; Santa Cruz, Santa Cruz, CA) and visualized as described above. Where appropriate, immunoblots were subjected to densitometric analysis using the Image Pro Plus 4.5.0 software (Media Cybernetics, Silver Spring, MD) (Gönczi et al., 2008; Szegedi et al., 2009).

Immunocytochemistry

Cells were fixed in acetone, permeabilized by 0.1% Triton X-100 (Sigma-Aldrich), and then incubated with the above primary antibodies against CB₁ or CB₂ (1:200 dilution, Cayman). For fluorescence staining, slides were then incubated with FITC-conjugated secondary antibodies (dilution 1:200) (Bodó et al., 2005; Dobrosi et al., 2008; Tóth et al., 2009).

Quantitative real-time PCR

Q-PCR was performed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using the 5′ nuclease assay as detailed in our previous reports (Dobrosi et al., 2008; Tóth et al., 2009, 2011). PCR amplification was performed by using TaqMan primers and probes (Applied Biosystems) (see Supplementary data online for Assay IDs).

Determination of viable cell numbers and proliferation

The number of viable cells (hence the rate of proliferation) was determined by measuring the conversion of the tetrazolium salt MTT (Sigma-Aldrich) to formazan by mitochondrial dehydrogenases. In addition, proliferation was also assessed by the CyQuant fluorimetric Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s protocol (Bodó et al., 2005; Kiss et al., 2008; Szegedi et al., 2009) (see Supplementary data online for further details).

Determination of apoptosis

A decrease in the mitochondrial membrane potential is one of the earliest markers of apoptosis. Mitochondrial membrane potential of NCL-SG3 cells was determined using a MitoProbe DiIC₁(5) Assay Kit (Invitrogen), where the decrease in fluorescence intensity reflects apoptosis. In addition, apoptosis was also determined by fluorimetric measurement of activation of pro-apoptotic caspases using a Poly-Caspases Detection Kit (Invitrogen) (Dobrosi et al., 2008; Tóth et al., 2009, 2011) (see Supplementary data online for further details).

Determination of cytotoxicity (necrosis)

Necrotic cell death was determined by measuring the glucose-6-phosphate-dehydrogenase (G6PD) release (G6PD Release Assay Kit; Invitrogen). Moreover, as the activity of the glucose-6-phosphate-dehydrogenase released from necrotic cells decreases over 24–36 hours, the cytotoxic effects of long-term treatment protocols were assessed by the fluorimetric determination of Sytox Green accumulation to the nuclei of necrotic cells with ruptured plasma membranes (Invitrogen) (Tóth et al., 2009, 2011) (see Supplementary data online for further details).

Determination of intracellular lipids

For semiquantitative detection of cellular lipids, cells were fixed in 4%paraformaldehyde, washed in 60% isopropanol (both Sigma-Aldrich), and stained in freshly prepared Oil Red O solution (in 60% isopropanol) (Sigma-Aldrich). Nuclei were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and coverslips were mounted in mounting medium (DAKO, Glostrup, Denmark). In addition, for quantitative measurement of lipid content, a Nile Red (Sigma-Aldrich) fluorimetric method was employed as described before (Wróbel et al., 2003; Alestas et al., 2006; Dobrosi et al., 2008; Tóth et al., 2009) (see Supplementary data online for further details).

RNA interference

NCL-SG3 cells at 50–70% confluence were transfected with specific Stealth RNAi oligonucleotides (40 nm; all from Invitrogen) against CB₁ (ID No. HSS102082) or CB₂ (ID No. HSS102087) using Lipofectamine 2000 Transfection Reagent (Invitrogen). For controls, RNAi Negative Control Duplexes (scrambled RNAi; Invitrogen) were employed. The efficacy of RNAi-driven “knockdown” was daily evaluated by Q-PCR and western blotting for 4 days (see Supplementary Figure S1 online and Supplementary data online for further details).

Ca²⁺ imaging

Changes in intracellular Ca²⁺ concentration upon drug applications were detected by fluorimetric Ca²⁺ imaging (Bodó et al., 2005; Gönczi et al., 2008; Tóth et al., 2009). Cells were seeded in 96-well black-well/clear-bottom plates (Greiner Bio One, Frickenhausen, Germany) at a density of 10,000 cells per well and then were incubated with culturing medium containing the cytoplasmic calcium indicator 2 μmFluo-4 AM (Invitrogen) at 37 °C for 40 minutes. The cells were washed four times with and finally cultured in Hank’s solution containing 1% bovine serum albumin and 2.5 mm Probenecid (both from Sigma-Aldrich) for 30 minutes at 37 °C. The plates were then placed to a FlexStation II³⁸⁴ FLIPR (Molecular Devices, San Francisco, CA) and changes in intracellular Ca²⁺ concentration (reflected by changes in fluorescence; lEX=494 nm, lEM=516 nm) induced by various concentrations of the drugs were recorded in each well (during the measurement, cells in a given well were exposed to only one given concentration of the agent).

Statistical analysis

When applicable, data were analyzed using a two-tailed unpaired t-test and P<0.05 values were regarded as significant differences. In addition, statistical differences were further verified using one-way analysis of variance with Bonferroni’s and Dunnett’s post-hoc probes, resulting in similar results (data not shown).

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Conflict of interest

The authors state no conflict of interest.

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Acknowledgments

This work was supported by L’Oréal Research. CG is a recipient of the János Bolyai research scholarship of the Hungarian Academy of Sciences.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper

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