Robert Ramer

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

CYCLOOXYGENASE-2 AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASES-1 CONFER THE ANTIMIGRATORY EFFECT OF CANNABINOIDS ON HUMAN TRABECULAR MESHWORK CELLS #

Cannabinoids have received considerable attention as potential antiglaucomatous drugs.

Recently, prostaglandins (PG) have been suggested to contribute to this effect. Within the factors conferring the development of glaucoma, depletion of the aqueous humor outflowregulating trabecular meshwork (TM) cells elicited by migration from the outflow system is considered to play a pivotal role. This study therefore investigates the impact of two cannabinoids, Δ 9 -tetrahydrocannabinol and R(+)-methanandamide, on the migration of human TM cells and the involvement of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), to this response. Using Boyden chamber assays cannabinoids were shown to elicit an antimigratory effect that was reversed by antagonists for CB 1 as well as CB 2 receptors and accompanied by upregulation of COX-2 and TIMP-1 expression and PGE 2 synthesis. Knockdown of cannabinoid-induced COX-2 or TIMP-1 expression by siRNA or inhibition of COX-2 activity by NS-398 led to a significant suppression of this antimigratory action. Migration was also diminished by the major COX-2 product PGE 2 and by recombinant TIMP-1. Experiments using selective E prostanoid (EP) receptor agonists and antagonists revealed that decreased migration by PGE 2, THC and MA was mediated via EP 2 and EP 4 receptors. Finally, the cannabinoid-mediated increases of TIMP-1 levels were abolished by NS-398 and PGE 2 was shown to elicit a concentrationdependent increase of TIMP-1. Collectively, this data demonstrate a COX-2-dependent upregulation of TIMP-1 conferring the antimigratory action of cannabinoids. A decreased migration reducing TM cell loss in glaucoma might be involved in the antiglaucomatous action of cannabinoids.

During past years cannabinoids have received considerable attention as potential antiglaucomatous drugs [START_REF] Jarvinen | Cannabinoids in the treatment of glaucoma[END_REF]. The intraocular pressure (IOP)-lowering action of Δ 9 tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, was first reported in 1971 [START_REF] Hepler | Marihuana smoking and intraocular pressure[END_REF]. In the following years, various human or animal studies have confirmed IOP decreases by marijuana, THC and classic cannabinoid derivatives [START_REF] Flom | Marijuana smoking and reduced pressure in human eyes[END_REF][START_REF] Purnell | Δ 9 -tetrahydrocannabinol, euphoria and intraocular pressur in man[END_REF][START_REF] Colasanti | Ocular hypothensive effect of marihuana canabinoids: Correlate of central action or separate phenomenon?[END_REF]. Likewise, the endogenous cannabinoid, anandamide, and its stable analog, R(+)-methanandamide (MA), have been shown to cause IOP reduction [START_REF] Pate | Ophthalmic arachidonylethanolamide decreases intraocular pressure in normotensive rabbits[END_REF][START_REF] Juntunen | Anandamide prodrugs. 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide[END_REF]. Despite this, the molecular mechanism underlying the IOP-lowering and antiglaucomatous action of cannabinoids remains to be established. On the basis of inhibitor studies with non-steroidal antiinflammatory drugs (NSAIDs), the IOP-lowering effects of anandamide and THC [START_REF] Pate | Effects of topical anandamides on intraocular pressure in normotensive rabbits[END_REF][START_REF] Green | Interaction between Δ 9 -tetrahydrocannabinol and indomethacin[END_REF], have been associated with the generation of prostaglandins (PG) which are known to facilitate the outflow of aqueous humor [START_REF] Weinreb | Effects of prostaglandins on the aqueous humor outflow pathways[END_REF]. However, despite one study showing an upregulation of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and a subsequent PG-dependent induction of matrix metalloproteinases (MMP) by cannabinoids in non-pigmented ciliary epithelial cells [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF], no further mechanistic studies have been performed in this context so far.

Within the factors controlling IOP a pivotal role is ascribed to the trabecular meshwork (TM), a smooth muscle-like tissue with contractile properties in the anterior chamber angle of the eye that regulates the aqueous humor outflow [for review see 12]. Malfunctioning of TM may lead to elevation of intraocular pressure and development of glaucoma [START_REF] Zhou | Alteration of cytoskeletal structure, integrin distribution, and migratory activity by phagocytic challenge in cells from an ocular tissue--the trabecular meshwork[END_REF]. Moreover, depletion of TM cells is considered to play a pivotal role within the factors conferring the development of primary open-angle glaucoma (POAG). In fact, the age-related TM cell loss is even more pronounced in patients with primary open-angle glaucoma (POAG) than in agematched normals [START_REF] Grierson | The effects of age and antiglaucoma drugs on the meshwork cell population[END_REF][START_REF] Alvarado | Trabecular meshwork cellularity in primary openangle glaucoma and non glaucomatous normals[END_REF][START_REF] Grierson | Age-related depletion of the cell population in the human trabecular meshwork[END_REF][START_REF] Grierson | The proliferative and migratory activities of trabecular meshwork cells[END_REF]. In this context detachment from the trabeculae and migration from the outflow system stimulated by factors present in aqueous humor has been suggested as one mechanism conferring meshwork cell depletion in POAG [START_REF] Grierson | Age-related depletion of the cell population in the human trabecular meshwork[END_REF][START_REF] Grierson | The proliferative and migratory activities of trabecular meshwork cells[END_REF][START_REF] Grierson | Characteristics of meshwork cells and age changes in the outflow system of the eye: their relevance to primary open angle glaucoma[END_REF][START_REF] Hogg | Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro[END_REF].

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

Recently, the TM has been implicated as a target of cannabinoid action. Accordingly, various cannabinoids including anandamide [START_REF] Njie | N-arachidonylethanolamide-induced increase in aqueous humor outflow facility[END_REF], 2-arachidonylglycerol [START_REF] Njie | Aqueous humor outflow effects of 2arachidonylglycerol[END_REF], noladin ether [START_REF] Njie | Noladin ether acts on trabecular meshwork cannabinoid (CB 1 ) receptors to enhance aqueous humor outflow facility[END_REF],

WIN55212-2 [START_REF] Mcintosh | Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells[END_REF] and JWH015 [START_REF] Zhong | CB 2 cannabinoid receptors in trabecular meshwork cells mediate JWH015-induced enhancement of aqueous humor outflow facility[END_REF] were shown to act on trabecular meshwork cells to enhance aqueous humor outflow. In two further studies, cannabinoids have been demonstrated to exhibit an inhibitory action on the migration of TM cells by virtue of CB 1 and/or CB 2 receptor activation [START_REF] Kumar | CB 1 cannabinoid receptor-mediated changes of trabecular meshwork cellular properties[END_REF][START_REF] He | Molecular and cellular changes induced by the activation of CB 2 cannabinoid receptors in trabecular meshwork cells[END_REF].

The present study investigates the mechanisms underlying the antimigratory action of cannabinoids on human TM cells. Based on our previous investigations demonstrating cannabinoids as potent inductors of COX-2 expression in diverse cell types [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF][START_REF] Ramer | Ceramide is involved in R(+)methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells[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][START_REF] Hinz | R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts[END_REF][START_REF] Eichele | R(+)-methanandamide-induced apoptosis of human cervical carcinoma cells involves a cyclooxygenase-2-dependent pathway[END_REF][START_REF] Ramer | Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1[END_REF] and on the above mentioned interference of NSAIDs with the IOP-lowering action of cannabinoids, we were particularily interested in the role of COX-2 and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), in this process. Here we show that the antimigratory action of the phytocannabinoid THC and the stable endocannabinoid analogue MA on human TM cells is conferred by a COX-2dependent upregulation of TIMP-1. Although more studies are needed, a decreased COX-2driven migration and consequent reduced TM cell loss could be one factor contributing to the antiglaucomatous action of cannabinoids.

A c c e p t e d M a n u s c r i p t 6

MATERIALS AND METHODS

Materials

MA and recombinant human TIMP-1 were purchased from Calbiochem (Bad Soden, Germany and Triton ® X-100 were bought from Applichem (Darmstadt, Germany). Aprotinine, leupeptine, phenylmethylsulfonyl fluoride and THC were obtained from Sigma (Taufkirchen, Germany).

2.2.

Cell culture

An immortalized simian virus (SV40) transformed human trabecular meshwork (HTM) cell line was kindly provided by Prof. Ernst Tamm, Regensburg, Germany [START_REF] Junglas | Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells[END_REF]. Cells were maintained in DMEM supplemented with 10% heat-inactivated 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

Migration assay

The effect of test substances on the migration of TM cells was determined using Boyden chambers according to the manufacturer´s instructions (BD Biosciences, Oxford, UK). In this assay, cellular motility is monitored by transmigration through transwell inserts (8 µM pore size) towards a chemoattractant. In brief, 1 x 10 5 cells in a volume of 500 µl serum-free DMEM were seeded into the upper sides of the transwell inserts and treated with test substances or vehicles for the indicated times. DMEM containing 10% FCS was used as a chemoattractant in the companion plate. Following incubation at 37°C and 5% CO 2 atmosphere for 24 h, the non-migrated cells on the upper surface of the inserts were removed with a cotton swab. For calculation of migration, the viability of the migrated cells on the lower side of uncoated chambers was determined 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]-1.6-benzene
disulfonate) by mitochondrial succinate-tetrazolium-reductase in metabolically active cells.

To exclude the possibility that the effect of cannabinoids on migration was an unspecific cytotoxicity-related phenomenon, cell viability was analyzed after cannabinoid exposure in quadruplicate. For this purpose, cells were seeded into 48-well plates at 1 x 10 5 cells per well to match conditions of migration assays. Viability was measured subsequently using the WST-1 test.

Wound-healing assay

To visualize migration human TM cells were seeded on 4-well chamber slides coated with 40.5 µg Matrigel per well and were allowed to grow to complete confluence. A plastic pipette tip was used for gently scratching the cell monolayer to create a cleared area. Subsequently, cells were washed extensively with phosphate-buffered saline to remove cellular debris.

Thereafter, cells were incubated with the indicated substances and the respective vehicles daily, and wound closure was monitored for 72 h. Cell images of marked regions along the wound area were obtained using an inverted microscope attached to a camera. To gain

2.5.

Quantitative RT-PCR analysis

Human TM cells were seeded into 24-well plates at a density of 1 x 10 5 cells per well and were grown to confluence. Following incubation of cells with the respective test compounds or its vehicles for the indicated times, cell culture media were removed and cells were lysed for subsequent RNA isolation. Total RNA was isolated using the RNeasy total RNA Kit (Qiagen, Hilden, Germany). β-Actin-(internal standard), COX-2 and TIMP-1 mRNA levels were determined by quantitative real-time RT-PCR using the TaqMan ® RNA-to-C T ™ Kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer´s instruction.

Primers and probe for human ß-actin, COX-2, mPGES-1, mPGES-2 and TIMP-1 were Gene Expression Assay™ products. (Applied Biosystems, Darmstadt, Germany).

Western Blot Analysis

For analysis of COX-2 and ß-actin protein levels human TM cells were seeded into 6-well plates at a density of 2 x 10 5 cells per well and were grown to confluence. Following incubation with test substances or its vehicles for the indicated times, cell lysates were used for further analyses. Cells were 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 phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 10 µg/ml aprotinin], homogenized by sonication, and centrifuged at 10,000 g for 5 min. Cell culture media were used for Western blot analysis.

Total protein in the cell culture media were measured using the bicinchoninic acid assay (Pierce, Rockford, IL, USA).

For analysis of TIMP-1 protein levels human TM cells were seeded into 24-well plates at a density of 2 x 10 5 cells per well and were grown to confluence. Following the respective A c c e p t e d M a n u s c r i p t 9 incubation time, cell culture media were collected and centrifuged at 500 x g. Cell culture media were used for subsequent Western blot analysis of TIMP-1. Total protein was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA).

Proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel. Following transfer to nitrocellulose and blocking of the membranes with 5% milk powder, blots were probed with specific antibodies raised to COX-2 (BD Biosciences, Heidelberg, Germany), ßactin (Calbiochem, Bad Soden, Germany) or TIMP-1 (New England BioLabs GmbH, Frankfurt, Germany). Membranes were probed with horseradish peroxidase-conjugated Fabspecific anti-mouse IgG (New England BioLabs GmbH, Frankfurt, Germany) for detection of TIMP-1 and COX-2 protein expression. Antibody binding was visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Freiburg, Germany). Densitometric analysis of 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.

In case of the COX-2 blots equal loading of lysates was ensured by hybridisation of membranes with an antibody against ß-actin. To ensure that equal amounts of protein in cell culture media used for protein analysis of TIMP-1 had been transferred to the membrane, proteins on Western blot membranes were stained with Ponceau S. To ascertain equal protein loading in Western Blots of cell culture media obtained from upper Boyden chambers, a band with a size of about 65 kDa that appeared unregulated is shown as a loading control (LC) for protein analysis of cell culture media.

For determination of cannabinoid receptors, membrane fractions of proteins were obtained using the Proteo Extract™ Native Membrane Protein Extraction Kit according to the manufacturer´s instructions (Calbiochem, Bad Soden, Germany). Subsequently, membrane fractions were concentrated using Microcon YM-10 centrifugal filter units (Millipore GmbH, Schwalbach, Germany) with a 10-kDa cutoff with 12.000 x g for 45 minutes. The blots were probed with antibodies raised to the CB 1 -(Santa Cruz, Heidelberg, Germany) or the CB 2 receptor (Calbiochem, Bad Soden, Germany). (Qiagen GmbH, Hilden, Germany). The target sequences of the siRNAs were as follows: 5´gagcaatatgtcagtcactaa-3´ (CB 1 ), 5´-ttccggaatcatctacaccta-3´ (CB 2 ), 5´aactgctcaacaccggaattt-3´ (COX-2) and 5´-tcccatctttcttccggacaa-3´ (TIMP-1). The sequences of the siRNAs were controlled via BLAST search and did not show any homology to other known human genes. Transfections were performed according to the manufactures´s instructions. For migration assays cells grown to confluence were transfected with COX-2 siRNA (2.5 µg/ml), TIMP-1 siRNA (0.25 µg/ml) or non-silencing siRNA as respective negative control with an equal ratio (w/v) of RNA to transfection reagent for 24 h in DMEM supplemented with 10% FCS. Subsequently, cells were trypsinized, centrifuged, resuspended to a final density of 1 x 10 5 cells per 500 µl in serum-free DMEM containing the same amounts of siRNA or non-silencing siRNA to provide constant transfection conditions. Afterward, cells were incubated for another 24-h period and migration was analyzed as described above. Transfection efficacy was monitored in parallel experiments by Western blot analysis as described above. For siRNA transfection targeting cannabinoid receptors, cells grown to confluence were transfected with CB 1 , CB 2 or nonsilencing siRNA (2.5 µg/ml) in serum-free DMEM for 72 h. Subsequently, membrane fractions were purified as decribed above.

Determination of PGE 2

Human TM cells were seeded into 24-well plates at a density of 2 x 10 5 cells per well and were grown to confluence. Following incubation with test substances or its vehicles for the indicated times. PGE 2 concentrations in cell culture media were determined using a commercially available enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). PGE 2 amounts were normalized to protein levels and calculated as percent of vehicle control.

Antimigratory and COX-2-inducing action of THC in human TM cells

The effect of THC on the migration of TM cells was determined using Boyden chambers. In this assay cellular motility is monitored by transmigration through transwell inserts towards FCS-containing medium being used as a chemoattractant. As shown in Fig. 1A, migration of human TM cells was diminished by a 24-h incubation with THC. Analysis of earlier incubation times (i.e., 4, 8, 12 h) revealed no antimigratory effect of THC (data not shown). Inhibition of migration by THC was shown to occur in a concentration-dependent manner and was even significant at concentrations as low as 0.01 µM (Fig. 1A). To rule out the possibility that decreased migration by THC was an unspecific cytotoxicity-related phenomenon, cellular viability was measured. However, under experimental conditions very similar to those used for migration assays (1 x 10 5 cells per well, 24-h incubation) incubation with THC left viability of TM cells virtually unaltered (Fig. 1A). Moreover, the antimigratory action of THC was confirmed using the wound-healing assay. According to the phase-contrast images presented in Fig. 1B, THC concentration-dependently inhibited TM cell migration into the wounded area.

In experiments addressing the impact of THC on the expression of COX-2 at the time of its antimigratory effect, the phytocannabinoid exhibited a concentration-dependent induction of COX-2 at the protein level (Fig. 1C). In further analyses an increase of COX-2 mRNA and PGE 2 levels by THC was shown (Fig. 1D). Following a 24-h treatment of cells with THC were used at a concentration of 1 µM which has been reported to be within the range of concentrations inhibiting CB 1 -and CB 2 -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. 2A, THCinduced inhibition of TM cell migration was totally prevented by both antagonists. This effect appeared to be further substantiated by the combination of CB 1 -and CB 2 antagonists (Fig. 1A). In addition, an interference of AM-251 and AM-630 with the antimigratory effect of THC was observed in the wound-healing assay (Fig. 2B). With respect to COX-2 induction by THC, both CB receptor antagonists were shown to elicit a pronounced inhibition of THCelicited COX-2 expression as assessed by Western blotting experiments (Fig. 2C). Finally, the expression of CB 1 and CB 2 receptors in TM cells was proven in respective membrane fractions (Fig. 2D). To further substantiate the specificity of the bands shown in the Western blots, knockdown of either CB 1 and CB 2 receptors was performed by transfection of siRNA targeting cannabinoid receptors. As shown in Fig. 2D both CB 1 -and CB 2 siRNA substantially reduced the expression of its particular target, whereas leaving the expression of the respective non-targeted CB receptor virtually unaltered.

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

Role of COX-2 in the antimigratory effect of THC

In a first attempt to link THC-induced COX-2 expression to the observed decrease of migration, TM cells were treated with THC in the presence of NS-398 at a concentration proven to interfere with THC-induced PGE 2 synthesis (Fig. 1D). As shown in Fig. 1A andB, NS-398 suppressed the antimigratory action of THC in both the Boyden chamber as well as in the wound-healing assay. The involvement of COX-2 in the antimigratory effect of THC was confirmed by experiments showing that transfection of cells with COX-2 siRNA completely inhibited the diminished migration by THC (Fig. 3C). At the concentration tested COX-2 siRNA was demonstrated to significantly interfere with THC-induced COX-2 protein expression (Fig. 3C). Control experiments revealed no significant effect of non-silencing siRNA on both the antimigratory and COX-2-inducing action of THC (Fig. 3C).

Antimigratory effect of the major COX-2 product PGE 2 and role of diverse EP receptors in this action

To further confirm the pivotal role of COX-2 in the antimigratory action of THC, additional experiments were performed using the major COX-2 product PGE 2 . According to Fig. 4A, PGE 2 decreased the migrative behaviour of TM cells in a concentration-dependent and significant manner. By contrast, agonists to the FP receptor (PGF 2α , latanoprost) and the TP receptor (U-46619; stable analog of prostaglandin H 2 that mimickes thromboxan A 2 activity [START_REF] Abramovitz | The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs[END_REF]) did not confer inhibition of TM cell migration (Fig. 4B). Analysis of the time-course of the antimigratory effect of 10 µM PGE 2 revealed a much faster action as compared to THC. The respective percentual values relative to control (100%) were as follows: 86.4%  6.3% at 4 h; 63.9%  4.9% at 8 h (P < 0.01); 47.2%  5.9% at 12 h (P < 0.001) and 38.4%  1.3% at 24 h (P < 0.001). On the basis of this data, the 24-h incubation protocol conferring the maximum inhibition by PGE 2 was used for further analyses.

In a first approach to determine the EP receptor subtype involved in PGE 2 -mediated decrease of TM cell migration, different EP receptor agonists were tested. According to Fig. To confirm a possible involvement of EP 2 and EP 4 receptor signaling in PGE 2 -mediated decrease of TM cell migration, additional experiments were performed using the EP 1 /EP 2 receptor antagonist, AH-6809, and the selective EP 4 receptor antagonist, GW627368X. As shown in Fig. 4D and4E, both compounds significantly suppressed the antimigratory action of PGE 2 . In contrast, the EP 1 receptor antagonist SC19220 was inactive in this respect (Fig. 4E). To provide evidence for a PGE 2 -dependent mechanism underlying the antimigratory action of THC, the same receptor antagonists were coincubated with THC to address this issue. According to Fig. 4F the antimigratory effect of THC was reversed by GW627368X

and AH-6809 (Fig. 4F). Again, this effect was not observed in the presence of SC19220 (Fig. 4F).

3.5.

Role of TIMP-1 in the antimigratory effect of THC

To provide evidence for a downstream target of COX-2 in conferring the antimigratory action of THC on human TM cells, the role of TIMP-1 in this process was focused on in further experiments. Western blot analysis of cell culture media revealed a profound upregulation of TIMP-1 in the presence of 1 µM THC after a 24-h incubation period (Fig. 5A,C,D).

In a first attempt to link the observed THC-mediated antimigratory effect with a potential upregulation of TIMP-1, TM cells were transfected with TIMP-1 siRNA. Monitoring of TIMP-1 secretion into the cell culture media confirmed a profound inhibition of TIMP-1 expression in cells transfected with TIMP-1 siRNA as compared to control samples (Fig. 5A). As further shown in Fig. 5A, knock-down of TIMP-1 expression led to an almost complete abrogation of the THC-mediated decrease of migration. By contrast, cultures treated with a non-silencing sequence exhibited the same migration pattern as control cells treated with transfection agent only (Fig. 5A). The functional relevance of TIMP-1 upregulation was substantiated by demonstrating that recombinant TIMP-1 significantly inhibited TM cell migration at concentrations not interfering with the viability of TM cells (Fig. 5B).

The proposed function of TIMP-1 as a downstream target of COX-2 was focused on in experiments using NS-398. In fact, inhibition of COX-2 activity by this compound was associated with a significant reduction of THC-induced TIMP-1 protein levels (Fig. 5C).

Moreover, the COX-2 product PGE 2 was shown to elicit a concentration-dependent induction of TIMP-1 levels in cell culture media of TM cells (Fig. 5D). Finally and in line with the migration and COX-2 data provided earlier (Fig. 2A,B,C), inhibitor experiments revealed TIMP-1 induction by THC as an effect reversible by both CB 1 and CB 2 receptor antagonists (Fig. 5E).

3.6.

Confirmation of the proposed antimigratory mechanism by use of another cannabinoid

To determine whether the contribution of COX-2 and TIMP-1 to impaired migration of TM cells was unique for THC or also shared by another cannabinoid, additional experiments were performed with MA, a stable anandamide analogue. According to the data presented in Fig. 6 and Table 1, MA elicited a concentration-dependent (Fig. 6A) and CB 1 /CB 2 receptordependent (Table 1) decrease of TM cell migration. As shown for THC, the antimigratory effect of MA was confined to the investigated 24-h incubation time point and was not observed earlier (i.e., at 4, 8, 12 h) (data not shown). This functional effect was accompanied by a CB 1 /CB 2 receptor-dependent upregulation of COX-2 expression and activity (Fig. 6B,C) as well as TIMP-1 protein expression (Fig. 6F). As shown in Fig. 6C the MA-induced increased release of PGE 2 into cell culture media was abolished in the presence of NS-398. Again, inhibition of MA-induced COX-2 activity by NS-398 (Table 1) or knockdown of TIMP-1 expression by TIMP-1 siRNA (Fig. 6D) led to a reversal of the cannabinoid-elicited decrease in TM cell migration. As shown before for THC, the MA-mediated increase of TIMP-1 levels was likewise abolished by NS-398 (Fig. 6E).

The upregulation of TIMP-1 protein levels as shown in Fig. 6D,E 1). As shown before for PGE 2 and THC, SC19220, on the other hand, was inactive in this respect (Table 1).

A c c e p t e d M a n u s c r i p t 18 [START_REF] Purnell | Δ 9 -tetrahydrocannabinol, euphoria and intraocular pressur in man[END_REF].

DISCUSSION

Recent investigations have provided evidence that cannabinoids mediate its IOP-lowering action, at least in part, through the release of endogenous PGs. However, the molecular mechanisms as well as the functional consequence of cannabinoids´ PG-elevating action are poorly understood.

The results from the present study show that newly expressed COX-2 confers the antimigratory effect of the phytocannabinoid THC and the stable anandamide analogue MA on human TM cells. There are several lines of evidence supporting this notion: First, significant elevations of COX-2 mRNA and protein as well as increased PGE 2 levels were observed at the time of the antimigratory effect. Second, inhibition of cannabinoid-induced PGE 2 formation with the selective COX-2 inhibitor NS-398 was associated with a reversal of the antimigratory action. Third, transfection of cells with COX-2 siRNA mimicked the inhibitory effect of NS-398 on THC-elicited decrease of migration. Fourth, an antimigratory effect was also elicited by PGE 2 , the major prostanoid of the COX-2 pathway [START_REF] Hinz | Cyclooxygenase-2 expression in lipopolysaccharidestimulated human monocytes is modulated by cyclic AMP, prostaglandin E 2 , and nonsteroidal anti-inflammatory drugs[END_REF].

In case of THC, the decrease of migration was even significant at concentrations as low as 0.01 µM (28% inhibition) and 0.1 µM (37% inhibition). With reference to the fact that in humans average peak plasma concentrations of 0.046 µM and 0.030 µM can be obtained after oral doses of 15 and 20 mg THC [START_REF] Wall | Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men and women[END_REF], the effects of THC on cell migration were observed at therapeutically relevant concentrations.

In the present study PGE [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF] and neuroglioma cells [START_REF] Ramer | Ceramide is involved in R(+)methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells[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][START_REF] Hinz | R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts[END_REF][START_REF] Ramer | R(+)-methanandamide induces cyclooxygenase-2 espression in human neuroglioma cells via a non-cannabinoid receptor-mediated mechanism[END_REF] and for MA in human cervical carcinoma cells [START_REF] Eichele | R(+)-methanandamide-induced apoptosis of human cervical carcinoma cells involves a cyclooxygenase-2-dependent pathway[END_REF]. Thus, dependent on the cell type, cannabinoids may use either receptor-dependent or -independent pathways to confer increased synthesis of Interestingly, besides its COX-2-inducing potency, THC also elicited a substantial increase of the mRNA expression of mPGES-1 and mPGES-2 mRNA both acting downstream to COX activity by producing PGE 2 . Regarding mPGES-1 this finding was expected given that mPGES-1 upregulation is mostly coupled to COX-2 induction [START_REF] Koeberle | Curcumin blocks prostaglandin E 2 biosynthesis through direct inhibition of the microsomal prostaglandin E 2 synthase-1[END_REF][START_REF] Murakami | Cellular prostaglandin E 2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2[END_REF]. In case of mPGES-2 this enzyme is known to be constitutively expressed in a variety of cells and coupled to COX-1 and COX-2 with modest preference for COX-2 [START_REF] Murakami | Cellular prostaglandin E 2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2[END_REF]. However, mPGES-2 has also been shown to be upregulated by diverse stimuli including angiotensin II and UVB light [START_REF] Jia | Microsomal prostaglandin synthase-1-derived prostaglandin E2 protects against angiotensin II-induced hypertension via inhibition of oxidative stress[END_REF][START_REF] Black | UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes[END_REF].

To provide evidence for a downstream target of COX-2/PGE 2 in conferring its antimigratory A relationship between COX-2 and TIMP-1 has also been established in other cell types. In support of the data presented here, an upregulation of TIMP-1 expression by COX-2dependent PGs has been observed in human monocytes [START_REF] Zhang | Differential regulation of monocyte matrix metalloproteinase and TIMP-1 production by TNF-alpha, granulocyte-macrophage A c c e p t e d M a n u s c r i p t 28 CSF, and IL-1 beta through prostaglandin-dependent and -independent mechanisms[END_REF], human dendritic cells [START_REF] Baratelli | Prostaglandin E 2 -dependent enhancement of tissue inhibitors of metalloproteinases-1 production limits dendritic cell migration through extracellular matrix[END_REF] and human non-pigmented ciliary epithelial cells [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF]. In the latter cell type, cannabinoids elicited increased expression of TIMP-1 along with that of MMP-9 via a mechanism requiring prior induction of COX-2 [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF]. Upregulation of TIMP-1 expression by cannabinoids has also been demonstrated in cervical carcinoma and lung cancer cells [START_REF] Ramer | Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1[END_REF], albeit a possible COX-2dependent pathway was not focussed on in these cells. Moreover, the antimigratory function Remarkably, the TIMP-1-dependent antimigratory action described in this study was observed using uncoated Boyden chambers as experimental setup. This fact needs further discussion given that TIMP-1 is traditionally regarded to impact the turnover of extracellular matrix by inhibiting the activity of matrix-degrading MMPs thereby suppressing vascular tumor growth, angiogenesis, migration and invasion [for review see 54]. A possible explanation for this apparent discrepancy may be provided by the fact that, in addition to their enzymatic activities, the TIMP-1-sensitive MMP-2 and MMP-9 can also influence cellular migration by influencing cytoskeletal organization through an association with adhesion receptors [START_REF] Sancéau | Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells[END_REF]. In line with this notion, several other groups have also found alterations in migration elicited by a modulation of the MMP/TIMP system even when uncoated chambers were used [START_REF] Guedez | Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis[END_REF][START_REF] Zeng | Prolonged butyrate treatment inhibits the migration and invasion potential of HT1080 tumor cells[END_REF][START_REF] Sancéau | Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells[END_REF][START_REF] Cheung | Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9[END_REF]. A second explanation may lie in the outcome of recent observations showing that TIMP-1 and other members of the TIMP family are multifunctional and can act either directly through cell surface receptors or indirectly through modulation of protease activity [START_REF] Stetler-Stevenson | Tissue inhibitors of metalloproteinases in cell signaling: metalloproteinase-independent biological activities[END_REF].

Collectively, our data indicate a novel mechanism by which cannabinoids could prevent POAG-associated TM cell migration and subsequent cell loss. As in vitro analysis of TM cell migration has the severe limitation of being remote from the physiological and pathologic events in vivo, further in vivo analyses are necessary to confirm our observations. Apart from the clarification of the signaling events elicited by cannabinoids in human TM cells and a novel pathway possibly contributing to the antiglaucomatous action of cannabinoids, our study extends previous investigations on the role and function of intraocular COX-2 in glaucoma. According to a post-mortem analysis of human eyes, the expression of the PGsynthesizing COX-2 is lost in the non-pigmented ciliary epithelium of patients with end-stage POAG [START_REF] Maihöfner | Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes[END_REF]. In line with this notion, the amounts of outflow-facilitating PGE 2 are significantly lower in aqueous humor of patients with POAG or steroid-induced glaucoma as compared to cataract patients [START_REF] Maihöfner | Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes[END_REF]. On the basis of these results, we recently proposed an induction of antiglaucomatous drugs [START_REF] Rösch | R(+)-methanandamide and other cannabinoids induce the expression of cyclooxygenase-2 and matrix metalloproteinases in human nonpigmented ciliary epithelial cells[END_REF][START_REF] Hinz | Latanoprost induces matrix metalloproteinase-1 expression in human non-pigmented ciliary epithelial cells through a cyclooxygenase-2-dependent mechanism[END_REF] as a mechanisms contributing to its IOP-lowering action. The data presented here confirm this assumption.

In summary, we have demonstrated for the first time that cannabinoids are inductors of a COX-2/TIMP-1 pathway in human TM cells that confers an antimigratory action. Although more studies are needed, activation of COX-2 expression and subsequent formation of TIMP-1-inducing PGs could be an important mechanism by which cannabinoids exert its antiglaucomatous action.

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

A c c e p t e d M a n u s c r i p t 2 Keywords:

 2 Figures: 6

A c c e p t e d M a n u s c r i p t 8 contrast

 8 cells were stained with Diff-Quick ® according to the manufacturer´s instructions (Medion Diagnostics GmbH, Büdingen, CH). Wound-healing experiments were performed in duplicate.

A c c e p t e d M a n u s c r i p t 10 2. 7 .

 107 siRNA transfectionsHuman TM cells were transfected with siRNA targeting CB 1 , CB 2 , COX-2, TIMP-1 or nonsilencing siRNA (Eurogentec, Seraing, Belgium; Cat. No. OR-0030-neg) using RNAiFect ®

A c c e p t e d M a n u s c r i p t 11 2. 9 .

 119 StatisticsComparisons between groups were performed with Student´s t test. All statistical analyses were undertaken using Prism 4.00 (GraphPad Software, San Diego, CA).

  (1 µM), average PGE 2 concentrations measured in cell culture medium were 1.16 + 0.25 nmol/L (mean + SEM of n = 4 experiments). To examine the source of PGE 2 release, cells were treated with THC in the presence of the selective COX-2 inhibitor NS-398. NS-398 almost completely abolished THC-induced PGE 2 release, confirming an involvement of the COX-2isozyme in this response (Fig.1D). As COX-2-dependent regulation of PGE 2 release has been shown to be associated with upregulation of microsomal prostaglandin E 2 synthase-1 (mPEGS-1)[START_REF] Koeberle | Curcumin blocks prostaglandin E 2 biosynthesis through direct inhibition of the microsomal prostaglandin E 2 synthase-1[END_REF], we additionally analysed the regulation of mPGES-1 and mPGES-2 mRNA.

  A c c e p t e d M a n u s c r i p t 13 Real-time RT-PCR analyses performed after a 12-h treatment of TM cells with 1 µM THC revealed an induction of both mPGES-1 and mPGES-2: 142.1% + 8.1% vs. 100% + 10.0% (vehicle control) for mPGES-1 (means + SEM of n = 4 experiments, P < 0.05), and 161.7% + 12.9% vs. 100% + 5.8% (vehicle control) for mPGES-2 (means + SEM of n = 3-4 experiments, P < 0.05) 3.2. Involvement of cannabinoid receptors in the antimigratory and COX-2-inductive action of THC To investigate whether cannabinoid receptors are involved in THC-mediated reduction of TM cell migration and the concomitant induction of COX-2 expression, the impact of antagonists of CB 1 receptor (AM-251) and CB 2 receptor (AM-630) on THC´s action was tested. Inhibitors

A c c e p t e d M a n u s c r i p t 15 4C

 15 , the antimigratory action of PGE 2 was mimicked by butaprost (EP 2 receptor agonist) and 11-deoxy PGE 1 (EP 2 /EP 4 receptor agonist), but not by the EP 1 /EP 3 agonists 17-phenyl-ωtrinor PGE 2 (EP 1 > EP 3 ) and sulprostone (EP 3 > EP 1 ).

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

 16 upregulation of TIMP-1 protein levels as shown in Fig.5A, C, and D was reflected by a concomitant upregulation of TIMP-1 mRNA expression (161.8% ± 7.3% [THC at 1 µM] vs. 100.0% ± 5.3% [vehicle control]; means ± SEM of n = 10 experiments, P < 0.001) after a 24h incubation period.

A c c e p t e d M a n u s c r i p t 17 After a 24

 1724 -h treatment of cells with 10 µM MA, average PGE 2 concentrations measured in cell culture medium were 5.69 + 0.32 nmol/L (mean + SEM of n = 4 experiments).

A c c e p t e d M a n u s c r i p t 20 PGs

 20 which may on the one hand depend on extracellular activation of membrane-bound receptors and might otherwise rely on a lipid raft-dependent intracellular uptake[START_REF] Hinz | R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts[END_REF].

  action on TM cells, the expression of TIMP-1 was focused on in further experiments. RT-PCR and Western blot analyses revealed an induction of TIMP-1 by cannabinoids in human TM cells. The proposed function of TIMP-1 as a downstream target of COX-2 was corroborated by several experimental outcomes. First, inhibition of COX-2 activity by NS-398 was associated with a significant reduction of cannabinoid-induced TIMP-1 levels. Second, a role for COX-2 as effector of TIMP-1 expression was supported by the observation that PGE 2 mimicked the stimulatory effect of cannabinoids on TIMP-1 expression. Third, the functional relevance of TIMP-1 upregulation by cannabinoids was substantiated by demonstrating a concentration-dependent and significant decrease of TM cell migration by recombinant TIMP-1.

A c c e p t e d M a n u s c r i p t 21 of

 21 TIMP-1 shown here is in line with other studies demonstrating a correlation between increased TIMP-1 levels and impaired cellular migration[START_REF] Baratelli | Prostaglandin E 2 -dependent enhancement of tissue inhibitors of metalloproteinases-1 production limits dendritic cell migration through extracellular matrix[END_REF][START_REF] Fernandez | Inhibition of endothelial cell migration by gene transfer of tissue inhibitor of metalloproteinases-1[END_REF][START_REF] Guedez | Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis[END_REF][START_REF] Akahane | TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms[END_REF][START_REF] Zacchigna | AAVmediated gene transfer of tissue inhibitor of metalloproteinases-1 inhibits vascular tumor growth and angiogenesis in vivo[END_REF][START_REF] Zeng | Prolonged butyrate treatment inhibits the migration and invasion potential of HT1080 tumor cells[END_REF].

A c c e p t e d M a n u s c r i p t 22 intraocular

 22 COX-2 by certain established (latanoprost) and potential (cannabinoids)

1

 1 Figure 1

Figure 2

 2 Figure 2

A c c e p t e d M a n u s c r i p t 31 Migrated

 31 cells were stained with Diff-Quick ® (Medion Diagnostics GmbH, Büdingen, CH) and documented under a 200 x magnification (B). D, Expression of CB 1 and CB 2 receptors in human TM cells. Membrane fractions obtained from cells treated with vehicle, CB 1 -, CB 2 -or nonsilencing siRNA at a final concentration of 2.5 µg/ml were used for monitoring cannabinoid receptors (D). Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means + SEM of n = 8 (A) experiments. ***P < 0.001, vs. corresponding vehicle control; ### P < 0.001 vs. THC-treated cells, Student´s t-test. Values above the blot (C) are means + SEM obtained from densitometric analysis of n = 4 blots and represent percent control in comparison with vehicle-treated cells (100%) in the absence of test substance. Equal loading of lysates is indicated by hybridisation of membranes with an antibody against ß-actin.

Figure 3

 3 Figure 3

A c c e p t e d M a n u s c r i p t 35

 35

  Fig. 1 Fig. 1

  Figure

[

  Fig.6

  ). AM-251, AM-630, latanoprost acid, NS-398 and PGE 2 were bought from Alexis

	Deutschland GmbH (Grünberg, Germany). AH-6809, butaprost, 11-deoxy PGE 1 ,
	GW627368X, 17-phenyl-ω-trinor PGE 2 , PGF 2α , SC-19220, sulprostone and U-46619 were
	obtained from Cayman Chemical (Ann Arbor, MI, USA). 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. Matrigel was obtained from BD Biosciences (Oxford, UK). Dimethyl sulfoxide (DMSO), EDTA, glycerol, HEPES, NaCl, Ponceau S, sodium dodecyl sulfate-polyacrylamide

  2 elicited inhibition of TM cell migration, whereas agonists to FP and TP receptors did not confer a comparable effect. PGE 2 is known to be a ligand of four PGE 2 Therefore, a decreased migration was elicited by the EP 2 receptor agonist butaprost and by the EP 2 /EP 4 receptor agonist 11-deoxy PGE 1 , whereas EP 1 /EP 3 receptor agonists (17-phenyl-ω-trinor PGE 2 , sulprostone) were virtually inactive in this respect. In support of a causal link between EP 2 /EP 4 receptor activation and diminished migration by PGE 2 , the EP 1 /EP 2 receptor antagonist, AH-6809, and the selective EP 4 receptor antagonist, GW627368X, were shown to significantly reduce the antimigratory effect of PGE 2 . In contrast, the selective EP 1 antagonist SC-19220 was devoid of a significant inhibitory effect in this respect. Importantly, all three EP antagonists influenced the antimigratory action of both tested cannabinoids in the same manner thereby confirming a causal link between cannabinoid-induced PGE 2 release and inhibition of migration via EP 2/4 receptor activation.In contrast to the delayed antimigratory effect of the tested cannabinoids which both require synthesis of endogenous PGE 2 , the antimigratory effect of exogenously added PGE 2

	M a n u s c r i p t
	Additional experiments using selective antagonists of CB 1 -(AM-251) or CB 2 receptors (AM-A c c e p t e d 630) revealed that both CB 1 and CB 2 confer cannabinoid-induced COX-2 expression and
	receptor subtypes, which mediate stimulation of phosphoinositol turnover with elevation in subsequent decreased migration. With respect to the observed CB receptor dependency of
	intracellular free calcium (EP 1 -and some EP 3 receptors isoforms), activation of adenylyl COX-2 expression by both cannabinoids, our data contradict previous observations from our
	cyclase activity resulting in elevation of intracellular cAMP (EP 2 -and EP 4 receptors) or group showing a receptor-independent induction of COX-2 expression by both cannabinoids
	in human non-pigmented ciliary epithelial

inhibition of adenylyl cyclase (EP 3 receptor)

[START_REF] Negishi | Two isoforms of prostaglandin E receptor EP 3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization[END_REF][START_REF] Coleman | A novel inhibitory prostanoid receptor in piglet saphenous vein[END_REF]

. Using agonists and antagonists of the different EP receptor subtypes, evidence was provided suggesting that EP 2 -and EP 4 receptors, but not EP 1 -and EP 3 receptors, are involved in the antimigratory action of PGE 2 on 19 human TM cells. occurred much earlier suggesting an immediate action by direct EP receptor activation. In our hands, relatively high concentrations of exogenous PGE 2 were necessary to confer inhibition of migration. Given a a significant antimigratory effect of PGE 2 at 0.1 µM, the endogenous levels of PGE 2 produced by THC-and MA-stimulated TM cells were 86-and 18fold lower. This apparent difference has been observed in several studies. Only recently, Sirianni et al.

[START_REF] Sirianni | Inhibition of cyclooxygenase-2 down-regulates aromatase activity and decreases proliferation of Leydig tumor cells[END_REF]

investigating the impact of COX-2 on aromatase activity presented data suggesting that a high endogenous production of PGE 2 requires the addition of high exogenous amounts of PGE 2 to observe a particular regulation.

Table 1

 1 Effect of AM-251 (CB 1 antagonist; 1 µM), AM-630 (CB 2 antagonist; 1 µM), NS-398 (selective COX-2 inhibitor; 1 µM), GW627368X (EP 4 receptor antagonist, 1 µM), AH-6809 (EP 1 /EP 2

	receptor antagonist, 10 µM) and SC19220 (EP 1 receptor antagonist, 1 µM) on the
	antimigratory effect of MA. Cells were pretreated with antagonists/inhibitors for 30 min and
	incubated with MA (10 µM) for another 24 h. Percent control represents comparison with
	vehicle-treated cells (100%) in the absence of test substance of n = 4 experiments. **P <
	0.01; ***P < 0.001, vs. corresponding vehicle control; ## P < 0.01; ### P < 0.001 vs. MA-treated
	cells, Student´s t-test.
		Migration (%)
	Vehicle	100.0 + 12.8
	MA	23.8 + 14.4 **
	MA + AM-251	98.5 + 6.1 ##
	MA + AM-630	96.6 + 10.8 ##
	MA + AM-251 + AM-630	104.6 + 5.6 ##
	Vehicle	100.0 + 3.3
	MA	38.0 + 4.1 ***
	MA + NS-398	95.3 + 11.4 ##
	NS-398	100.7 + 1.8
	Vehicle	100 + 3.6
	MA	36.4 + 4.4 ***
	MA + GW627368X	83.9 + 8.6 ##
	MA + AH6809	97.1 + 6.0 ###
	MA + SC19220	21.3 + 4.8

# This study was supported by grants from Deutsche Forschungsgemeinschaft (SFB 539 TP BI.6)


nmol/µg protein TdR exposure time (min)

but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC-MS/MS which sugars accumulated, their subcellular localization (using 3 H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with 3 H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane.

Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis.

Introduction

The platelet derived endothelial cell growth factor (PD-ECGF) or thymidine phosphorylase (TP) is often overexpressed in human cancers, which has been related to a higher microvessel density, a higher tumor stage and the induction of metastasis [START_REF] De Bruin | Role of platelet derived endothelial cell growth factor/ thymidine phosphorylase in health and disease[END_REF][START_REF] Bronckaers | The dual role of thymidine phosphorylase in cancer development and chemotherapy[END_REF][START_REF] Liekens | Targeting platelet-derived endothelial cell growth factor/thymidine phosphorylase for cancer therapy[END_REF][START_REF] Koukourakis | Different patterns of stromal and cancer cell thymidine phosphorylase reactivity in non-small-cell lung cancer: impact on tumour neoangiogenesis and survival[END_REF]. In various in vitro studies, TP had a chemotactic effect on endothelial cells [START_REF] Ishikawa | Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor[END_REF][START_REF] Hotchkiss | Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3[END_REF][START_REF] Hotchkiss | Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration[END_REF][START_REF] Seeliger | Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy[END_REF]. In vivo, TP induced angiogenesis using various cancer cell lines that were transfected with TP [START_REF] Moghaddam | Thymidine phosphorylase is angiogenic and promotes tumor growth[END_REF][START_REF] Sengupta | Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms[END_REF]. The role of TP in angiogenesis seems evident, but its exact mechanism is still unknown and it is postulated to be related to the sugars that are formed from thymidine (TdR) degradation by TP.

Deoxy-α-D-ribose-1-phosphate (dR-1-P) and thymine are the first products that are formed by the phosphorylysis of TdR by TP (Fig. 1) [START_REF] De Bruin | Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells[END_REF]. After its formation, dR-1-P rapidly disappears, possibly by conversion to 2-deoxy-D-ribose (dR) or 2-deoxyribose-5-phosphate (dR-5-P) [START_REF] Carta | Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells[END_REF]. dR-5-P can be converted to glyceraldehyde-3-phosphate (G3P), which can enter the glycolytic or pentose phosphate pathway. dR can form advanced glycation end products (AGE) or reactive oxygen species (ROS) by Schiff base reactions [START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF]. The Schiff base is formed from either dR or dR-5-P, which can be coupled to an intracellular protein by nonenzymatic condensation between the sugar's aldehyde group and a lysine residue. This

Schiff base can subsequently rearrange to an Amadori product, such as an α-hydroxyketone, which may also form an enediol intermediate [START_REF] Wolff | The autoxidation of glyceraldehyde and other simple monosaccharides[END_REF].These unstable intermediates react via nonenzymatic reactions to form AGEs. During these reactions, specifically in the transition metal-catalyzed auto-oxidation, free radicals are produced [START_REF] Wolff | Glucose autoxidation and protein modification. The potential role of "autoxidative glycosylation" in diabetes[END_REF][START_REF] Monnier | Nonenzymatic glycosylation, the Maillard reaction and the aging process[END_REF][START_REF] Bierhaus | AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept[END_REF]. The formation of AGE from these reactions could be responsible for the angiogenic activity of TP [START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF].

The enzymatic activity of TP is indispensable for the angiogenic effect, since a competitive inhibitor of TP could block the angiogenic effect [START_REF] Haraguchi | Angiogenic activity of enzymes[END_REF]. Therefore, substrate and metabolites from the reaction are often used for studying the angiogenic effect of TP [START_REF] Stevenson | Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis[END_REF].

A c c e p t e d M a n u s c r i p t 4

The metabolite dR has previously shown angiogenic activity [START_REF] Hotchkiss | Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3[END_REF][START_REF] Sengupta | Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms[END_REF] and could elongate the sprouting in the aortic ring assay [START_REF] Stevenson | Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis[END_REF]. In addition, dR induced endothelial cell migration in various studies [START_REF] Ishikawa | Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor[END_REF][START_REF] Seeliger | Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy[END_REF]. The association between TP activity and angiogenesis is based on the potential accumulation of TdR-derived sugars. However, little is known about the cellular metabolism of dR-1-P. Although dR is considered the main factor causing the angiogenic switch, the real identity and extent of formation of TdR-derived sugars remains unclear. dR-1-P rapidly disappears after its formation and its conversion could be reduced by addition of dR-5-P [START_REF] De Bruin | Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells[END_REF]. The aim of the present study was to characterize the specific sugars that can be formed by the TP reaction, their subcellular localization and whether they are secreted by the cancer cells, enabling endothelial cells to migrate and invade towards tumor sites.

Materials and Methods

Cell culture and chemicals

The human colon carcinoma cell line Colo320 TP1 was a variant of Colo320 (obtained from the ATCC), transfected with TP, as described previously [START_REF] De Bruin | Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity[END_REF]. RT112/TP was a kind gift of Dr. Bicknell (Oxford, UK). Cells were cultured as monolayers in DMEM, supplemented with 10% heat inactivated FCS and 20 mM Hepes in 25 cm 2 culture flasks (Greiner Bio-One, Frickenhausen, Germany). Cells were maintained in a humidified 5% CO 2 atmosphere at 37°C. TPI was provided by Taiho Pharmaceutical, Co., Ltd. (Tokushima, Japan). TdR, thymine, dR, dR-1-P, dR, dR-5-P and G3P were obtained from Sigma Aldrich Chemicals (Zwijndrecht, the Netherlands). These chemicals were dissolved in PBS in stock solutions of 1-20 mM and stored at -20ºC. 13 C 6 -glucose-6-phosphate (prepared as previously described [START_REF] Huck | Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency[END_REF]) and 13 C 2 -xylulose were obtained from Omicron biochemicals (South Bend, IN, USA).

Ethoxyamine was obtained from Acros (Geel, Belgium) and pyridine from Merck (Darmstadt, Germany). 5'-3 H-TdR (specific activity of 18.8 mCi/mmol, conc. 12.9 µg/ml) was purchased

Treatment of the cells

For measurement of TdR and its sugars, Colo320TP1 and RT112/TP cells were seeded at 2x10 6 cells/flask. After 48 h, cells were exposed to 100 µM TdR for 0 min, 15 min, 30 min and 60 min. TPI (10 µM) was added 24 h prior to TdR exposure, after which samples were incubated for 60 min. After treatment, cells were trypsinized, washed in PBS and centrifuged for 5 min at 323 g at room temperature (RT). The medium and the cell pellets were stored separately at -80ºC until sample preparation.

Measurement of TP enzymatic activity and of TdR and thymine

HPLC measurement of TP enzymatic activity was performed as described previously [START_REF] De Bruin | Sulfasalazine down-regulates the expression of the angiogenic factors platelet-derived endothelial cell growth factor/thymidine phosphorylase and interleukin-8 in human monocytic-macrophage THP1 and U937 cells[END_REF] and is based on the detection of both TdR and thymine [START_REF] Laurensse | A sensitive non-radioactive assay for pyrimidine nucleoside phosphorylase using reversed-phase high performance liquid chromatography[END_REF]. After incubation with thymidine, 25 µl 80% trichloroacetic acid (TCA) was added to the samples, which were left on ice for 20 min. Subsequently, samples were centrifuged at 14000 g at 4ºC for 10 min. The supernatant was transferred to a new vial, and the pH was neutralized. Samples were mixed and centrifuged for 1 min at 14000 g. The upper aqueous layer was used for analysis of TdR and thymine by HPLC analysis for nucleosides with UV detection as described previously [START_REF] Laurensse | A sensitive non-radioactive assay for pyrimidine nucleoside phosphorylase using reversed-phase high performance liquid chromatography[END_REF].

A c c e p t e d M a n u s c r i p t 6

Measurement of sugar-phosphates

For sample preparation, the cell pellet was dissolved in 500 µl MQ and subsequently samples were sonificated 3 x for 3 seconds on ice. 5 µM 13 C 6 -glucose-6-phosphate was used as an internal standard. The samples were centrifuged through a filter with 10 kDa cut off (Ultracel YM-10; #42407; Amicon Micron, Millipore, Billerica, MA, USA). Subsequently, the samples were injected into the LC-MS/MS as described previously [START_REF] Wamelink | Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism[END_REF]. The amount of sugars was expressed based on the total protein content of each sample that was measured before centrifugation through the 10 kDa cut off filter. Protein concentration was determined using the BioRad Protein assay (#500-0006; Bio-Rad Laboratories, Veenendaal, The Netherlands) according to manufacture's instruction.

Measurement of dR

For preparation of the samples for the dR measurement, the cell pellet was dissolved in 150 µL distilled water. 13 C 2 -xylulose was used as internal standard. 17 µl of 20 µM 13 C 2 -xylulose was added to 50 µl of the sample. To deproteinize the sample, 200 µl methanol was added.

After 15 min, the sample was centrifuged for 10 min at 10000 g at 4ºC. Following this, the supernatant was transferred to a new vial and evaporated to dryness under a slight stream of nitrogen. Ethoxime derivatives of dR were formed by treating the residue with 2 mg ethoxyamine in 100 µl pyridine at 60ºC for 30 min. After cooling down to RT, the hydroxygroups were acetylated by adding 100 µl acetic anhydride at 80ºC for 60 min. This solution was evaporated to dryness and the residue was redissolved in ethylacetate. (1:21) of which the batch of [5'-3 H]-TdR was mixed with 1 mM unlabeled TdR). After incubation for 1, 6 or 24 h or 1 h plus 24 h [5'-3 H]-TdR-free medium at 37ºC, cell fractions were separated using a ProteoExtract® Subcellular Proteome Extraction Kit according to manufacture's instructions (Calbiochem, San Diego, CA). Of every cell fraction, including from the medium above the cells after the designated incubation times, 5 µl was counted. To determine to which fraction (protein or non-protein fraction) secreted [5'-3 H]-TdR-derived metabolites accumulated, 100 µl of the medium above the cells after the retention was precipitated with 60 µl 35% TCA for 20 min on ice. Subsequently, samples were centrifuged for 5 min at 300 g at 4ºC and 5 µl of the supernatant (non-protein fraction) was counted. In addition, the pellet (protein fraction) was recovered in 200 µl MQ, of which 5 µl was counted.

Western blotting

Colo320, Colo320 TP1, RT112 and RT112/TP cells were washed twice with ice-cold PBS and lysed in lysis buffer (Cell Signalling Technology Inc., Denver, USA). Cell lysates were scraped, transferred into a vial and centrifuged at 11000 g at 4ºC for 10 min. Supernatants were transferred to a new vial and protein amounts were determined by the Bio-Rad assay, according to the manufacturer's instruction (Bio-Rad Laboratories, Veenendaal, the Netherlands). From each condition 30 µg of protein was separated on a 10% SDS-PAGE and electroblotted onto polyvinylidenedifluoride (PVDF) membranes (Millipore Immobilon TM -FL PVDF, 0.45 µm). Subsequently, the membranes were blocked for 1 h at room temperature (RT) in Odyssey blocking buffer (Odyssey blocking buffer #927-40003, Westburg, Leusden,

The Netherlands) and incubated overnight at 4C with the primary antibodies (dilution 1:1000-10000 in Odyssey blocking buffer 1:1 diluted with PBS-T (PBS with 0.05% Tween- ). The membrane was washed 5 times in PBS-T and incubated with the secondary antibodies (1:10000) for 1 h at RT in the dark. After incubation, the membrane was washed in PBS-T and followed by 5 min washing in PBS without Tween-20 to decrease the background signal. Subsequently, the bands were scanned using an Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, Nebraska USA), 0 mm offset, 84 µm resolution and with high quality [START_REF] Mathews | Imaging systems for westerns: chemiluminescence vs. infrared detection[END_REF].

Statistical analysis

Potential differences between controls and TdR exposed cells were evaluated using the twotailed Student's t-test for paired data. Changes were considered significantly different when p<0.05.

Results

TdR conversion to thymine

In order to determine the extent of TdR conversion, TdR and thymine levels were measured, after incubation with 100 µM TdR for 15, 30 and 60 min. As expected, the parental Colo320 and RT112 cells did not or hardly convert TdR to thymine, since these cells do not or hardly express TP (Fig 2) [START_REF] De Bruin | Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity[END_REF] TP was highly expressed in both Colo320 TP1 and RT112/TP cells (Fig 2 ), while no band could be detected in Colo320 parental cells and a very faint band was found in RT112 cells. Colo320 and RT112 cells converted thymidine at a rate of 0 ± 0 and 3 ± 0.04 nmol/ million cells / h, respectively. Colo320 TP1 and RT112/TP converted thymidine at a rate of 518 ± 40 nmol/million cells/h and 418 ± 34 nmol/ million cells / h (Fig. 3A). After 1 h incubation of intact cells with TdR, the extracellular thymine concentrations were 24 and 30 µM, respectively. The TK protein, which activates TdR for DNA synthesis, was similarly expressed in the TP-transfected cells (data not shown).

A c c e p t e d M a n u s c r i p t 9

Intracellular accumulation of dR-5-P and dR-

TP degrades TdR into one molecule of thymine and one dR-1-P, which can be metabolized to other sugars (Fig. 1). We measured the intracellular levels of the potential metabolites dR-1-P, dR-5-P, dR and G3P in Colo320, RT112 and the transfected variants, Colo320 TP1 and RT112/TP. In the cells that did not express TP, no sugars accumulated at all (data not shown), which is in agreement with the absence of thymine formation. In general, the sugars accumulated to a much higher extent in RT112/TP cells, compared to Colo320 TP1 cells (Fig. 3A and3B). dR-1-P increased rapidly, and was about 10 fold higher in RT112/TP cells compared to Colo320 TP1 cells. dR-1-P was rapidly metabolized to dR-5-P and dR. In both Colo320TP1 and RT112/TP cells, dR-1-P was the major intracellular metabolite (Fig. 3B). In RT112/TP cells, the accumulation of dR-5-P was lower than the level of dR-1-P. dR accumulated intracellularly to a low extent in Colo320TP1 cells and was only 4 fold increased compared to the control dR level. In RT112/TP cells, dR hardly accumulated. In Colo320 TP1 cells, G3P increased only at very low levels, with a 2 fold increase compared to the control.

G3P did not accumulate in RT112/TP cells.

Addition of TPI alone to the cultures did not alter the production of the tested sugars.

TPI completely blocked the conversion of TdR in Colo320 TP1 and RT112/TP cells (Fig. 3A), which is in agreement with the formation of the sugars, which were not formed at all (Fig. 3A and3B).

dR is secreted from the cells

dR is an important angiogenic sugar that can be secreted by the cells [START_REF] Hotchkiss | Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration[END_REF][START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF]. Therefore, we determined the level of dR in the medium. dR-1-P and dR-5-P can not cross the membrane, because of the negative charge of the phosphate group on these sugars. In the medium above A c c e p t e d M a n u s c r i p t 10 Colo320 TP1 and RT112/TP cells, dR increased in time (Fig. 3C) and was found at higher levels than the other sugars intracellularly (Table 1). Of the total thymine detected, 10 and 13 % was measured as extracellular dR after 1 h by Colo320 TP1 and RT112/TP cells, respectively (Table 1). TPI alone did not have any effect on the intracellular levels of dR, while TPI prevented dR from being formed (Fig. 3C).

Thymidine-derived sugars and accumulation levels

Since the thymine levels are representative for the total amount of sugars that are formed, we compared each value with the amount of thymine (in which thymine was set at 100 % for each time point) (Table 1). In Colo320 TP1 cells, the total amount of measured sugars was much lower than that of RT112/TP cells (Fig. 3A). In Colo320 TP1 cells, about 20% of the total TdR-derived sugars presented intracellularly as dR-1-P, dR-5-P, dR and G3P and extracellular dR. This did not increase after a longer exposure time to TdR, indicating that the sugars are rapidly metabolized to other products. In RT112/TP cells, about 50 % of the TdRderived sugars were measured (Table 1), indicating a slower metabolism of these sugars into other products compared to that in Colo320 TP1 cells.

Accumulation in the cytoskeleton and nucleus

In order to determine in which cellular compartment the converted products of TdR accumulated, Colo320 TP1 and RT112/TP cells were exposed to [5'-3 H]-TdR for different time periods. Subsequently, subcellular cell fractions were separated and the amount of radioactivity was determined in each compartment (Fig. 4).

In Colo320 TP1 cells, the level of [5'-3 H]-products in the cytosolic compartment decreased in time, until it was hardly detectable after 24 h. Products in the membrane fraction increased to some extent after 6 h, while after 24 h hardly any radioactivity was detectable in A c c e p t e d M a n u s c r i p t 11 this fraction. In the nuclear fraction, the radioactivity was increased after 24 h, which was about 55% of the total intracellular radioactivity. This is possibly in part due to metabolic activation of TdR by thymidine kinase, resulting in incorporations into the DNA and nuclear trapping. Interestingly, TdR-derived radioactivity was present in the cytoskeletal protein fraction, which increased in time up to about 45% after 24 h.

In RT112/TP cells, the radioactivity in the cytosolic fraction decreased in time, with about 45% of [5'-3 H]-products still detectable after 24 h (Fig. 4). This is in agreement with the lower metabolism of the sugars in these cells, as measured by LC-MS/MS (Table 1).

Comparable to Colo320 TP1 cells, the radioactive products in the nuclear fraction increased in RT112/TP cells, although at lower levels; up to 25 % after 24 h. Both the membrane and cytoskeleton fraction increased in time, with a maximum of about 25 % and 10 % after 24 h incubation, respectively.

When the [5'-3 H]-TdR was washed away following 1 h incubation, the radioactivity was found to a high extent in the membrane (9.6 and 13.6 %) and cytoskeletal (73.5 and 39.4 %) compartments of both Colo320 TP1 and RT112/TP cells, respectively (data not shown).

Hardly any of the radioactive label retained in the nuclear fraction, which may be related to a lack of activation of TdR by TK within 1 h exposure. In summary, the TdR-sugars are used in the cytoskeletal and membrane, which may possibly be related to protein-glycation.

Secreted products are in the non-protein fraction

To determine whether products were secreted after conversion of TdR, Colo320 TP1 and RT112/TP cells were exposed to [5'-3 H]-TdR for 1 h, after which the medium was refreshed and cells were washed to remove the excess of [5'-3 H]-TdR. The amounts of secreted products were determined by analyzing the amount of radioactivity in the medium fraction. To analyze whether the secreted products were in the protein or in the non-protein fraction, proteins were precipitated by adding TCA (Fig. 4B). In both Colo320 TP1 and RT112/TP cells, the largest part of the secreted radioactive products was found in the nonprotein fraction. This indicates that the sugars may not be used to a high extent for proteinglycation of secreted proteins, but may be more important for processes intracellularly.

Discussion

In this study we describe TdR phosphorylysis to dR-1-P, which is rapidly followed by its isomerization to dR-5-P or degradation to dR of which the latter was extensively secreted. To our knowledge, the formation of these sugars using human (eukaryotic) cells has not been described earlier. We observed different levels of accumulation of TdR-derived sugars in the two cell types. In addition, the converted TdR was found in the cytoskeleton and to some extent in the cell membrane.

Previously we demonstrated that dR-1-P formed from TdR rapidly disappeared from Colo320 TP1 cells [START_REF] De Bruin | Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells[END_REF]. dR-1-P can be isomerized to dR-5-P mediated by phosphopentomutase (E.C. 5.4.2.7) and is degraded to dR by a phosphatase [START_REF] Sengupta | Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms[END_REF]. dR-5-P can be split into G3P plus acetaldehyde by deoxyriboaldolase (E.C. 4.1.2.4) [START_REF] Carta | Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells[END_REF][START_REF] Moriwaki | Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues[END_REF]. The formed acetaldehyde may be converted into acetyl-CoA by aldehyde oxidase and acetyl-CoA synthetase [START_REF] Moriwaki | Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues[END_REF]. G3P can enter several metabolic pathways, including the glycolytic and the pentose phosphate pathway, and possibly therefore did not accumulate in the cells in our study. Initially, these steps were only described in e.g. Bacillus ceresus [START_REF] Sgarrella | Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties[END_REF]. Enzymatic activity needed for this pathway was also identified in epithelial anmoitic WISH cells [START_REF] Carta | Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells[END_REF].

The sugars may also be substrate in the formation of AGE (unpublished data), which may be dR-1-P can also be formed by conversion of purine deoxynucleosides by purine nucleoside phosphorylase (PNP) [START_REF] Tozzi | Pentose phosphates in nucleoside interconversion and catabolism[END_REF]. Therefore theoretically, PNP may also be an angiogenic enzyme. However, to the best of our knowledge, a role for PNP in angiogenesis has never been described before. Important differences between TP and PNP involve their function: TP overexpression in tumor sites has clearly been related with angiogenesis [START_REF] Bronckaers | The dual role of thymidine phosphorylase in cancer development and chemotherapy[END_REF],

while PNP is involved in immune function [START_REF] Pieters | Adenosine deaminase and purine nucleoside phosphorylase in childhood lymphoblastic leukemia: relation with differentiation stage, in vitro drug resistance and clinical prognosis[END_REF][START_REF] Van Laarhoven | Purine metabolism in relation to leukemia and lymphoid cell differentiation[END_REF]. In addition, PNP is hardly overexpressed in cancer [START_REF] Pieters | Adenosine deaminase and purine nucleoside phosphorylase in childhood lymphoblastic leukemia: relation with differentiation stage, in vitro drug resistance and clinical prognosis[END_REF][START_REF] Van Laarhoven | Purine metabolism in relation to leukemia and lymphoid cell differentiation[END_REF][START_REF] Bardot | Purine and pyrimidine metabolism in human gliomas: relation to chromosomal aberrations[END_REF], making it less likely to be an angiogenic enzyme. It might be possible that other products than the sugars are involved in angiogenesis as well, such as β-Aminoiso-butyric acid, which is another downstream metabolite of thymine [START_REF] Stevenson | Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis[END_REF]. However, several enzymes involved in thymine catabolism (e.g. DPD and β-ureidopropionase) are usually low or not detectable in many tumors [START_REF] Westra | Predictive value of thymidylate synthase and dihydropyrimidine dehydrogenase protein expression on survival in adjuvantly treated stage III colon cancer patients[END_REF][START_REF] Johnson | Importance of dihydropyrimidine dehydrogenase (DPD) deficiency in patients exhibiting toxicity following treatment with 5-fluorouracil[END_REF][START_REF] Thomas | Genetic regulation of dihydropyrimidinase and its possible implication in altered uracil catabolism[END_REF]. In our and other panels of colon cancer cell lines, DPD activity was low to not detectable [START_REF] Peters | Dihydropyrimidine dehydrogenase in livers from mouse and rat, and in human colon tumors, and mucosa in relation to anabolism of 5-fluorouracil[END_REF][START_REF] Beck | A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil[END_REF], in contrast to pancreatic and breast cancer [START_REF] Beck | A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil[END_REF][START_REF] Shimoda | Thymidylate synthase and dihydropyrimidine dehydrogenase are upregulated in pancreatic and biliary tract cancers[END_REF][START_REF] Anan | Increased dihydropyrimidine dehydrogenase activity in breast cancer[END_REF]. Therefore, it is unlikely that β-Amino-iso-butyric acid is involved in the angiogenic activity of TP.

In the present study, we used cancer cells to determine the sugar metabolism in order to stimulate angiogenesis. TP expression in endothelial cells may also be important for their angiogenic properties. However, TP expression is very low in endothelial cells [START_REF] Bijnsdorp | Increased migration by stimulation of thymidine phosphorylase in endothelial cells of different origin[END_REF][START_REF] Pula | Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures[END_REF]. In addition, TP expression is often reported to be in either cancer cells or other tumor stromal cells such as fibroblasts. The angiogenic role of TP is possibly more related to the secretion of angiogenic factors (i.e. dR or chemoattractants such as IL-8) by cancer cells, rather than an Although TP is clearly associated with angiogenesis, the exact link between TdRrelated sugars and angiogenesis is still missing. Cells with a high TP can secrete angiogenic factors at high levels, including the vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), but the role of sugars in the activation of these factors remains unclear [START_REF] Hotchkiss | Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3[END_REF][START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF]. TdR-derived sugars accumulated in the cell fractions of the membrane and cytoskeleton, which may be related to glycation of proteins by the formation of AGE. This may lead to the hypothesis that protein glycation plays a role in the activation of the transcription of angiogenic factors, such as IL-8 and VEGF [START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF] which can subsequently be secreted. The secreted fraction consisted of non-proteins. This indicates that dR may be the main TdR-sugar-product that is secreted by TP-expressing cells. Possibly, protein-glycation of secreted proteins is not the main regulator of angiogenesis. Thus, glycation of proteins that remain intracellularly are possibly important for angiogenesis, by activating transcription of angiogenic enzymes. Since dR can be secreted by the cancer cells, it can be taken up by the endothelial cells, in which it can also undergo Schiff base reactions, forming AGE inside the endothelial cells, stimulating their angiogenic properties (Fig. 1).

A possible role of sugars in the angiogenic potential of TP was studied through its metabolite dR, which can stimulate endothelial cell migration and angiogenesis. dR is a strongly reducing sugar that can generate oxygen radical species during the early stages of protein glycation [START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF]. dR can bind to an amino group of proteins by a non-enzymatic reaction. This leads to the formation of a Schiff base, which can subsequently rearrange to an α-hydroxyketone. During this reaction, free oxygen radicals are produced. Thus, through the formation of dR, TP induces oxidative stress in TP-overexpressing tumor cells. This may result in the secretion of angiogenic factors, such as VEGF or IL-8 [START_REF] Brown | Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors[END_REF]. dR secreted by the

Figure 2 protein

 2 Figure 2

A c c e p t e d M a n u s c r i p t 2 ABSTRACT

 2 Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear,

A c c e p t e d M a n u s c r i p t 5 from

 5 Moravek Biochemicals Inc. (Brea, CA, USA). The exact moiety where the sugar of the TdR is labelled is indicated in Fig. 1. The thymidine phosphorylase antibody was purchased from R&D systems Inc. (Minneapolis, MN USA) and the thymidine kinase antibody was purchased from QED Bioscience (San Diego, CA, USA). The secondary antibody, goat-mouse-IRDye (800CW;#926-32210 and 680;#926-32220) was obtained from Westburg (Leusden, the Netherlands).

1 - 2

 12 µl was injected into the GC-MS operating under positive chemical ionization in the single ion monitoring mode.

A c c e p t e d M a n u s c r i p t 7 2. 6

 76 Separation of cell compartmentsColo320 TP1 and RT112/TP cells (1.5x10 6 cells) were exposed to 200 µM TdR (hot:cold

A c c e p t e d M a n u s c r i p t 8 20

 20

A c c e p t e d M a n u s c r i p t 12 Both

 12 Colo320 TP1 and RT112/TP cells secreted radioactive products, with higher levels for RT112/TP (24%) than for Colo320 TP1(15%) cells.

A c c e p t e d M a n u s c r i p t 13 used

 13 for protein glycation, e.g. adding sugar moieties to proteins by non-enzymatic reactions[START_REF] Cho | The road to advanced glycation end products: a mechanistic perspective[END_REF][START_REF] Monnier | Nonenzymatic glycosylation, the Maillard reaction and the aging process[END_REF]. However, whether protein glycation of AGEs are formed in the cells used in our study, remains to be identified. Brown et al. previously reported a possible Schiff base reaction in RT112 cells, and demonstrated the presence of reactive oxygen species by expression levels of the oxidative stress marker heme oxygenase-1 (HO-1)[START_REF] Brown | Cooperative stimulation of vascular endothelial growth factor expression by hypoxia and reactive oxygen species: the effect of targeting vascular endothelial growth factor and oxidative stress in an orthotopic xenograft model of bladder carcinoma[END_REF].

A c c e p t e d M a n u s c r i p t 14 autocrine

 14 stimulation by endothelial cells themselves. Therefore, the model that we used in the present study was intended to reflect the stimulation of angiogenesis by cancer cells.

A c c e p t e d M a n u s c r i p t 15 cancer

 15 cells may in this way directly or indirectly (by its conversion to other sugars) stimulate angiogenesis. dR was secreted by the cancer cells in our study, indicating that dR can directly play a role in the angiogenic potential of TP.In conclusion, dR-1-P and subsequently dR-5-P and dR are formed from TdR conversion by TP. Both metabolites may stimulate angiogenesis, e.g. by the stimulation of angiogenic factors, while secreted dR may attract endothelial cells to form new blood vessels in the tumor tissue. The sugars formed from TdR will accumulate in the membrane and cytoskeleton of the cells, indicating that the sugars are used for further cellular metabolism, possibly mediating angiogenesis in a more indirect manner.