Robert Ramer

Jutta Merkord

Helga Rohde

Dr Burkhard Hinz
email: burkhard.hinz@med.uni-rostock.de

CANNABIDIOL INHIBITS CANCER CELL INVASION VIA UPREGULATION OF TISSUE INHIBITOR OF MATRIX METALLOPROTEINASES-1 #

Keywords: cannabidiol, Matrigel cell invasion, tissue inhibitor of matrix metalloproteinases-1, human cancer cells, experimental lung metastasis

Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB 1 and CB 2 receptors as well as to transient receptor potential vanilloid 1 (TRPV1).

The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness.

Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls.

Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.

Introduction

During the last decade several cell culture and animal studies have provided a great body of evidence for an antitumorigenic action of cannabinoids on the levels of cancer cell proliferation [START_REF] Jacobsson | Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors[END_REF][START_REF] Blázquez | Cannabinoid receptors as novel targets for the treatment of melanoma[END_REF], apoptosis [START_REF] Galve-Roperh | Antitumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation[END_REF][START_REF] Hinz | Up-regulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells[END_REF], metastasis [START_REF] Portella | Inhibitory effects of cannabinoid CB 1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis[END_REF], and angiogenesis [START_REF] Blázquez | Inhibition of tumor angiogenesis by cannabinoids[END_REF][START_REF] Blázquez | Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas[END_REF][START_REF] Pisanti | Antiangiogenic activity of the endocannabinoid anandamide: correlation to its tumor-suppressor efficacy[END_REF]. Although cannabinoid-based drugs such as ∆ 9 -tetrahydrocannabinol (THC) are clinically used to palliate wasting, emesis and pain in cancer patients, a shortcoming for these and forthcoming indications clearly lies in the psychoactive adverse side effects of cannabinoids.

For this reason, the interest in the non-psychoactive cannabinoid-mimetic compound cannabidiol has substantially increased in recent years. Meanwhile, a formulation including a 1:1 ratio of THC and cannabidiol has been approved for the pharmacotherapy of multiple sclerosis-related spasticity and pain in Canada [START_REF] Wade | Do cannabis-based medicinal extracts have general or specific effects on symptoms in multiple sclerosis? A doubleblind, randomized, placebo-controlled study on 160 patients[END_REF].

The precise mechanism by which cannabidiol exerts its biological effects is still a matter of debate. Although cannabidiol displays a low affinity to CB 1 and CB 2 receptors [START_REF] Thomas | Comparative receptor binding analyses of cannabinoid agonists and antagonists[END_REF], several effects of cannabidiol including modulation of cytokine release and macrophage chemotaxis [START_REF] Sacerdote | The nonpsychoactive component of marijuana cannabidiol modulates chemotaxis and IL-10 and IL-12 production of murine macrophages both in vivo and in vitro[END_REF], antiproliferative [START_REF] Massi | Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines[END_REF] as well as proapoptotic properties [START_REF] Mckallip | Targeting CB 2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease[END_REF] have been shown to be mediated via CB 1 and/or CB 2 receptors. In other investigations, cannabidiol has even been reported to display antagonistic effects on CB 1 and CB 2 receptor [START_REF] Thomas | Cannabidiol displays unexpectedly high potency as an antagonist of CB 1 and CB 2 receptor agonists in vitro[END_REF], as well as synergistic effects on THC-mediated hypoactivity, hypothermia and impairment of spatial memory [START_REF] Hayakawa | Cannabidiol potentiates pharmacological effects of Delta(9)-tetrahydrocannabinol via CB 1 receptordependent mechanism[END_REF].

On the other hand increasing evidence suggests an indirect receptor activation by cannabidiol. In this context an inhibitory action on fatty acid amidohydrolase activity that confers release of anandamide and increased receptor-mediated signalling by this endocannabinoid has been demonstrated in diverse systems [START_REF] Watanabe | Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids[END_REF][17][START_REF] De Filippis | Effect of cannabidiol on sepsis-induced motility disturbances in mice: involvement of CB receptors and fatty acid amide hydrolase[END_REF]. Additionally, the nonselective cation channel transient receptor potential 1 (TRPV1) was shown to be involved in cannabidiol-mediated antihyperalgesia [START_REF] Costa | Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation[END_REF] and inhibition of cancer cell proliferation [17].

Finally, diverse studies suggest the involvement of a hitherto undefined abnormal cannabidiol receptor in several actions of cannabidiol (for review see [START_REF] Ryberg | The orphan receptor GPR55 is a novel cannabinoid receptor[END_REF]). In context with the antitumorigenic action of cannabinoids, modulation of cancer cell invasion has recently emerged as a topic of increasing interest [START_REF] Mcallister | Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells[END_REF][START_REF] Blázquez | Cannabinoids inhibit glioma cell invasion by down-regulating matrix metalloproteinase-2 expression[END_REF][START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF]. Cancer cell invasion is a complex process of cell locomotion through degraded matrix components of microenvironmental spaces surrounding a tumor thereby contributing to tumor cell metastasis and angiogenesis. The level of tumor invasiveness and malignancy is mainly determined by a sensitive balance between collagen-and proteoglycan-degrading matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs) (for review see ref. [START_REF] Curran | Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis[END_REF]). Among the four distinct members of the TIMP family, elevated TIMP-1 was shown to mediate the anti-invasive effects of several anticarcinogenic drugs [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF][START_REF] Khokha | Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells[END_REF][26][START_REF] Park | Arsenic trioxide (As 2 O 3 ) inhibits invasion of HT1080 human fibrosarcoma cells: Role of nuclear factor-kappaB and reactive oxygen species[END_REF][START_REF] Park | Inhibitory effects of a benz(f)indole-4,9-dione analog on cancer cell metastasis mediated by the downregulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells[END_REF][START_REF] Ramer | Upregulation of tissue inhibitor of matrix metalloproteinases-1 confers the anti-invasive action of cisplatin on human cancer cells[END_REF]. Furthermore, decreased TIMP-1 levels were demonstrated to correlate with high cancer invasiveness [START_REF] Khokha | Antisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells[END_REF][START_REF] Chan | Intestinal trefoil factor promotes invasion in non-tumorigenic Rat-2 fibroblast cell[END_REF].

The present study focussed on a potential role of TIMP-1 upregulation in the anti-invasive action of cannabidiol and on a possible involvement of cannabinoid receptors and TRPV1 in this process. In view of recent studies demonstrating p38 and p42/44 mitogen-activated protein kinases (MAPK) activation as intracellular signaling pathways leading to induction of TIMP-1 [START_REF] Tong | Mitogen-activated protein kinases Erk1/2 and p38 are required for maximal regulation of TIMP-1 by oncostatin M in murine fibroblasts[END_REF][START_REF] Han | Induction by IL-1 beta of tissue inhibitor of metalloproteinase-1 in human orbital fibroblasts: modulation of gene promoter activity by IL-4 and IFN-gamma[END_REF] as well as findings showing a cannabinoid receptor-dependent activation of MAPKs [START_REF] Galve-Roperh | Antitumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation[END_REF][START_REF] Sanchez | Activation of A c c e p t e d M a n u s c r i p t 26 phosphoinositide 3-kinase/PKB pathway by CB 1 and CB 2 cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction[END_REF][START_REF] Herrera | p38 MAPK is involved in CB 2 receptor-induced apoptosis of human leukaemia cells[END_REF], our study also assessed a role of both MAPKs in cannabidiol-modulated invasion and TIMP-1 expression. Finally, the impact of cannabidiol on cellular invasion was confirmed by studying metastasis in vivo.

Materials and methods

Materials

(-)-Cannabidiol was purchased from Tocris (Bad Soden, Germany). AM-251, AM-630, capsazepine, PD98059 and SB203580 were bought from Alexis Deutschland GmbH (Grünberg, Germany). Dulbecco´s Modified Eagle´s medium (DMEM) with 4 mM L-glutamine and 4.5 g/L glucose was from Cambrex Bio Science Verviers S.p.r.l. (Verviers, Belgium).

Fetal calf serum (FCS) and penicillin-streptomycin were obtained from PAN Biotech (Aidenbach, Germany) and Invitrogen (Karlsruhe, Germany), respectively. DMSO, HEPES, NaCl, EDTA, Triton ® X-100, and glycerol were bought from Applichem (Darmstadt, Germany). PMSF, leupeptin, aprotinin and picrinic acid, and formaldehyde were obtained from Sigma (Taufkirchen, Germany).

Cell culture

HeLa, C33A and A549 cells were maintained in DMEM supplemented with 10% heatinactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were grown in a humidified incubator at 37°C and 5% CO 2 . All incubations were performed in serum-free medium. Phosphate-buffered saline was used as a vehicle for the tested substances with a final concentration of 0.1% (v/v) ethanol (for cannabidiol) or 0.1% (v/v) dimethyl sulfoxide (DMSO) (for AM-251, AM-630, capsazepine, PD98059 and SB203580).

Matrigel invasion and migration assays

The effect of test substances on the invasiveness of cells was determined using a modified Boyden chamber technique with Matrigel-coated membranes according to the manufacturer´s instructions (BD Biosciences, Oxford, UK). In this assay, tumor cells must overcome a reconstituted basement membrane by a sequential process of proteolytic degradation of the substrate and active migration. In brief, the upper sides of the transwell inserts (8 µm pore size) were coated with 28.4 µg Matrigel (BD Biosciences, Oxford, UK) per For calculation of migration, the viability of cells on the lower side of uncoated invasion chambers was determined by the WST-1 test. Invasion was expressed as the invasion index, which is calculated as the absorbance at 490 nm of cells that invaded through Matrigelcoated Boyden chambers divided by absorbance of cells that migrated through uncoated control inserts with equal treatment ([invasion/migration] x 100%). For corroboration of the calculated invasion indices in one experiment (Fig. 3C), HeLa cells that had invaded through Matrigel-coated membranes were fixed and stained with Diff-Quick (Medion Diagnostics GmbH, Dügingen, Switzerland) and visualized using a microscope at 200 x magnification. To exclude the possibility that the effect of cannabidiol on invasion was an unspecific cytotoxicity-related phenomenon, cell viability was analyzed after cannabidiol exposure. For this purpose, cells were seeded into 48-well plates at 5 x 10 5 cells per well to match conditions of invasion assays or at 2.5 x 10 5 , 1 x 10 5 , 0.5 x 10 5 , and 0.1 x 10 5 per well for testing lower cell densities in a volume of 500 µl DMEM per well and treated with 10 µM cannabidiol or ethanol vehicle for 72 hours. Viability was measured subsequently using the WST-1 test.

The antiinvasive action of cannabidiol was confirmed in another experimental setup. To this end, HeLa cells were seeded at a final concentration of 2.5 x 10 6 cells in 10-cm dishes in DMEM containing 10% FCS. After 24 hours cells were washed and treated with cannabidiol or vehicle in serum-free DMEM for an additional 48-hour incubation period. Subsequently, trypsinized and pelleted cells were counted, suspended to a final concentration of 5 x 10 5 cells in 500 µl serum-free DMEM and subjected into each insert without further addition of test substances. Quantification of Matrigel invasion was performed after another 24-hour incubation of HeLa cells in the Boyden chamber.

Quantitative RT-PCR analysis

HeLa cells were seeded into 48-well plates at a density of 5 x 10 5 cells per well. Following incubation of cells with cannabidiol or its vehicle for the indicated times, supernatants were removed and cells were lysed for subsequent RNA isolation using the RNeasy total RNA Kit (Qiagen, Hilden, Germany). β-Actin-(internal standard) and TIMP-1 mRNA levels were determined by quantitative real-time RT-PCR as described [START_REF] Hinz | Latanoprost induces matrix metalloproteinase-1 expression in human nonpigmented ciliary epithelial cells through a cyclooxygenase-2-dependent mechanism[END_REF]. Primers and probe for human TIMP-1 was an Assay-on-demand™-product (Applied Biosystems, Darmstadt, Germany).

Western blot analysis

For determination of TIMP-1, MMP-2 and MMP-9 protein levels in Fig. 2B, cells grown to A c c e p t e d M a n u s c r i p t 9 confluence in 48-well-plates were incubated with test substances or vehicles. Afterward, cell culture supernatants were centrifuged at 500 x g and used for Western Blot analysis. For all other blots, TIMP-1 was determined in supernatants collected from the upper Boyden chambers at the end of the respective invasion experiment. Total protein in the supernatants was measured using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). For Western blot analysis of p38, phospho-p38, p42/44, and phospho-p42/44, cells grown to confluence in 6-well-plates were incubated with cannabidiol or its vehicle for the indicated times. Afterward, cells were washed, harvested, lysed in solubilization buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton ® X-100, 10% (v/v) glycerol,1 mM PMSF, 1 µg/ml leupeptin and 10 µg/ml aprotinin), homogenized by sonication, and centrifuged at 10,000 x g for 5 min.

Supernatants were used for Western blot analysis. All proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide (Applichem, Darmstadt, Germany) gel. Following transfer to nitrocellulose and blocking of the membranes with 5% milk powder, blots were probed with specific antibodies raised to TIMP-1, MMP-2 and MMP-9 (all antibodies from Oncogene Research Products, San Diego, CA) or p38, phospho-p38, p42/44 and phospho-p42/44 (New England BioLabs GmbH, Frankfurt, Germany). Membranes were probed with horseradish peroxidase-conjugated Fab-specific anti-mouse IgG for detection of TIMP-1 and MMPs (New England BioLabs GmbH, Frankfurt, Germany) or anti-rabbit IgG for analysis of MAPK protein expression (New England BioLabs GmbH, Frankfurt, Germany). Densitometric analysis of TIMP-1 and MMP band intensities was achieved by optical scanning and quantifying using the Quantity One 1-D Analysis Software (Biorad, Muenchen, Germany).

Vehicle controls were defined as 100% for evaluation of changes in protein expression. To ensure that equal amounts of protein in cell culture supernatants used for protein analysis of TIMP-1 and MMPs had been transferred to the membrane, proteins on Western blot membranes were stained with the fluorescent dye Roti-Green (Carl Roth, Karlsruhe, Germany) except for Fig. 2B, were membranes were stained with Ponceau red. To ascertain equal protein loading in Western Blots of supernatants obtained from upper Boyden chambers, a band with a size of about 65 kDa that appeared unregulated is shown as a

SiRNA transfections

HeLa, C33A, and A549 cells were transfected with siRNA targeting the indicated sequence of TIMP-1 using RNAiFect ® as the transfection reagent (Qiagen GmbH, Hilden, Germany) or negative control RNA (Eurogentec, Seraing, Belgium; Cat. No. OR-0030-neg). The target sequence of the TIMP-1 siRNA (Qiagen GmbH, Hilden, Germany) was as follows: 5´tcccatctttcttccggacaa-3´. A BLAST search revealed that the sequence selected did not show any homology to other known human genes. Transfections were performed according to the manufactures´s instructions. For invasion assays, cells grown to confluence were transfected with 0.25 µg/mL siRNA or non-silencing siRNA as negative control with an equal ratio (w/v) of RNA to transfection reagent for 24 hours in DMEM supplemented with 10% FCS. Subsequently, cells were treated with trypsin for 3 minutes at 37°C in a humidified incubator, centrifuged at 200 x g, resuspended to a final density of 5 x 10 5 cells in 500 µl of serum-free DMEM containing the same amounts of siRNA or non-silencing siRNA to provide constant transfection conditions, and seeded for invasion analysis as described above.

Mouse model of tumor metastasis

Athymic nude mice (NMRI-nu/nu) were purchased from Charles River GmbH (Sulzfeld, Germany). Mice were given injections of A549 cells (1 x 10 6 per 100 µl in PBS) through the lateral tail vein (day 1) and, after 24 hours (day 2), were treated with cannabidiol (5 mg/kg body weight) or with vehicle intraperitoneally. Treatment protocols for the evaluation of the antitumorigenic action of cannabidiol in vivo developed by other groups revealed 5 mg cannabidiol per kg as an appropriate dose for this purpose [17]. In our experiments cannabidiol or vehicle were administered every 72 hour. Mice were sacrificed on day 28 and one lung per animal was evaluated for experimental metastases. To contrast lung nodules, lungs were fixed in Bouin´s fluid (picrinic acid 15 ml, formol 5 ml, acetate 1 ml), and metastatic nodes were scored under a stereoscopic microscope in an investigator-blinded

Statistics

All statistical analyses were undertaken using GraphPad Prism 3.00 (GraphPad Software, San Diego, CA). Results were considered to be statistically significant at P < 0.05.

To assess the half maximal effective concentration (EC 50 ) of cannabidiol in eliciting an antiinvasive response, concentration response curves were fitted by a log(agonist) vs. response regression with variable slope.

Results

Time-course and concentration-dependency of cannabidiol´s inhibitory effect on HeLa cell invasion

HeLa cells showed a diminished invasion within the first 24 hours of incubation with 10 µM cannabidiol that continued to increase over the investigated 72-hour incubation period (Fig. 1A). To exclude an effect based on cell motility, transmigration through uncoated Boyden chambers was evaluated after 48 and 72 hours. As shown in Fig. 1B, cannabidiol did not show a significant inhibitory effect on this parameter.

Moreover, cannabidiol caused a concentration-dependent decrease of invasion that was even significant at concentrations as low as 0.01 µM (Fig. 1C). Cellular viability measured under comparable experimental conditions (i.e., 48 well plates with 5 x 10 5 cells per well) was virtually unaltered in the presence of either cannabidiol concentration tested (Fig. 1C). As recently shown, the phytocannabinoid THC and the hydrolysis-stable endocannabinoid analog R(+)-methanandamide revealed a toxic influence on HeLa cells that was dependent on cellular density [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF]. A similar pattern was observed for cannabidiol in HeLa cells. As compared with vehicle-treated cells, cell viability was significantly (P < 0.001, Student´s ttest) lowered by a 72-hour treatment with 10 µM cannabidiol at cellular densities lower than of 2.5 x 10 5 cells (Table 1).

To confirm the antiinvasive action of cannabidiol in another experimental setup, additional experiments were performed with HeLa cells that were first treated with cannabidiol or vehicle for 48 hours and trypsinized, counted and adjusted to a final cellular density of 0.5 x 10 5 cells per insert thereafter. Quantification of invasiveness was performed after a further incubation of cells in Boyden chambers for another [START_REF] Curran | Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis[END_REF]

Involvement of cannabinoid receptors and TRPV1 in cannabidiol´s anti-invasive action

As previously demonstrated, HeLa, A549 and C33A cells express CB 1 and CB 2 receptors as well as TRPV1 [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF]. To investigate the role of cannabinoid receptors and TRPV1 in cannabidiol-mediated reduction of HeLa cell invasiveness, the impact of antagonists of CB 1 receptor (AM-251), CB 2 receptor (AM-630) and TRPV1 (capsazepine) on the cannabidiolreduced invasion was tested. Inhibitors were used at a concentration of 1 µM which has been reported to be within the range of concentrations inhibiting CB 1 -, CB 2 -and TRPV1-dependent events [START_REF] Jacobsson | Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors[END_REF][START_REF] Mukherjee | Species comparison and pharmacological characterization of rat and human CB 2 cannabinoid receptors[END_REF]. As shown in Fig. 1D as well as in Table 2, cannabidiol-induced inhibition of HeLa cell invasion was prevented in the presence of AM-251, AM-630 or the combination of both antagonists that were added to the cultures 1 hour prior to cannabinoid stimulation.

Likewise, the TRPV1 antagonist capsazepine fully abolished the anti-invasive action of cannabidiol (Fig. 1D, Table 2). Cannabinoid receptor and TRPV1 antagonists tested without cannabidiol treatment left basal invasiveness virtually unaltered (data not shown).

Effect of cannabidiol on the expression of TIMP-1, MMP-2 and MMP-9

In experiments addressing the impact of cannabidiol on the expression of TIMP-1, cannabidiol at a concentration of 10 µM exhibited a time-dependent induction of TIMP-1 mRNA that became first evident after a 6-hour incubation period (Fig. 2A). In line with the invasion data, cannabidiol increased TIMP-1 protein levels in a concentration-dependent manner (Fig. 2B). Analysis of MMP-2 and MMP-9 revealed no alteration by either concentration of cannabidiol (Fig. 2B).

Impact of cannabinoid receptor-and TRPV1 antagonists on cannabidiol-elicited TIMP-1 induction

Experiments using cannabinoid receptor-and TRPV1 antagonists were performed to determine the receptor targets of cannabidiol involved in the induction of TIMP-1 expression.

According to Western blot analyses from cell culture supernatants obtained from the upper Boyden chambers of the respective invasion experiments, cannabidiol-induced TIMP-1

A c c e p t e d M a n u s c r i p t 14 expression was substantially reduced by antagonists to both CB 1 and CB 2 receptors (Fig. 2C). Furthermore, cannabidiol-induced TIMP-1 expression was abrogated by the TRPV1 antagonist capsazepine (Fig. 2C).

Effect of TIMP-1 knockdown on the anti-invasive action of cannabidiol

To further confirm a causal link between cannabidiol-mediated TIMP-1 induction and the accompanied decreased invasion, the expression of TIMP-1 was prevented by transfecting cells with TIMP-1 siRNA. As recently shown by our group, RNA interference with 0.25 µg/ml TIMP-1 siRNA elicits a reduction of cannabinoid-stimulated TIMP-1 expression without altering the basal level of HeLa cell invasiveness [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF]. To study the functional impact of cannabidiol-induced TIMP-1 expression on the observed decrease of invasiveness, TIMP-1 knockdown experiments were therefore performed with a final concentration of 0.25 µg/ml TIMP-1 siRNA.

Referring to Fig. 3A and Table 2, knockdown of TIMP-1 expression led to a significant abrogation of cannabidiol-mediated decrease of invasion, whereas cultures treated with a non-silencing sequence exhibited the same invasion pattern as controls treated with transfection agent only. Monitoring of TIMP-1 secretion into the culture medium of the upper Boyden chamber confirmed a substantial inhibition of cannabidiol-induced TIMP-1 expression in cannabidiol-treated cells additionally transfected with TIMP-1 siRNA (Fig. 3A).

The impact of TIMP-1 siRNA transfection on tumor cell invasion was also confirmed by staining cells invaded through Matrigel-coated membranes after removal of non-invaded cells from the upper surface of the transwell membrane. The pictures in Fig. 3B demonstrate a significantly lower number of cells invaded in the presence of cannabidiol and a reversal of this effect when cells were concomitantly treated with TIMP-1 siRNA. Again, non-interfering sequences elicited an invasive behavior comparable to controls lacking siRNA.

Effect of blockade of MAPK pathways on cannabidiol´s anti-invasive and TIMP-1-inducing action

In further experiments addressing the role of p38 and p42/44 MAPKs in the anti-invasive and TIMP-1-inducing action of cannabidiol, inhibitors of p38 MAPK activity (SB203580, 10 µM) and of p42/44 MAPK activation (PD98059, 10 µM) substantially inhibited both cannabidiol effects (Fig. 4A,B, Table 2). As previously described, SB203580 and PD98059 alone did not modulate baseline TIMP-1 expression and Matrigel invasion of HeLa cells [START_REF] Ramer | Upregulation of tissue inhibitor of matrix metalloproteinases-1 confers the anti-invasive action of cisplatin on human cancer cells[END_REF].

Additional inhibitor experiments were performed to confirm a causal link between receptor activation and MAPK phosphorylations. As shown in Fig. 4C, cannabidiol induced activation of p38 and p42/44 MAPKs at two early time points following stimulation. According to Fig. 4D, both activations were substantially suppressed by CB 1 -and CB 2 antagonists as well as by the TRPV1 antagonist.

Role of TIMP-1 in the anti-invasive action of cannabidiol in other tumor cell lines

To exclude that the demonstrated effects are restricted to HeLa cells, experiments were also performed in human lung carcinoma cells (A549, Fig. 5) and in another human cervical carcinoma cell line (C33A, Fig. 6).

Like in HeLa cells, addition of cannabidiol resulted in a profound suppression of invasion through Matrigel-coated transwell plates in A549 (Fig. 5) and C33A cells (Fig. 6). Decreased invasiveness was accompanied by increased TIMP-1 secretion with both events being significantly suppressed by a 1-hour preincubation with antagonists of CB 1 (AM-251, 1 µM) and CB 2 receptors (AM-630, 1 µM) alone or in combination and by a TRPV1 antagonist (capsazepine, 1 µM) in A549 (Fig. 5A) and C333A cells (Fig. 6A).

Furthermore, in both cell lines the blockade of p38 or p42/44 pathways led to a significant reconstitution of the cannabidiol-mediated decrease of Matrigel invasion (Fig. 5B and6B).

Like in HeLa cells, TIMP-1 levels in cell culture supernatants of the upper transwell inserts revealed an inverse correlation between invasiveness and TIMP-1 levels (Fig. 5B and6B).

A c c e p t e d M a n u s c r i p t 16

To further confirm the causal link between cannabidiol-mediated decrease of invasiveness and elevated TIMP-1 expression, additional RNA interference experiments were performed by targeting TIMP-1 expression with 0.25 µg/ml siRNA and equal concentrations of nonsilencing siRNA in both cell lines after a 72-hour incubation with 10 µM cannabidiol. As expected, transfection of A549 and C33A cells with TIMP-1 siRNA led to inhibition of cannabidiol´s effects on invasion and corresponding TIMP-1 expression (Fig. 5C and6C).

Measurement of cellular viability with the WST-1 test revealed no significant toxicity by cannabidiol in A549 cells (P = 0.411) and only a weak toxic effect in C33A cells (P = 0.039).

For viability analysis under conditions comparable to invasion assays, cells were treated with 10 µM cannabidiol for 72 hours at a density of 5 x 10 5 cells per well in a 48-well plate. The toxic effect became highly significant (P < 0.001, Student´s t-test) when cells of both lines were seeded at cell numbers of 2.5 x 10 5 per well (Table 1).

Impact of cannabidiol on lung metastases

We further investigated the impact of cannabidiol on tumor cell metastasis in vivo in a mouse model. For this purpose, A549 cells were injected intravenously through the lateral tail vein of athymic nude mice. According to Fig. 7, the number of lung metastatic nodules was significantly lower in cannabidiol-treated animals as compared to vehicle-treated controls.

The decrease of nodules was from an average of 6 nodules in vehicle-treated nude mice to an average of 1 nodule in cannabidiol-treated mice yielding an 84% inhibition of metastasis.

A c c e p t e d M a n u s c r i p t 17

Discussion

An increasing number of data obtained both in vivo and in vitro implicates a possible role for cannabinoids as systemic cancer therapeutics. Although cannabinoids virtually lack side effects of contemporarily used anticancer drugs such as chemotherapeutics-induced emesis and collateral toxicity, cannabinoids´ therapeutical use is limited by their psychotropic side effects. Therefore, recent research focussed on non-psychoactive cannabinoid-mimetic compounds such as cannabidiol.

In the present study we identified a cannabinoid receptor-and TRPV1-triggered expression of TIMP-1 as an important mediator of the anti-invasive action of cannabidiol. Cannabidiol was shown to decrease HeLa cell invasion in a time-and concentration-dependent manner.

Following a 72-hour incubation period, the decreased invasiveness was significant at a cannabidiol concentration as low as 0.01 µM which elicited a 33% inhibition when compared to vehicle control. Although this effect was less pronounced as compared to an equimolar concentration of THC which yielded a 68% inhibition of invasion in the same experimental setup [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF], the anti-invasive effects of cannabidiol described here occurred in a range of therapeutically relevant concentrations. Accordingly, referring to clinical data on healthy volunteers obtained from studies with Sativex™ (1:1 ratio of THC and cannabidiol) applied at a 10-mg buccal dose or at self-titrated doses during chronic therapy, cannabidiol peak plasma concentrations of 0.01 µM or up to 0.05 µM can be achieved in vivo [38]. Moreover, mean cannabidiol plasma levels of 0.036 µM were analyzed following a 6-week oral treatment with cannabidiol at doses of 10 mg/kg/day [START_REF] Consroe | Assay of plasma cannabidiol by capillary gas chromatography/ion trap mass spectroscopy following high-dose repeated daily oral administration in humans[END_REF].

With reference to a number of data reporting an anti-invasive effect of TIMP-1 on cancer cell invasion [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF][START_REF] Khokha | Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells[END_REF][26][START_REF] Park | Arsenic trioxide (As 2 O 3 ) inhibits invasion of HT1080 human fibrosarcoma cells: Role of nuclear factor-kappaB and reactive oxygen species[END_REF][START_REF] Park | Inhibitory effects of a benz(f)indole-4,9-dione analog on cancer cell metastasis mediated by the downregulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells[END_REF][START_REF] Ramer | Upregulation of tissue inhibitor of matrix metalloproteinases-1 confers the anti-invasive action of cisplatin on human cancer cells[END_REF][START_REF] Khokha | Antisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells[END_REF][START_REF] Chan | Intestinal trefoil factor promotes invasion in non-tumorigenic Rat-2 fibroblast cell[END_REF], the present work focussed on the causal link of cannabinoid receptorand TRPV1-mediated activation of MAPK pathways and subsequent TIMP-1 induction as upstream effectors of cannabidiol-mediated suppression of cancer cell invasion. Several lines of evidence presented in this study suggest TIMP-1 induction as a major event underlying cannabidiol´s anti-invasive action. First, antagonists of CB 1 and CB 2 alone or in A c c e p t e d M a n u s c r i p t 18 combination as well as an antagonist of TRPV1 caused a significant suppression of both cannabidiol-induced TIMP-1 expression and activation of the MAPKs p38 and p42/44, and a significant reconstitution of the subsequent suppression of cancer cell invasion. Second, inhibitors of p38 and p42/44 MAPKs pathway that have previously been demonstrated as upstream regulators of TIMP-1 [START_REF] Tong | Mitogen-activated protein kinases Erk1/2 and p38 are required for maximal regulation of TIMP-1 by oncostatin M in murine fibroblasts[END_REF][START_REF] Han | Induction by IL-1 beta of tissue inhibitor of metalloproteinase-1 in human orbital fibroblasts: modulation of gene promoter activity by IL-4 and IFN-gamma[END_REF] elicited a comparable reconstitution of cancer cell invasiveness and inhibition of TIMP-1 induction by cannabidiol. Third, and most convincing, posttranscriptional silencing of cannabidiol-induced TIMP-1 was associated with a removal of its anti-invasive action. A TIMP-1-dependent anti-invasive effect involving activation of cannabinoid receptors, TRPV1 and MAPKs was also proven in another human cervical cancer (C33A) as well as in a human lung cancer cell line (A549), implying that the observed mechanism is of general importance and not restricted to one cell line. Additionally and in line with its anti-invasive action, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. This finding is in agreement with a previous report in which cannabidiol treatment inhibited breast cancer metastasis [17].

An invasion-associated effect that is mainly determined by the ability of cancer cells to secrete matrix-degrading enzymes or to modulate their activity rather than on cellular motility was substantiated by the finding that reduced Matrigel invasion was not associated with a decrease of transmigration through uncoated Boyden chambers after cannabidiol treatment.

To rule out the possibility that decreased invasion by cannabidiol was an unspecific cytotoxicity-related phenomenon, control experiments were also performed to analyse cellular viability which revealed no significant cytotoxicity by cannabidiol under conditions of the experimental set-up of invasion monitoring. In line with recent data obtained with THC and R(+)-methanandamide [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF], cannabidiol showed a significant and progressive toxicity only when the density of HeLa, A549 and C33A cells was reduced. Such "inoculum effect" has been described for a number of chemotherapeutic drugs such as tamoxifen [START_REF] Brandt | Tamoxifen induces suppression of cell viability and apoptosis in the human hepatoblastoma cell line HepG2 via downregulation of telomerase activity[END_REF], doxorubicin and vincristine [START_REF] Kobayashi | Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects[END_REF]. Kobayashi et al. [START_REF] Kobayashi | Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects[END_REF] demonstrated that at high cell densities, a decreased cellular uptake of chemotherapeutics results in attenuated availability A c c e p t e d M a n u s c r i p t 19 of drug molecules at its intracellular binding sites. Finally, the antiinvasive effect of cannabidiol was confirmed to be comparable in another experimental setting, where HeLa cells treated with cannabidiol or vehicle for 48 hours were readjusted to equal numbers of cells prior to the 24-hour incubation in Matrigel inserts.

There are several issues raised in this study that have to be addressed in future experiments. First, the effect of cannabidiol on TIMP-1 was specific in that the expression of other regulable components of this systems, i.e. MMP-2 and MMP-9, were not influenced by cannabidiol on the level of protein expression. This finding stands in contrast to a recent study from our laboratory reporting a cannabinoid receptor-independent downregulation of MMP-2 by THC and R(+)-methanandamide [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF]. Second, in view of the low affinity of cannabidiol to either CB 1 and CB 2 receptors [START_REF] Thomas | Comparative receptor binding analyses of cannabinoid agonists and antagonists[END_REF], the involvement of cannabinoid receptors in the anti-invasive and TIMP-1-stimulatory action of cannabidiol was rather surprising. However, in line with our data cannabidiol has been reported to exert several of its effects, including modulation of cytokine release and macrophage chemotaxis [START_REF] Sacerdote | The nonpsychoactive component of marijuana cannabidiol modulates chemotaxis and IL-10 and IL-12 production of murine macrophages both in vivo and in vitro[END_REF] as well as antiproliferative [START_REF] Massi | Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines[END_REF] and proapoptotic properties [START_REF] Mckallip | Targeting CB 2 cannabinoid receptors as a novel therapy to treat malignant lymphoblastic disease[END_REF] in a cannabinoid receptor-dependent manner. One possible explanation for the discrepancy between the low receptor affinity and the apparent involvement of cannabinoid receptors in cannabidiol-mediated effects may be given by cannabidiol´s inhibitory action on fatty acid amidohydrolase activity that confers release of anandamide and increased receptor-mediated signalling by this endocannabinoid [START_REF] Watanabe | Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids[END_REF][17][START_REF] De Filippis | Effect of cannabidiol on sepsis-induced motility disturbances in mice: involvement of CB receptors and fatty acid amide hydrolase[END_REF]. Additionally, the non-selective cation channel TRPV1 seems to be involved in cannabidiol´s anti-invasive effect which is in line with recent findings demonstrating antihyperalgesic [START_REF] Costa | Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation[END_REF] and antiproliferative effects [17] of cannabidiol to be mediated via TRPV1. Third, more research is needed to understand the complete mode of action underlying the anti-invasive effect of cannabidiol. Thus, besides TIMP-1, other components such as a modulation of the urokinase plasminogen activator system (Ramer and Hinz, unpublished data) may contribute to the anti-invasive action as well.

Collectively, this is the first report on a cannabinoid receptor-and TRPV1-dependent antiinvasive action of cannabidiol that adresses the aspect of modulation of the matrix

  -well plates. Trypsinized and pelleted cells were suspended to a final concentration of 5 x 10 5 cells in 500 µl serum-free DMEM in each insert and treated with a final concentration of 10 µM cannabidiol (except Figure1Band the data in Table2) or ethanol vehicle for various incubation times. In Table2 thehalf maximal effective concentration (EC 50 ) of cannabidiol in eliciting inhibition of Matrigel invasion was used for confirming key invasion experiments. To address the role of cannabinoid-activated receptors, specific antagonists to cannabinoid receptors (AM-251, AM-630) or TRPV1 (capsazepine) were used at a final concentration of 1 µM. In further experiments, inhibitors of p38 MAPK (SB203580) or p42/44 MAPK activation (PD98059) were used at a final concentration of 10 µM. Both receptor antagonists and inhibitors of MAPK pathways were tested vs. DMSO vehicles by adding to the cultures 1 hour prior to starting the incubation with cannabidiol. Cannabidiol was added to the cultures without washing out the antagonists. DMEM containing 10% FCS was used as a chemoattractant in the companion plate. Following incubation in a humidified incubator at 37°C and 5% CO 2 for the indicated times, the noninvading cells on the upper surface of the inserts were removed with a cotton swab, and the viability of the cells on the lower surface was measured by the colorimetric WST-1 test (Roche Diagnostics, Mannheim, Germany). This cell viability test is based on the cleavage of the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6benzene disulfonate) by mitochondrial succinate-tetrazolium-reductase in metabolically active cells.

A

  c c e p t e d M a n u s c r i p t 10 loading control (LC) for protein analysis of supernatants.

  histopathological examination lung samples were fixed in 4% formaline. Paraffin sections were stained with hematoxylin and eosin.

  A c c e p t e d M a n u s c r i p t12

  A c c e p t e d M a n u s c r i p t13

A 1

 1 Figure legends

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 4 30 Figure 6

 4306 Figure 4

Figure 7

 7 Figure 7

  Fig.1

  Fig.4

31 Tables Table 1

 Tables1 Influence of cellular density on the viability of cannabidiol (CBD)-treated HeLa, A549 and C33A cells. Cells were seeded with the indicated number of cells per well of a 48-well plate in serum free DMEM and treated with 10 µM cannabidiol or vehicle, respectively. After a 72hour incubation time, cellular viability was measured by WST-1. Values are means + SEM of n = 4 experiments vs. corresponding vehicle control. * P < 0.05, *** P < 0.001, vs. corresponding vehicle control (Student´s t-test).

				32
	Seeded cell number 5.0 x 10 5	Vehicle 100 + 8.9	HeLa CBD 98.3 + 7.6	Cellular viability (%) A549 Vehicle CBD 100 + 8.4 90.7 + 6.3 M a n u s c r i p t C33A Vehicle CBD 100 + 5.2 82.8 + 4.1*
	2.5 x 10 5 1.0 x 10 5 0.5 x 10 5 0.1 x 10 5 A c c e p t e d 100 + 2.3 62.5 + 2.6*** 100 + 4.1 54.8 + 1.1*** 100 + 1.1 2.0 + 0.2*** 100 + 3.3 1.5 + 0.6*** 100 + 6.9 1.8 + 0.1*** 100 + 1.4 1.5 + 0.7*** 100 + 4.2 3.3 + 0.2*** 100 + 17.5 1.0 + 0.8***	100 + 6.0 100 + 8.4 100 + 6.2 100 + 1.3	56.2 + 3.6*** 12.1 + 2.6*** 11.4 + 0.8*** 10.6 + 4.4***

Table 2

 2 Involvement of cannabinoid receptors, TRPV1, p38 and p42/44 MAPKs and TIMP-1 in the anti-invasive action of 0.025 µM cannabidiol (CBD) on HeLa cells. Effect of a 1-hour pretreatment of cells with AM-251 (CB 1 antagonist; 1 µM), AM-630 (CB 2 antagonist; 1 µM) and capsazepine (TRPV1 antagonist; 1 µM) on the anti-invasive action of 0.025 µM cannabidiol (upper set). Effect of a 1-hour pretreatment with 10 µM SB203580 (inhibitor of p38 MAPK activity) and PD98059 (inhibitor of p42/44 MAPK activation) on the anti-invasive action of 0.025 µM cannabidiol (middle set), and effect of TIMP-1 siRNA (0.25 µg/mL) or nonsilencing siRNA (0.25 µg/mL) on the antiinvasive action of 0.025 µM cannabidiol (lower set). Cells were incubated with cannabidiol for 72 hours. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance of n = 4 experiments. *** P < 0.001; vs. corresponding vehicle control; ### P < 0.001, vs. cannabidiol (Student´s t-test).

	Invasion Index (%)

Acknowledgements

We thank Ilona Klammfuß and Kathrin Sievert-Küchenmeister (Institute of Experimental Surgery, University of Rostock, Germany) for their excellent technical assistance.

# This study was supported by grants from the Deutsche Krebshilfe e. V. (Bonn, Germany), Deutsche Forschungsgemeinschaft (SFB 539 TP BI.6) and the FORUN programme of the Medical Faculty of the University of Rostock

To analyze MAPK phosphorylations, immunoblots were probed with antibodies directed against the phosphorylated form of p38 or p42/44. Equal loading of lysates was ensured by probing membranes with antibodies against the nonphosphorylated forms of p38 and p42/44 MAPKs.


Annalisa Bruno

Luigia Di

Isabella Coletta

Giorgina Mangano

Maria Alessandra Alisi

Lorenzo Polenzani

Claudio Milanese

Paola Anzellotti

Emanuela Ricciotti

Melania Dovizio

Andrea Di

Stefania Tacconelli

Marta L Capone

Ph.D Paola Patrignani
email: ppatrignani@unich.it

Andrea Di Francesco

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Keywords: mPGE synthase-1, PGE 2, monocytes, inflammation, human whole blood

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Prostanoids are biologically active derivatives of arachidonic acid(AA) released from membrane phospholipids by phospholipases [START_REF] Funk | Prostaglandins and leukotrienes: advances in eicosanoid biology[END_REF]. AA is transformed into prostaglandin(PG)H 2 , through the activity of cyclooxygenase(COX) enzymes (i.e. COX-1 and COX-2), and subsequently PGH 2 is metabolized by terminal synthases to the biologically active prostanoids, i.e. prostacyclin(PGI 2 ), PGD 2 , PGF 2α , PGE 2 and thromboxane(TX)A 2 [START_REF] Funk | Prostaglandins and leukotrienes: advances in eicosanoid biology[END_REF]. In particular, the isomerization of PGH 2 to PGE 2 is catalyzed by three different isomerases: a cytosolic PGE synthase(cPGES) and two membranebound PGESs, mPGES-1 and mPGES-2 [START_REF] Kudo | Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis[END_REF]. Of these isomerases, cPGES and mPGES-2 are constitutive enzymes whereas mPGES-1 is mainly an induced isoform.

Several lines of evidence suggest that the mechanism of action of traditional non-steroidal antiinflammatory drugs(tNSAIDs) and NSAIDs selective for COX-2 (named coxibs) for the treatment of pain and inflammatory joint disease, is mainly through the inhibition of COX-2-dependent PGE 2 [START_REF] Fitzgerald | The coxibs, selective inhibitors of cyclooxygenase-2[END_REF]. Inhibition of constitutively expressed COX-1 in gastrointestinal(GI) tract and presumably in platelets by tNSAIDs seems to play a role in increased risk of upper GI bleeding/perforation [START_REF] Patrono | Cyclooxygenase-selective inhibition of prostanoid formation: transducing biochemical selectivity into clinical read-outs[END_REF]. In fact, coxibs which spare COX-1 are associated with lower GI toxicity [START_REF] Fitzgerald | The coxibs, selective inhibitors of cyclooxygenase-2[END_REF]. Although the inhibition of COX-2-dependent PGE 2 generation by tNSAIDs and coxibs is effective in ameliorating symptoms of inflammation and pain, a small but consistent increased risk of myocardial infarction has been detected in NSAID users [START_REF] Grosser | Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities[END_REF][START_REF] Rodríguez | Role of dose potency in the prediction of risk of myocardial infarction associated with nonsteroidal anti-inflammatory drugs in the general population[END_REF][START_REF] Patrignani | NSAIDs and cardiovascular disease[END_REF]. Such adverse reactions are probably linked to the inhibition of COX-2-derived PGI 2 in vascular endothelial cells in the absence of an almost complete and persistent inhibition of platelet COX-1-derived TXA 2 [START_REF] Grosser | Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities[END_REF][START_REF] Rodríguez | Role of dose potency in the prediction of risk of myocardial infarction associated with nonsteroidal anti-inflammatory drugs in the general population[END_REF][START_REF] Patrignani | NSAIDs and cardiovascular disease[END_REF]. Thus, a great interest is in the development of new anti-inflammatory drugs with a safer profile. Inhibitors of mPGES-1 are being developed for relief of pain -similarly to NSAIDs -but with the potential advantage of not affecting or perhaps causing a beneficial impact on the cardiovascular system [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. Inhibitors of mPGES-1 might circumvent adverse GI side-effects of unselective COX inhibitors. However, in the stomachs of mice lacking mPGES-1 a shift from PGE 2 to all other prostanoids, i.e. TXA 2 , PGD 2 , PGF 2 , and PGI 2 , was detected thus making extremely difficult to predict the clinical consequences of mPGES-A c c e p t e d M a n u s c r i p t 5 1 inhibition in the GI tract [START_REF] Boulet | Deletion of microsomal prostaglandin E 2 (PGE 2 ) synthase-1 reduces inducible and basal PGE 2 production and alters the gastric prostanoid profile[END_REF]. Further studies addressing this issue in appropriate animal models are required. mPGES-1 belongs to the MAPEG(membrane-associated proteins involved in eicosanoid and glutathione metabolism) superfamily [START_REF] Jakobsson | Identification of human prostaglandin e synthase: a microsomal, glutathione-dependent, inducible enzyme constituting a potential novel drug target[END_REF]. Similar to COX-2, mPGES-1 is highly up-regulated by proinflammatory stimuli and participates in elevated PGE 2 generation in inflammation [START_REF] Jakobsson | Identification of human prostaglandin e synthase: a microsomal, glutathione-dependent, inducible enzyme constituting a potential novel drug target[END_REF]. It is functionally coupled with COX-2 [START_REF] Murakami | Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2[END_REF], but differences in the promoter region between mPGES-1 and COX-2 [START_REF] Sampey | Microsomal prostaglandin E synthase-1: the inducible synthase for prostaglandin E2[END_REF] suggest divergent transcriptional mechanisms responsible for the inducible regulation of COX-2 and mPGES-1.

Deletion of mPGES-1 is associated with a profound inhibition of PGE 2 in vitro and in vivo and with a redirection of the PGH 2 substrate to other PG synthases, such as PGI 2 and PGD 2 , which might participate to the safe cardiovascular profile [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. Discrepant results have been found on the possible shift of PGH 2 metabolism to TXA 2 [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Trebino | Redirection of eicosanoid metabolism in mPGES-1-deficient marophages[END_REF]. Whether redirection of PGH 2 metabolism towards other PG synthases might be influenced by the extent of reduction of mPGES-1 and by cell-specific features is unknown.

Development of biomarkers to predict drug responses is useful for a rational selection of doses for efficacy and toxicity. Endogenous mediators of inflammation might be used as biomarkers for therapeutic effects [START_REF] Huntjens | Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors[END_REF][START_REF] Patrignani | Risk management profile of etoricoxib: an example of personalized medicine[END_REF]. Thus, the assessment of COX-2 inhibition by the evaluation of PGE 2 levels in whole blood in response to E. coli lipopolysaccharide (LPS) (an index of inducible COX-2 activity in monocytes) both in vitro and ex vivo (i.e. after dosing) [START_REF] Huntjens | Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors[END_REF][START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF] is considered a promising biomarker of drug response (analgesia, anti-inflammatory effects) in humans [START_REF] Huntjens | Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors[END_REF][START_REF] Patrignani | Risk management profile of etoricoxib: an example of personalized medicine[END_REF]. In fact, it has been shown that inhibition of LPS-induced PGE 2 by approximately 80% is sufficient to translate into clinical efficacy (analgesia) in humans [START_REF] Huntjens | Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors[END_REF].

The design, synthesis and characterization of a series of carbazole benzamides led us to the identification of several mPGES-1 inhibitors, among which AF3442[N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide] was identified as a lead compound and selected for further characterization [START_REF] Alisi | 3-aminocarbazole compounds, pharmaceutical composition containing the same and method for the preparation thereof[END_REF]. In the present study we aimed to characterize the contribution of mPGES-1 to PGH 2 metabolism in LPS-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids and expression of COX-isozymes and down-stream synthases in the presence of pharmacological inhibition by AF3442. We choose to study the effect of the compound in human monocytes because they represent an important cell-target of the clinical efficacy of mPGES-1 inhibitors. Interestingly, we found that inhibition of PGE 2 generation at clinically relevant ranges was not associated with any significant change of other prostanoids in human monocytes in vitro.

The functional relevance of this observation deserves to be investigated in vivo.

A c c e p t e d M a n u s c r i p t 7

Materials and Methods

Materials

AF3442 (Figure 1) was synthesized and characterized in the Angelini Research Center [START_REF] Alisi | 3-aminocarbazole compounds, pharmaceutical composition containing the same and method for the preparation thereof[END_REF]. MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) was purchased from Calbiochem (San Diego, CA, USA). RPMI-1640 and fetal calf serum (FCS) were purchased from GIBCO-Invitrogen (Carlsbad, CA, USA). Interleukin-1 (IL-1 ), penicillin/streptomycin, L-glutamine, LPS (derived from Escherichia Coli 026:B6), anti-rabbit peroxidase-conjugated IgG were from Sigma-Aldrich (St. Louis, MO, USA). PGH 2 substrate, COX-1, mPGES-1, mPGES-2, cPGES, TXS, PGIS polyclonal antibodies were from Cayman Chemical (Ann Arbor, MI, USA); -actin and anti-goat peroxidase-conjugated IgG were purchased from Santa Cruz (USA). COX-2 polyclonal antibody was kindly provided by Dr Stacia Kargman from Merk Frosst (Canada). Nucleofector Kit T was from Lonza (Cologne, AG, Germany). Ficoll-Paque and ECL plus were from GE Healthcare Life Sciences (Bucks, UK). Polyvinylidene fluoride (PVDF) membrane was purchased from Bio-Rad Laboratories (Hercules, CA).

Cloning and expression of human mPGES-1 in A549 cells

Cloning of human mPGES-1was performed isolating mRNA from A549 (human lung adenocarcinoma) cells cultured in RPMI-1640 supplemented with 2% (v/v) FCS and stimulated with IL-1β (10ng/ml) for 24 h. It was reverse transcribed into cDNA. Polymerase Chain Reaction amplification was then performed with specific primers [5'-CGGATCCGCCACCATGCCTGCCCACAGCCTG -3' (FW) and 5'-GCTCTAGAGTCACAGGTGGCGGGCCGC -3' (REV) containing BamHI and XbaI restriction sites respectively] and fragments of the expected 459 bp size were obtained, ligated in the bacterial expression vector pcDNA3.1 and transformed into JM109 competent cells. Plasmids were isolated and miniprep products from a few colonies were subjected to restriction analysis that confirmed the presence of the expected fragment pattern. Two clones were sequenced in comparison with a sequence from EMBL Databank (accession number AF027740) and a conservative mutation in A c c e p t e d M a n u s c r i p t 8 position 183 was found (a C replacing a T); however, the protein sequence alignement of the 2 clones with that of the reference mPGES-1 submitted in GenBank resulted in a 100% aminoacid identity. A549 cell lines were transfected in order to obtain a stable cell line overexpressing mPGES-1 enzyme. Transfection was performed in cuvettes by Nucleofector II (AMAXA) instrument using Nucleofector Kit T according to the manufacturer's instructions. The recombinant vector pcDNA3.1 (Neomicine resistant) ligated with human mPGES-1 was used for transfection.

Transfection efficiency was assessed transfecting cells with a GFP (green fluorescent protein)expressing vector and checking cell fluorescence by fluorescence microscopy after 4 and 24 h from transfection. After 48 h the medium was replaced with medium containing 400µg/ml G418 (neomycin) as selecting agent. Cells were grown until neomycin selection in untransfected cells was confirmed. Clones were amplified and subsequently tested for mPGES-1 expression by Western blot [START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF] and for PGE 2 production by a validated radioimmunoassay [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF]. Clone D8L was selected for its mPGES-1 high expression level.

Preparation of human mPGES-1-containing microsomal fraction

A549 and A549/human mPGES-1 (A549 clone D8L) cells maintained in culture were centrifuged, washed once with PBS and suspended at 10x10 6 cells/ml in lysis buffer [TrisHCl 15mM; sucrose 0.25M; EDTA 0.1mM; phenylmethylsulfonyl fluoride (PMSF) 2mM; Aprotinin 10µg/ml; Leupeptin 25µg/ml; glutathione 1mM]. The samples were kept on ice for 10 minutes. Then, the cells were lysed by 4 x 30 seconds sonication pulses in a BANDELIN Soniplus sonifier at 70% of maximum power, frequency 9. Cell debris was removed by centrifugation at 10 000g for 15 minutes at 4°C. The supernatant was centrifuged at 250 000g for 1 h and the membrane pellets were finally suspended in 0.1ml of PGEG plus buffer (i.e. potassium phosphate, 10mM, pH 7, glycerol, 20%, EDTA, 0.1mM, glutathione, 1mM) containing PMSF 2mM, Aprotinin 10µg/ml, Leupeptin 25µg/ml, for each milliliter of lysis buffer. Total protein amount was determined by the Bradford method (Bio-Rad Protein Assay) according to the manifacturer's instructions. Microsomal

Inhibition of recombinant human mPGES-1

Aliquots of microsomal preparation from mPGES-1-overexpressing A549 clone D8L (30 µg/ml protein concentration) were used for assessing the enzymatic activity using MultiPROBE II Automated Liquid Handling System (Perkin Elmer, Waltham, MA, USA) in the presence of 2.5mM reduced glutathione and 10µM PGH 2 substrate. AF3442 (0.01-10 M) was pre-incubated with reaction mixture containing microsomal preparation for 15 minutes at 4°C. Reaction was started by substrate addition (PGH 2 ) and stopped after 5 minutes at 4°C with 100μl of stop solution (40mM ferric chloride and 0.4mM citric acid). Samples were then subjected to PGE 2 quantification by previously described radioimmunoassay technique [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF]. MK-886 (0.1-100 M), an inhibitor of MAPEG proteins, such as leukotriene C 4 synthase and 5-lipoxygenase activating protein (FLAP), affecting mPGES-1 activity at higher concentrations [START_REF] Mancini | Cloning, expression, and up-regulation of inducible rat prostaglandin E synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis[END_REF][START_REF] Riendeau | Inhibitors of the inducible microsomal prostaglandin E 2 synthase (mPGES-1) derived from MK-886[END_REF] was assessed, as positive control.

Isolation of human monocytes and pharmacological treatment

Human monocytes were separated from buffy coat by Ficoll-Paque, as previously described [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF].

The contribution of COX-1 to prostanoid biosynthesis was minimized by pretreating the buffy coat with aspirin (300 M). Mononuclear cell layer (obtained by Ficoll-Hypaque density gradient centrifugation) was treated with EDTA (2mM) for 15 minutes at 37°C to remove platelets specifically adherent to the monocytes [START_REF] Praticó | Generation of 8-epiprostaglandin F2alpha by human monocytes. Discriminate production by reactive oxygen species and prostaglandin endoperoxide synthase-2[END_REF]. Cell suspensions routinely contained >90% monocytes [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF]. Monocytes (1.5-2x10 6 cell/ml) cultured in RPMI-1640 supplemented with 0.5% (v/v) of FCS, 1% (v/v) penicillin/streptomycin and L-glutamine (2mM), were incubated with LPS (10 g/ml ) (derived from Escherichia Coli 026:B6) at 37°C for 24 h in the presence of DMSO vehicle or increasing concentrations (0.01-100 M) of the mPGES-1 inhibitor, AF3442. This compound was dissolved in DMSO and 2 l of vehicle or stock solution was added to 1ml of cell suspension. PGE 2 , A c c e p t e d M a n u s c r i p t 10 TXB 2 (the stable hydrolysis product of TXA 2 ), PGF 2 and 6-keto-PGF 1 (the hydrolysis product of PGI 2 ) levels were measured in cell culture media by previously described and validated radioimmunoassay techniques [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF][START_REF] Patrono | Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects[END_REF], while COX-2, COX-1, mPGES-1, mPGES-2, cPGES, TXS and PGIS protein levels were evaluated in cell lysates by previously described Western blot techniques [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF].

Isolation of human platelets

Platelet-rich plasma was prepared by centrifugation of citrated blood at 200g for 15 minutes and platelets were isolated from plasma as previously described [START_REF] Patrignani | COX-2 is not involved in thromboxane biosynthesis by activated human platelets[END_REF][START_REF] Evangelista | De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin[END_REF] and resuspended in RPMI-1640 supplemented with 0.5% (v/v) FCS. Platelet suspensions (5x10 8 /ml; containing <1 leukocyte per 10 000 platelets) were analyzed for the levels of COX-2, COX-1, mPGES-1, mPGES-2, cPGES and TXS in cell lysates by Western blot technique [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF].

Effects of AF3442 on prostanoid generation in human whole blood

Whole blood was drawn from 13 healthy volunteers (age range: 23-35 years). The study was approved by the local Ethics Committee and informed consent was obtained from each subject. The inhibitory effect towards constitutive PGE 2 generation was assessed by evaluating PGE 2 levels generated in whole blood allowed to clot for 60 minutes at 37°C. In clotting whole blood, PGE 2 is mainly produced by platelets in response to endogenously generated thrombin through the activity of COX-1 [START_REF] Patrono | Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects[END_REF][START_REF] Patrignani | COX-2 is not involved in thromboxane biosynthesis by activated human platelets[END_REF][START_REF] Evangelista | De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin[END_REF][START_REF] Patrignani | Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects[END_REF] and cPGES and/or mPGES-2. In fact, mPGES-1 is not detectable in platelets (Figure 2). The parallel measurement of TXB 2 (the major product of endogenous AA metabolism in platelets), PGF 2 and 6-keto-PGF 1 was performed to verify a possible off-target effect of AF3442 on COX-1. The effects of AF3442 on inducible PGE 2 generation were studied by assessing the levels of PGE 2 produced in heparinized human whole blood stimulated for 24 h with LPS (10 g/ml), a stimulus for the induction of both COX-2 and mPGES-1 in monocytes [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Sciulli | Effects of acetaminophen on constitutive and inducible prostanoid biosynthesis in human blood cells[END_REF]. The simultaneous measurements of TXB 2 , PGF 2 and 6-keto-PGF 1 were performed to verify the impact of AF3442 on COX-2 and the possible redirection of the accumulated PGH 2 substrate towards other PG synthases. For the assessment of the effects on constitutive PGE 2 generation, peripheral venous A c c e p t e d M a n u s c r i p t 11 blood samples were drawn from healthy volunteers when they had not taken any NSAID during the 2 weeks preceding the study. For the assessment of the effects on LPS-induced PGE 2 generation, heparinized peripheral venous blood samples were drawn from the same subjects pretreated with 300mg aspirin 48 h before sampling to suppress the contribution of platelet COX-1 activity.

Different concentrations (0.001-100μM) of AF3442 or DMSO vehicle (2 l) were incubated with whole blood samples and then allowed to clot at 37°C for 1 h or with heparinized whole blood samples in the presence of LPS (10μg/ml) at 37°C for 24 h. Prostanoids were measured in serum or plasma by previously described and validated radioimmunoassays [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF][START_REF] Patrono | Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects[END_REF]. In these assays the least detectable concentration was 1-2 pg/ml for all prostanoids.

Western blot analysis

Microsomal fractions or cell lysates samples were loaded (30 or 25-50 µg/lane, respectively) onto 4-15% SDS-PAGE and transferred to PVDF membrane. Membranes were saturated with a solution of 5% non-fat milk in tris-buffered saline-0.1% Tween-20 (TBS-Tween-20), and then incubated with anti-COX-2, anti-COX-1, anti-mPGES-1, anti-mPGES-2, anti-cPGES, anti-TXS, anti-PGIS, or anti-β-actin polyclonal antibodies for 1 h at room temperature [START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF]. Then, the membranes were washed in TBS-Tween-20 and incubated with the secondary antibodies: anti-goat peroxidaseconjugated IgG for β-actin or anti-rabbit peroxidase-conjugated IgG for the other proteins. Finally, the membranes were washed in TBS-Tween-20 and then all blots were developed by using ECL plus detection according to the manufacturer's instructions.

Statistical Analysis

Results are expressed as mean±SEM. For the experiments of LPS-stimulated monocytes and whole blood, the production of PGE 2 , TXB 2 , PGF 2 and 6-keto-PGF 1 was subtracted from the levels of the prostanoids measured in the presence of vehicle (DMSO). Statistical analysis was performed with Student's t test or one-way ANOVA and Newman-Keuls multiple comparison test, using GraphPad Instat (version 3.00 for Windows). Values of P<0.05 were considered statistically

Effect of AF3442 on human recombinant mPGES-1

As shown in Figure 3B, AF3442 caused a concentration-dependent inhibition of PGE 2 generation by human recombinant mPGES-1 with and IC 50 of 0.06 M. In the same microsomal preparation, MK-886, used as a positive control, inhibited PGE 2 generation with an IC 50 value of 4.1 μM (not shown), a comparable value to that found by Mancini et al. [START_REF] Mancini | Cloning, expression, and up-regulation of inducible rat prostaglandin E synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis[END_REF] (IC 50 : 3.2 μM).

Expression of COX-isozymes and down-stream synthases and prostanoid generation in LPSstimulated human monocytes

As shown in Figure 4, in isolated human monocytes stimulated with LPS, COX-2, mPGES-1 were profoundly induced. A slight, but reproducible, increase of cPGES by only 2-fold was detected. In contrast, we detected a constitutive expression of COX-1, TXS, mPGES-2 which was not affected by LPS. PGIS was barely detectable (Figure 4). The spectrum of prostanoid generation (TXB 2 , PGE 2 , PGF 2 and 6-keto-PGF 1 ) in monocytes both unstimulated and LPS-stimulated is reported in the Table. LPS caused a significant increase of their generation. TXB 2 was the major product of AA (increased by 370-folds) while 6-keto-PGF 1 was the minor one (increased by 10-folds). PGE 2 and PGF 2 were increased at a similar extent (120-folds) (Table ).

Effects of AF3442 on prostanoid generation in LPS-stimulated human monocytes

Incubation of LPS-stimulated monocytes for 24 h with AF3442 caused a concentration-dependent reduction of PGE 2 biosynthesis with an IC 50 value of 0.41 M (Figure 5). At 1 M, AF3442 caused an inhibitory effect of 61±3.3% (P<0.01 versus DMSO vehicle) without significantly affecting the other prostanoids (TXB 2 , PGF 2 and 6-keto-PGF 1 : 25.1±5.1%, 21.1±4.1%, 14.8±4.8% of inhibition, respectively) (Figure 5). Similar effects were found at 10 M. In contrast, at 100 M, we detected a higher reduction in the biosynthesis of PGE 2 (71.2±2.4%, P<0.05 versus 1 and 10 M) (Figure 5). This additional inhibitory effect of PGE 2 was associated with a significant reduction of TXB 2 generation of 54.7±5.5% (P<0.01 versus DMSO vehicle). A lower reduction, but not A c c e p t e d M a n u s c r i p t 14 significant, was detected for PGF 2 and 6-keto-PGF 1 38.0±8.9% and 29.0±8.8%, respectively) (Figure 5). These data suggest a selective, but incomplete, inhibitory effect of AF3442 on mPGES-1 activity up to 10 M. At 100 M, PGE 2 reduction was associated with an inhibitory effect on TXB 2 suggesting a possible off-target effect on COX-2 activity and/or expression.

Effects of AF3442 on the expression of COX-isozymes and down-stream synthases in LPSstimulated human monocytes

As shown in Figure 6, AF3442 up to 10 M did not significantly affect the levels of COX-2, COX-1, mPGES-1, mPGES-2, cPGES and TXS. In contrast, at 100 M, the compound caused a significant (P<0.01 versus control) reduction only of COX-2 levels by 31.4±2.6% (assessed as ratio of their optical density normalized to the optical density of -actin).

Effects of AF3442 on prostanoid generation in human whole blood assays

To study whether the compound affected mPGES-1 activity even in the presence of plasma proteins, we assessed the effect of AF3442 on prostanoid generation in LPS-stimulated whole blood. Incubation of heparinized whole blood with LPS (10 g/ml) resulted in a significant (P<0.01) enhancement of PGE 2 , TXB 2 , PGF 2 and 6-keto-PGF 1 production (Table ). In this experimental model, PGE 2 and TXB 2 were the major prostanoid generated with PGE 2 that was higher than TXB 2 (P<0.05) (Table ). As reported in Figure 7A, AF3442 inhibited in a concentration-dependent fashion LPS-induced PGE 2 biosynthesis with an IC 50 of 29 M. This value was 70-fold higher than that found in isolated human monocytes (in the presence of low plasma protein level: 0.5% FCS) suggesting an important binding of the compound to plasma proteins. A significant and selective inhibition of mPGES-1 activity was detected at 10 and 100 M (38±14%, P<0.05 and 69±5%, P<0.01 versus vehicle, respectively). Up to 100 M, the other prostanoids were not significantly affected (Figure 7A).

A c c e p t e d M a n u s c r i p t

Then, we assessed the impact of AF3442 on thrombin-stimulated prostanoids which are generated mainly from platelet COX-1 and constitutive down-stream synthases. In fact, in platelets inducible COX-2 and mPGES-1 were not detectable (Figure 2). As shown in Figure 7B, the compound did not affect thrombin-stimulated PGE 2 and also other prostanoids.

Altogether, these results showed that the compound up to 100 M -causing a clinically relevant degree of inhibition of PGE 2 by approximately 70% -is a selective inhibitor of mPGES-1 in human whole blood.

A c c e p t e d M a n u s c r i p t 16

Discussion

Inhibitors of mPGES-1 are potential, novel anti-inflammatory drugs with the possible advantage of not affecting or perhaps causing a beneficial impact on cardiovascular homeostasis [START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. However, redirection of the PGH 2 substrate to other PG synthases in the presence of mPGES-1 deletion has been shown both in vivo and in vitro [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Trebino | Redirection of eicosanoid metabolism in mPGES-1-deficient marophages[END_REF]. This phenomenon might occur at different extent in different cell types, thus, translating into the generation of prostanoids with opposite effects for the cardiovascular system [START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. It is unknown whether this effect occurs also in the presence of an incomplete inhibition of mPGES-1, as it would take place after drug treatment. Thus, we studied the contribution of mPGES-1 to PGH 2 metabolism in LPS-stimulated isolated human monocytes and whole blood in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor, AF3442 [START_REF] Alisi | 3-aminocarbazole compounds, pharmaceutical composition containing the same and method for the preparation thereof[END_REF]. We aimed to explore this effect in human monocytes because: i) they are the target for clinical efficacy; ii) they play a key role in atherogenesis through the generation of PGE 2 , a mediator of inflammation, and TXA 2 , a potent proaggregatory and vasoconstrictor agent [START_REF] Egan | Cyclooxygenases, thromboxane, and atherosclerosis: plaque destabilization by cyclooxygenase-2 inhibition combined with thromboxane receptor antagonism[END_REF].

AF3442 affected LPS-induced mPGES-1 activity of human monocytes even in the presence of plasma proteins, i.e. in whole blood (Figure 7A). In whole blood at 100 M, it caused a clinically relevant degree of inhibition of PGE 2 by approximately 70% which was not associated with any significant change of the biosynthesis of other prostanoids.

AF3442 is a molecule belonging to the carbazole class of compounds [START_REF] Alisi | 3-aminocarbazole compounds, pharmaceutical composition containing the same and method for the preparation thereof[END_REF]. It was very potent towards recombinant human mPGES-1 (IC 50 : 0.06 M) and at 1 M, it completely suppressed the enzyme activity. In LPS-stimulated monocytes (in the presence of low concentrations of proteins, i.e. 0.5% FCS), AF3442 1 M caused a maximal, selective inhibition of PGE 2 of 61%. An almost comparable selective degree of inhibition of PGE 2 generation (by 69%) was obtained at 100 M of the compound in LPS-stimulated whole blood which suggests an extensive plasma protein binding by the compound. An important issue is whether this extent of inhibition of LPS-stimulated whole blood PGE 2 by AF3442 might be clinically relevant. We have previously looked at the degree of inhibition of LPS-stimulated whole blood PGE 2 by circulating peak concentrations of therapeutic A c c e p t e d M a n u s c r i p t doses of several NSAIDs [START_REF] Rodríguez | Role of dose potency in the prediction of risk of myocardial infarction associated with nonsteroidal anti-inflammatory drugs in the general population[END_REF]. Our results show that low-dose ibuprofen (1200 mg/day) caused a maximal inhibition of 75% [START_REF] Rodríguez | Role of dose potency in the prediction of risk of myocardial infarction associated with nonsteroidal anti-inflammatory drugs in the general population[END_REF]. Moreover, it has been shown that therapeutic plasma concentrations of analgesic doses of NSAIDs correlated with the IC 80 (drug concentration which inhibits enzyme activity by 80%) for inhibition of LPS-stimulated whole blood PGE 2 production in vitro [START_REF] Huntjens | Pharmacokinetic-pharmacodynamic correlations and biomarkers in the development of COX-2 inhibitors[END_REF]. Altogether these findings suggest the possible clinical efficacy of AF3442 which should be confirmed by in vivo studies.

Failure of AF3442 to completely suppress inducible PGE 2 generation could be explained by the contribution of other PGES, such as mPGES-2 which may couple to COX-2, to the synthesis of this prostanoid [START_REF] Ueno | Coupling between cyclooxygenases and terminal prostanoid synthases[END_REF]. Alternatively, it is possible that some PGH 2 isomerized to PGE 2 , nonenzymatically [START_REF] Nugteren | Chemical and enzymatic conversions of prostaglandin endoperoxide PGH 2[END_REF][START_REF] Soler | Human vascular smooth muscle cells but not endothelial cells express prostaglandin E synthase[END_REF]. However, in murine macrophages, in presence of deletion of mPGES-1, PGE 2 generation was reduced by more than 90% demonstrating that mPGES-1 activity is a key factor in LPS-induced PGE 2 production, in this cell-type [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Trebino | Redirection of eicosanoid metabolism in mPGES-1-deficient marophages[END_REF].

Unexpectedly, we showed that in the presence of an incomplete suppression of mPGES-1, shunting of COX-2-derived PGH 2 to other prostanoids did not occur. This finding was showed both in whole blood and in isolated monocytes.

In human monocytes, LPS induced the generation of TXB 2 , PGE 2 , PGF 2 and extremely low levels of PGI 2 . TXB 2 was the dominant product of monocytes exceeding by10-fold PGE 2 .

High levels of expression of TXS, together with efficiency of coupling between TXS and COX-2 [START_REF] Ueno | Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2[END_REF][START_REF] Capone | Pharmacodynamic of cyclooxygenase inhibitors in humans[END_REF] might be the determinants which contribute to the dominant generation of inducible TXA 2 in isolated monocytes. In fact, it has been shown that TXS efficiently couples to COX-2 when small amounts of AA are gradually supplied over a long period, such as in response to LPS [START_REF] Ueno | Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2[END_REF][START_REF] Capone | Pharmacodynamic of cyclooxygenase inhibitors in humans[END_REF]. In this scenario, mPGES-1 is not the dominant pathway of PGH 2 metabolism; thus its inhibition translates into a limited accumulation of PGH 2 which explains our failure to detect redirection towards TXB 2 and other prostanoids. This is different from the results obtained in thioglycollateelicited peritoneal macrophages stimulated in vitro with LPS [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Trebino | Redirection of eicosanoid metabolism in mPGES-1-deficient marophages[END_REF] showing that PGE 2 is the major prostanoid and deletion of mPGES-1 caused redirection towards TXB 2 and PGI 2 , but TXB 2 A c c e p t e d M a n u s c r i p t 18 predominated. However, Boulet et al. [START_REF] Boulet | Deletion of microsomal prostaglandin E 2 (PGE 2 ) synthase-1 reduces inducible and basal PGE 2 production and alters the gastric prostanoid profile[END_REF] studied the shunting of PGH 2 to alternative prostanoid pathways in the presence of complete and incomplete loss of mPGES-1 gene and they did not detect a significant redirection towards TXB 2 in peritoneal macrophages under similar experimental conditions. The reason of these discrepant results is unclear. It has been suggested that the use of mice with different genetic background might have played a role [START_REF] Trebino | Redirection of eicosanoid metabolism in mPGES-1-deficient marophages[END_REF].

We propose that differential expression of influx and efflux transport proteins may influence PGH 2 redirection towards other PG synthases. Excess PGH 2 may be immediately released outside the cell after intracellular biosynthesis -probably through the action of MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) [START_REF] Skazik | Differential expression of influx and efflux transport proteins in human antigen presenting cells[END_REF] or simple diffusion -and then it may be reuptaken, probably via the prostaglandin transporter [START_REF] Kanai | Identification and characterization of a prostaglandin transporter[END_REF][START_REF] Park | Prostaglandin E 2 synthesis and secretion: the role of PGE 2 synthases[END_REF]. Thus, shunting of PGH 2 to other prostanoids may be dependent on the expression of transport proteins in different cell types. This hypothesis will be explored in a specific study.

In order to verify the occurrence of PGH 2 redirection in the presence of mPGES-1 inhibition in a more physiologic system, we studied the effect of AF3442 in LPS-stimulated whole blood where TXB 2 , PGE 2 , PGF 2 and 6-keto-PGF 1 are mainly from monocytes through the activity of induced COX-2 [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF]. In this system, PGE 2 is the major prostanoid generated which was only slightly more abundant than TXB 2 (Table ). The lower concentration of PGE 2 generated in isolated monocytes versus whole blood may suggest the contribution of extramonocyte mPGES-1 to the generation of PGE 2 in whole blood. In particular, neutrophil mPGES-1, which has been shown to be constitutively expressed [START_REF] Mosca | Regulation of the microsomal prostaglandin E synthase-1 in polarized mononuclear phagocytes and its constitutive expression in neutrophils[END_REF], might act a part. However, even in whole blood where PGE 2 was the major prostanoid, we did not detect significant redirection of PGH 2 metabolism towards other prostanoids. These results may suggest that accumulated PGH 2 is rapidly released and not reuptaken by blood cells. Released PGH 2 may bind to plasma proteins with avidity thus preventing transcellular metabolism of PGH 2 between blood cells and leading to its degradation. Deletion of mPGES-1 retards atherogenesis and limits aortic aneurysm formation in hyperlipidaemic mice [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. However, it does not predispose to thrombogenesis and has a limited A c c e p t e d M a n u s c r i p t 19 impact on blood pressure compared to inhibition of COX-2 [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF]. These favourable effects for the cardiovascular system, in the presence of deletion of mPGES-1, may at least in part be due to augmented excretion of major metabolites of PGD 2 (PGD-M) and PGI 2 (PGI-M) [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease[END_REF][START_REF] Wang | Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress and angiotensin II-induced abdominal aortic aneurysm formation[END_REF].

Enhanced synthesis of PGI 2 in mPGES-1 deleted murine macrophages and vascular smooth muscle cells in vitro, was found by Wang et al. [START_REF] Wang | Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis[END_REF]. We did not confirm this phenomenon in human monocytes by the novel mPGES-1 inhibitor in vitro. However, it should be pointed out that in these cells PGI 2 was generated at very low-concentrations which is consistent with the very low levels of expression of PGIS detected in monocytes (Figure 4). The occurrence of increased biosynthesis of PGI 2 in vivo by pharmacological inhibition of mPGES-1 should be verified.

In human monocytes, in the absence of plasma proteins, a concentration of AF3442 100 M, i.e. 100-fold higher than that causing a maximal inhibition of mPGES-1, was associated with a small off-target effect on COX-2 expression. Whether down-regulation of COX-2 levels by 35% involved a transcriptional and/or a posttranscriptional mechanism remains to be investigated.

In conclusion, we have demonstrated that a selective mPGES-1 inhibitor reduced monocyte PGE 2 generation at clinically relevant ranges (i.e. approximately 70%) also in presence of plasma platelets. Platelet-rich plasma was prepared by centrifugation of citrated blood at 200g for 15 minutes and platelets were isolated from plasma as previously described [START_REF] Patrignani | COX-2 is not involved in thromboxane biosynthesis by activated human platelets[END_REF][START_REF] Evangelista | De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin[END_REF] and resuspended in RPMI-1640 supplemented with 0.5% (v/v) FCS (5x10 8 platelets per ml; containing <1 leukocyte per 10 000 platelets). Platelets were lysed and 25 g of total proteins were analyzed for COX-1, COX-2, TXS, cPGES, mPGES-2, mPGES-1 and -actin levels by SDS-PAGE and Western blot analysis [START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF]. The blot used for detection of COX-2 was stripped (stripping solution was from Chemicon International, USA) and reprobed for TXS. β-actin was used as protein loading control. The figure is representative of 3 different experiments. Monocytes were separated from buffy coat which was pretreated with aspirin 300 M for 5 minutes to suppress the contribution of COX-1 to prostanoid generation. Increasing concentrations of AF3442 (0.01-100µM) or DMSO vehicle were incubated with human monocytes (1.5-2x10 6 cell/ml) in the presence of LPS (10 g/ml) for 24 h at 37°C. PGE 2 , TXB 2 , PGF 2 and 6-keto-PGF 1 levels were assayed in culture media by specific radioimmunoassays [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF][START_REF] Francesco | Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells[END_REF][START_REF] Patrono | Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects[END_REF] incubated with 1ml of whole blood samples (drawn from healthy volunteers who did not take any NSAIDs for 2 weeks before blood collection) that were allowed to clot for 60 minutes to assess in vitro selective inhibiting effect of AF3442 towards COX-1-dependent prostanoids mainly of platelet origin [START_REF] Patrono | Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects[END_REF][START_REF] Patrignani | Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects[END_REF]. Since platelets do not express mPGES-1, PGE 2 generation may occur by the activity of cPGES and/or mPGES-2. Thus, this assay allowed to verify the possible effects of the compound towards other PGE synthases. PGE 2 , TXB 2 , PGF 2 and 6-keto-PGF 1 levels were measured by specific radioimmunoassays. Results were reported as % of control (DMSO vehicle) (mean±SEM)

  Abbreviations:

  at -80°C until used. The presence of the mPGES-1 protein in the microsomal preparation was confirmed by Western blot technique (Figure3A).

  -response curves were fitted (using PRISM, GraphPad, version 5.00 for Windows, San Diego, CA) and IC 50 values were reported.

  proteins. In human monocytes, mPGES-1-dependent PGH 2 metabolism represents a major metabolic route of PGE 2 generation, while other PGESs or non-enzymatic formation of PGE 2 represent minor pathways of this prostanoid biosynthesis. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH 2 metabolism towards other terminal PG synthases in monocytes. The functional consequences of the phenotype induced by pharmacological inhibition of mPGES-1 deserve to be investigated in vivo in suited experimental animals.

Figure 1 .Figure 2 .

 12 Figure 1. Structure of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]

Figure 3 .

 3 Figure 3. Effect of AF3442 on microsomal preparations of A549 (human lung

Figure 4 .

 4 Figure 4. Expression of COX-isozymes and down-stream synthases in LPS-stimulated human monocytes. Human monocytes were separated from buffy coat and 1.5-2x10 6 cell/ml were cultured

Figure 5 .

 5 Figure 5. Effects of AF3442 on prostanoid generation in LPS-stimulated human monocytes.

  . Results were reported as percentage of control (DMSO vehicle) (mean±SEM) from 3 separate experiments. §P<0.05, PGE 2 1 M and 10 M versus 100 M; **P<0.01, PGE 2 0.01-100 M and TXB 2 100 M versus control.

Figure 6 .

 6 Figure 6. Effects of AF3442 on the levels of COX-isozymes and down-stream synthases in LPS-stimulated human monocytes. Monocytes (1.5-2x10 6 cell/ml) were incubated with LPS

Figure 7 .

 7 Figure 7. Effects of AF3442 on LPS-and thrombin-induced prostanoids in human whole

A

  c c e p t e d M a n u s c r i p t 27 towards mPGES-1 and the possible redirection of PGH 2 metabolism to other PG synthases. Results were reported as % of control (DMSO vehicle) (mean±SEM) from 3 separate experiments. *P<0.05, PGE 2 (% of control ) at AF3442 10 M versus vehicle (DMSO); **P<0.01, PGE 2 (% of control ) at AF3442 100 M versus vehicle (DMSO); §P<0.01, PGE 2 versus PGF 2 , TXB 2 and 6keto-PGF 1 (% of control ), at AF3442 100 M. (B) Effects of AF3442 on prostanoid generation by whole blood COX-1-dependent pathway. AF3442 (0.001-100 M) or DMSO vehicle were

from 3

 3 separate experiments for AF3442 0.01-100 M and from 2 experiments for AF3442 0.001 M.

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Acknowledgements

This study was supported with a grant by A.C.R.A.F. to Paola Patrignani. The authors wish to thank Dr. Nicola Cazzolla, Dr. Angelo Guglielmotti and Dr. Giuseppe Biondi for revising the manuscript and for excellent scientific assistance.

Footnotes

Conflict of interest