A Bronckaers

L Aguado

A Negri

M.-J Camarasa

J Balzarini

M.-J Pérez-Pérez

F Gago

S Liekens

& S Liekens

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine

Keywords: 6A5BU: 6-amino-5-bromouracil, 6AT: 6-aminothymine, 7-DX: 7-deazaxanthine, BVDU molecular mechanics-generalized Born surface area, NMA: normal mode analysis, PMSF: phenylmethylsulfonylfluoride, RESP: restrained electrostatic potential, rmsd: root mean square deviation, SASA: solvent accessible surface area, TFT: 5trifluorothymidine, TP: Thymidine phosphorylase, TP65: 9-[8-phosphonooctyl]-7deazaxanthine, TPI: 5-chloro-6-[ Thymidine phosphorylase, 5'-O-tritylinosine (KIN59), allosteric inhibitor, modeling, mutagenesis

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis

and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-tritylinosine (KIN59)

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A c c e p t e d M a n u s c r i p t 3 ABSTRACT Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V max /K m of mutant enzyme ~ 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site of TP that can be targeted by small molecules to inhibit the catalytic activity of TP.

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

Introduction

Thymidine phosphorylase (TP) is a key enzyme of the nucleoside salvage pathway [START_REF] Friedkin | The enzymatic synthesis of nucleosides. I. Thymidine phosphorylase in mammalian tissue[END_REF] that catalyzes the reversible conversion of thymidine and 2'-deoxyuridine to their respective bases and α-D-2-deoxyribose-1-phosphate. TP not only recognizes natural 2'-deoxynucleosides but is also able to catalyze the phosphorolysis of several nucleoside analogues endowed with antiviral or antitumor activity, such as 5-trifluorothymidine (TFT), 5-(E)-(2-bromovinyl)-2'deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine [START_REF] Nakayama | Thymidine phosphorylase. Substrate specificity for 5-substituted 2'-deoxyuridines[END_REF][START_REF] Desgranges | Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets[END_REF][START_REF] De Bruin | Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity[END_REF]. In addition to its catabolic role in the nucleoside salvage pathway, TP is also implicated in tumor progression as it has been shown to stimulate angiogenesis [START_REF] Haraguchi | Angiogenic activity of enzymes[END_REF][START_REF] Ishikawa | Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor[END_REF][START_REF] Miyadera | Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase[END_REF][START_REF] Moghaddam | Thymidine phosphorylase is angiogenic and promotes tumor growth[END_REF] and protect tumor cells from apoptosis [START_REF] Kitazono | Prevention of hypoxia-induced apoptosis by the angiogenic factor thymidine phosphorylase[END_REF][START_REF] Liekens | Targeting platelet-derived endothelial cell growth factor/thymidine phosphorylase for cancer therapy[END_REF][START_REF] Matsushita | The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors[END_REF]. A wide range of tumors, including breast, colon and oesophageal carcinomas overexpress TP, and high levels of TP have been correlated with microvessel density, metastasis and poor patient survival [START_REF] Liekens | Targeting platelet-derived endothelial cell growth factor/thymidine phosphorylase for cancer therapy[END_REF].

Thus, TP represents a promising chemotherapeutic target and TP inhibitors may (i) improve the antiviral and antitumor activity of certain nucleoside analogues, and (ii) inhibit the angiogenic and anti-apoptotic activities of TP. During the last decade, several classes of novel TP inhibitors have been developed [START_REF] Pérez-Pérez | Thymidine phosphorylase inhibitors: recent developments and potential therapeutic applications[END_REF]. For more than 30 years, the reference TP inhibitors were 6-aminouracil derivatives including 6-aminothymine (6AT) and 6-amino-5bromouracil (6A5BU) [START_REF] Langen | Inhibition of thymidine phosphorylase by 6-aminothymine and derivatives of 6-aminouracil[END_REF]. In 2000, Fukushima et al. reported on TPI (5-chloro-6-[1-(2iminopyrrolidinyl)methyl]uracil hydrochloride) as one of the most effective TP inhibitors to date, with an IC 50 value of 35 nM [START_REF] Fukushima | Structure and activity of specific inhibitors of thymidine phosphorylase to potentiate the function of antitumor 2'-deoxyribonucleosides[END_REF]. In fact, a combination of TPI and TFT is designated TAS-102 which is currently being evaluated in clinical trials for the treatment of various solid tumors, including metastatic colon and breast carcinomas [START_REF] Hong | Phase I study to determine the safety and pharmacokinetics of oral administration of TAS-102 in patients with solid tumors[END_REF][START_REF] Temmink | Therapeutic potential of the dual-targeted TAS-102 formulation in the treatment of gastrointestinal malignancies[END_REF].

Our research groups have described 7-deazaxanthine (7-DX), the first purine derivative with inhibitory activity against TP and angiogenesis in the 'chicken chorioallantoic membrane' (CAM) assay [START_REF] Balzarini | 7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase[END_REF], and also TP65 (9-[8-phosphonooctyl]-7-deazaxanthine), the A c c e p t e d M a n u s c r i p t 5 first multisubstrate inhibitor of TP that was shown to interact with both the thymidine and the phosphate binding site of TP [START_REF] Balzarini | Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase[END_REF][START_REF] Liekens | Antiangiogenic activity of a novel multi-substrate analogue inhibitor of thymidine phosphorylase[END_REF]. More recently, we identified the purine nucleoside 5'-O-tritylinosine as a novel TP inhibitor [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF][START_REF] Liekens | 5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis[END_REF], in which the presence of the trityl moiety was proven to be crucial for both its inhibitory activity against TP and its anti-angiogenic effect in the CAM assay. 5'-O-Tritylinosine is also a rather unique TP inhibitor because (i) it promotes the degradation of pre-existing immature vessels in the CAM assay, and (ii) in contrast to all previously described TP inhibitors, it inhibits TP in a non-competitive fashion with respect to both thymidine and phosphate. This latter finding implies that 5'-O-tritylinosine does not bind to the substrate-binding sites of TP, but rather interacts with a novel, yet unknown, allosteric site of TP that could also play a role in the biological activities of this protein.

It may be mentioned that diffraction-grade crystals of human TP in complex with TPI [START_REF] Norman | Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor[END_REF] were only obtained after limited treatment with trypsin, yielding two protein fragments consisting of residues 12-408 and 411-482. The structure was solved at 2.7 Å resolution, revealing a single protomer in the asymmetric unit, but the polypeptide stretch encompassing amino acids 407-414 could not be determined. In contrast, no proteolysis was required and well-ordered crystals with diffraction at 2.3 Å resolution were obtained when both thymidine (2 mM) and 5'-O-tritylinosine (1 mM) were present under the crystallization conditions [START_REF] El Omari | Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design[END_REF].

In this latter structure, two dimers appeared in the asymmetric unit, and residues 406-415 were visible as an arginine-rich surface loop that extends across the active site cleft and appears to stabilize the closed conformation of the enzyme and the dimer interface through hydrogen bonds. Taking these two crystal structures together, it is clear that the overall fold of human TP is similar to that previously reported for E. coli TP [START_REF] Walter | Threedimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution[END_REF][START_REF] Pugmire | Structural and theoretical studies suggest domain movement produces an active conformation of thymidine phosphorylase[END_REF] and comprises a large  domain connected by three loops to a smaller  domain. These three loops act as a hinge that allows the motion of one domain relative to the other so that the enzyme can interconvert from an open, inactive form to a closed conformation that is catalytically active. Intriguingly, although the presence of 5'-O-tritylinosine was instrumental for yielding good-quality A c c e p t e d M a n u s c r i p t 6 diffracting crystals of TP, no clear electron density was found for this non-competitive inhibitor, which in light of these findings may be regarded as a synthetic small-molecule, so called "crystallization chaperone" [START_REF] Kossiakoff | Understanding mechanisms governing protein-protein interactions from synthetic binding interfaces[END_REF]. For this reason, we have attempted to find a plausible binding site for 5'-O-tritylinosine on human TP and to provide a rationale for its behavior as a non-competitive inhibitr. Our proposal has been indirectly validated by site-directed mutagenesis of Asp-203, an amino acid that has turned out to be important not only for selective inhibition of the enzyme by the 5'-O-tritylinosine ligand but also for full catalytic competence of the enzyme.

2.

Methods and materials

Reagents

5'-O-Tritylinosine (earlier designated KIN59) was synthesized as described previously [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF][START_REF] Casanova | Microwave-assisted selective 5 '-O-trityl protection of inosine derivatives[END_REF]. The synthesis of 6AT and 6A5BU was performed as described [START_REF] Langen | Inhibition of thymidine phosphorylase by 6-aminothymine and derivatives of 6-aminouracil[END_REF]28]. Thymidine (dThd) and thymine (Thy) were purchased from Sigma (St-Louis, MO). The structural formulae of these compounds, as well as the reference inhibitor TPI, are depicted in Figure 1.

Molecular modeling.

Normal mode analysis.

The X-ray crystal structure of human TP (PDB code 2J0F) [START_REF] Norman | Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor[END_REF] at 2.31 Å resolution, was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb/). To probe the flexibility of this enzyme, an elastic network model was used in which all non-hydrogen protein atoms (within a cutoff of 10Å) were modeled as point masses and C atoms were connected by springs representing the interatomic force fields. Monomer A was analyzed as a large set of coupled harmonic oscillators using the normal mode analysis, deformation and refinement (NOMAD-Ref) server [START_REF] Lindahl | NOMAD-Ref: visulatization, deformation and refinement of macromolecular structures based on all-atom normal mode analysis[END_REF] and default parameters. The conformational changes were

Quantum mechanical methods, molecular mechanics force field and atom point charges.

The geometry of 5'-O-tritylinosine was first refined by means of the semi-empirical quantum mechanical program molecular orbital package (MOPAC) [START_REF] Stewart | MOPAC: a semiempirical molecular-orbital program[END_REF], using the AM1 Hamiltonian and PRECISE stopping criteria, and further optimized using a restricted Hartree-Fock (RHF) method and a 6-31G(d) basis set, as implemented in the ab initio program Gaussian 03 [START_REF] Frisch | Gaussian 03[END_REF].

The resulting wavefunctions were used to calculate electrostatic potential-derived (ESP)

charges employing the restrained electrostatic potential (RESP) methodology, as implemented in the assisted model building with energy refinement (AMBER) suite of programs (http://amber.scripps.edu/). The missing bonded and nonbonded parameters were assigned, by analogy or through interpolation from those already present in the AMBER database, in a way consistent with the AMBER force field [START_REF] Cornell | A second generation force-field for the simulation of proteins, nucleic-acids, and organicmolecules[END_REF]. The geometry and atom point charges for the transition state (TS) of the phosphorolysis reaction were retrieved from previous work [START_REF] Mendieta | Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations[END_REF].

Docking of 5'-O-tritylinosine into human TP.

A box encompassing both the active site and the loop regions was defined for the exploration of possible binding modes. The Lamarckian genetic algorithm implemented in AutoDock respectively. Rapid intra-and intermolecular energy evaluation of each configuration was achieved by having the receptor's atomic affinity potentials for aliphatic and aromatic carbon, oxygen, nitrogen, and hydrogen atoms precalculated in a three-dimensional grid with a spacing of 0.375 Å. A distance-dependent dielectric function was used in the computation of electrostatic interactions.

Molecular dynamics (MD) simulations.

The MD simulations were carried out using the AMBER 8.0 suite of programs [START_REF] Case | The Amber biomolecular simulation programs[END_REF]. A model of D203A TP was constructed using the mutagenesis module available in the molecular graphics program PyMOL (http://www.pymol.org/).The complexes of wild-type and D203A

TP with the transition state (TS) of the reaction [START_REF] Mendieta | Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations[END_REF] and of wild-type TP with KIN59 were neutralized by addition of sodium ions [START_REF] Aqvist | Ion water interaction potentials derived from free-energy perturbation simulations[END_REF] and immersed in truncated octahedrons of TIP3P

water molecules [START_REF] Jorgensen | Comparison of simple potential functions for simulating liquid water[END_REF]. Periodic boundary conditions were applied and electrostatic interactions were treated using the smooth particle mesh Ewald method [START_REF] Darden | Particle mesh Ewald: an N logN method for Ewald sums in large systems[END_REF] with a grid spacing of 1 Å.

The cutoff distance for the non-bonded interactions was 9 Å. The SHAKE algorithm [START_REF] Ryckaert | Numerical integration of cartesian equations of motion of a system with constraints: Molecular-dynamics of N-alkanes[END_REF] was applied to all bonds and an integration step of 2.0 fs was used throughout. Solvent molecules and counterions were relaxed by energy minimization and allowed to redistribute around the positionally restrained solute (25 kcal•mol -1 •Å -2 ) during 50 ps of MD at constant temperature (300 K) and pressure (1 atm). These initial harmonic restraints were gradually removed and the resulting systems were allowed to equilibrate further in the absence of any restraints for 10.0 ns during which system coordinates were collected every 2 ps for further analysis.

MM-GBSA binding calculations.

The binding free energy describing protein-ligand associations is the difference between the free energy, G, of the complex and that of the respective binding partners, in our case the

G bind = G (TP-TS) (i) -(G TP (i) + G TS (i)) (1)
The value of G for each species was calculated using the following energy decomposition scheme, which is implemented in the Molecular Mechanics/Generalized Born/Surface Area (MM-GBSA) method within AMBER 8 (script mm_pbsa.pl) [START_REF] Kollman | Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models[END_REF][START_REF] Srinivasan | Continuum solvent studies of the stability of DNA, RNA, and phosphoramidate -DNA helices[END_REF]:

G = E gas + G sol -TS E gas = E int + E ele + E vdw E int = E bond + E angle + E torsion (3) G sol = G GB + G nonpolar G nonpolar =  SASA
E gas is the gas-phase energy, which is calculated using the AMBER force field [START_REF] Duan | A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations[END_REF][START_REF] Lee | Distinguish protein decoys by using a scoring function based on a new AMBER force field, short molecular dynamics simulations, and the generalized born solvent model[END_REF] as the sum of internal energies (E int ), encompassing bond lengths (E bond ), bond angles (E angle ) and torsional angles (E torsion ), and coulombic (E ele ) and van der Waals (E vdw ) non-bonded energies.

The solvation free energy, G sol , is decomposed into polar and nonpolar contributions: the polar contribution (G GB ) is calculated by solving the generalized Born equation [START_REF] Onufriev | Modification of the generalized Born model suitable for macromolecules[END_REF] using dielectric constants of 1 and 78.5 for solute and solvent, respectively, whereas the nonpolar contribution, G nonpolar , is estimated from the solvent accessible surface area (SASA) [START_REF] Sitkoff | Accurate Calculation of Hydration Free-Energies Using Macroscopic Solvent Models[END_REF],

which is determined using a water probe with a radius of 1.4 Å. The surface tension constant g was set to 0.005 kcal/mol/Å 2 . T and S are the temperature and the total solute entropy, respectively. S is typically estimated on the basis of classical statistical formulae and NMA of representative snapshots of energy minimized structures from a MD trajectory. In the present investigation this cumbersome procedure was obviated so that, in a first approximation, the solute entropy estimate were not included in the calculation of the free energy changes by A c c e p t e d M a n u s c r i p t 10 assuming, on the basis of previous work [START_REF] Huo | Computational alanine scanning of the 1 : 1 human growth hormone-receptor complex[END_REF], that its contribution to ΔG bind would be negligible.

On the other hand, the use of a single trajectory to calculate G bind is conducive to a much better agreement with the experimental data than when multiple trajectories are used [START_REF] Moreira | Computational alanine scanning mutagenesis -An improved methodological approach[END_REF]. It is then expected that, by using a single trajectory, error cancellation will overcome the insufficient sampling of the conformational space [START_REF] Huo | Computational alanine scanning of the 1 : 1 human growth hormone-receptor complex[END_REF][START_REF] Gohlke | Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RaIGDS complexes[END_REF].

Site-directed mutagenesis of human TP

The pMOAL-10T vector containing the human TP gene (fused to glutathione Stransferase [GST]) was described and kindly provided by Prof. R. Bicknell (Oxford, UK) [START_REF] Moghaddam | Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity[END_REF].

The mutant TP enzyme containing the D203A mutation was derived from this vector. Sitedirected mutagenesis was performed using the Quick Change site-directed mutagenesis kit (Stratagene, Westburg, Leusden, The Netherlands) according to manufacturer's instructions.

The oligonucleotide primers that were used are 5' cgg aat cct ata tgc agc cag agc tgt gac agc cac cgt gg-3' and its reverse primer (Invitrogen, Merelbeke, Belgium). After linear amplification of the plasmid using Pfu DNA polymerase (Stratagene) in a temperature cycler program (30 min at 95 °C followed by 20 cycles of 30 min at 95 °C, 1 min at 55 °C and 12 min at 68 °C), wild-type (methylated) plasmid was digested with DpnI restriction enzyme (Stratagene) and the mutant (unmethylated) DNA was transformed into E. coli XL1-Blue super competent cells (Stratagene). The presence of the desired mutation in the TP gene was confirmed by sequencing on an ABI Prism 3100 sequencer (Applied Biosystems, Foster City, CA), using the ABI Prism Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems).

Purification of human recombinant TP

TP enzyme assays

The conversion of dThd to thymine was measured by high-pressure liquid chromatography (HPLC) analysis. Retention times of thymine and dThd were respectively 5.1 and 10.8 minutes. UV-based detection was performed at 267 nm.

In the kinetic assays, the K m and V max of wild-type TP and the mutant D203A TP were determined at varying concentrations of dThd, while the concentration of inorganic phosphate was kept constant at 25 mM. In these experiments, wild-type TP was used at a concentration of 0.5 µg/500 µl reaction mixture in the presence of 5, 3, 2, 1, 0. When the surroundings of this loop were probed for plausible binding sites for 5'-Otritylinosine using the reported X-ray crystal structure and the automated docking program AutoDock, no convincing solutions were found. 5'-O-Tritylinosine is a rather lipophilic compound possessing a bulky trityl moiety for which no electron density emerged in the diffraction maps. However, the molecule clearly helped stabilize the conformation of this loop in the crystal lattice [START_REF] El Omari | Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design[END_REF]. We reasoned this might be due to ligand (5'-O-tritylinosine) expulsion following protein packing and local hydrophobic collapse. We therefore decided to further focus on some prior conformational state(s) of the enzyme for our docking studies.

Since open and closed TP conformations are connected by hinge-dependent interdomain motions [START_REF] Mendieta | Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations[END_REF], as is also the case in the related pyrimidine nucleoside phosphorylase (PyNP)

[49], we used normal mode analysis (NMA) to reproduce this motion and generate a series of intermediate protein conformers that could help in the identification of a region capable of lodging the hydrophobic and polar substituents of 5'-O-tritylinosine.

By following the lowest frequency mode, a cavity was generated in the vicinity of the Gly-405-Val-419 loop that was mainly lined by the side chains of apolar residues Leu-155,

Val-166 and Ile-167, and positively charged residues Arg-146 and Arg-408, all of which can provide good anchoring points for the crucial trityl group of 5'-O-tritylinosine. Indeed, the automated docking program could find satisfactory docking solutions for 5'-O-tritylinosine making use of this hydrophobic cavity. Thus, in the optimal solution, and following complex relaxation using molecular dynamics (MD), the guanidinium of Arg-146 is found stacking on top of one of the phenyl rings, which in turn makes van der Waals contacts with the side chains of Leu-155 and Val-166, whereas another phenyl of the trityl group interacts with the side chains of Val-166 and Ile-167. As regards the remaining part of the inosine nucleoside, the 3' hydroxyl of the ribose is engaged in a hydrogen bond with the carboxamide group of Gln-187 (not shown), whereas the hypoxanthine moiety gets sandwiched between the tip of the Gly-405-Val-419 loop and the C-terminus of helix 9. In this location, the heteroaromatic (not shown) (Figure 3).

This docking proposal accounts for the mechanism of non-competitive inhibition, insofar as 5'-O-tritylinosine does not make use of the dThd or phosphate binding sites, and is qualitatively consistent with the known structure-activity relationship for this class of 5'-Otritylinosines [START_REF] Casanova | 5'-Otritylinosine and analogues as allosteric inhibitors of human thymidine phosphorylase[END_REF]. Indeed, it is known that one of the phenyl rings in the essential trityl substituent can accept a chlorine atom at the para position leading to enhanced potency, and also that incorporation of a methylcyclopropyl or methylcyclohexyl substituent at the N1 position of hypoxanthine results in increased inhibitory activity. The former finding can be explained because the chlorine atom can be held in one of the hydrophobic pockets made up by Leu-155, Val-166, and Ile-167 whereas the latter may originate from improved hydrophobic interactions between the cycloalkyl substituent and the side chain of Val-204.

Given the central role ascribed to Asp-203 in the stabilization of the Gly-405-Val-419 loop over the active-site cleft in the closed conformation, we decided to mutate this residue to alanine in order to assess the impact of this amino acid replacement on both enzyme activity and modulation of catalysis by TP inhibitors.

Enzymatic activity and kinetic parameters of wild-type and mutant D203A TP

The enzymatic activities of purified recombinant wild-type and mutant D203A TP were compared by using different amounts of enzyme (0.5 and 0.1 µg/500 µl reaction mixture for wild-type TP and 20, 5 and 0.5 µg/500 µl reaction mixture for mutant D203A TP). The enzymes were incubated for 0, 15, 30 and 60 minutes in the presence of 100 µM of dThd (Fig. 4). The mutant D203A TP converted markedly less substrate to thymine, as compared to the corresponding wild-type enzyme. The reaction proceeded linearly for up to at least 60 min at the lower TP concentrations and up to 15 min at the highest enzyme concentrations. Based on A c c e p t e d M a n u s c r i p t 16 the enzyme concentrations used, it was estimated that the mutant D203A TP converts dThd to thymine at least 20-fold less efficiently than wild-type enzyme in the presence of 100 µM dThd (Fig. 4). Thus, the mutation of aspartic acid to alanine at amino acid position 203 of TP resulted in a significant loss of efficiency to perform the phosphorolysis of dThd. Therefore, higher concentrations were used for the mutant D203A enzyme than wild-type TP to obtain reliable enzyme kinetic data that could be compared.

To determine the kinetic parameters for wild-type and mutant D203A TP the enzyme concentrations used were 0.5 µg/500 µl reaction mixture and 10 µg/500 µl reaction mixture, respectively. Under these experimental conditions, the enzyme-catalytic activity proceeded linearly during the incubation (15 min) time. Both enzymes were incubated in the presence of varying concentrations of dThd at a saturating concentration of inorganic phosphate. The K m value of the mutant D203A TP (9.8 mM) proved to be 23 times higher than that of the wildtype enzyme (Km: 414 µM), which suggests a markedly diminished affinity of the mutant enzyme for its natural substrate dThd. Moreover, a 2.5-fold reduction in the V max value (1,026 nmol/min/µg for wild-type TP versus 400 nmol/min/µg for mutant D203A TP) indicated that the mutation also affected (albeit to a lesser extent) the rate of dThd phosphorolysis (Table 1).

Overall, the phosphorolytic capacity of the mutant D203A TP proved to be ~ 60-fold lower than that of wild-type TP using dThd as the natural substrate.

Simulation of the transition state of the reaction in the wild-type and mutant D203A TP enzymes

In light of the preceding results we sought to determine the structural basis for the decrease in catalytic performance of the mutant D203A TP relative to the wild-type enzyme using computational methods. Since the geometry of the phosphorolysis reaction transition state (TS) has been previously characterized in detail [START_REF] Mendieta | Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations[END_REF], unrestrained MD simulations of each enzyme in the presence of an active site-bound TS were undertaken to analyze the relative binding affinities in the two complexes.

In the wild-type enzyme the TS appears to be stabilized by a number of interactions involving both polar and apolar active-site residues (Figure 5): By hydrogen-bonding to the NH of Gly-149, the carboxylate of Asp-203 was thus seen to play a crucial role in facilitating the correct placement of the side-chain of Leu-148. This amino acid residue has been previously shown to be essential for TP activity [START_REF] Miyadera | Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase[END_REF], most likely because it provides a hydrophobic platform for the stacking of both the phenol ring of Tyr-199 (which is fixed in place with the aid of a hydrogen bond from the hydroxyl group to the backbone carbonyl of Arg-146) and the pyrimidine base of the substrate and competitive inhibitors. In fact, the major difference observed within the slightly more open active site in the simulated TS-TP complex for the mutant D203A enzyme involved the positioning of Leu-A c c e p t e d M a n u s c r i p t 18 148 and when the MM-GBSA method was used to calculate the binding energies of the TS within both enzymes along the simulation period, the binding energy corresponding to the mutant D203A TP was indeed found to be consistently decreased relative to the wild-type protein (Figure 6). Moreover, the loss of binding energy originated in the van der Waals term because the electrostatic and desolvation components were virtually identical (data not shown). Therefore, even though these binding energy values have to be regarded as qualitative rather than quantitative (since entropic effects, which are cumbersome to compute and subject to large numerical errors, were not included in the calculation), the trend is very clear and is in agreement with the experimental findings: the loss of the hydrogen bond between the carboxylate of Asp-203 and the backbone NH of Gly-149 results in a repositioning of the side chain of Leu-148 and in a more open active site, both of which are detrimental for dThd binding, TS stabilization and efficient catalysis.

Inhibition of wild-type and mutant D203A TP by competitive and non-competitive inhibitors

It has been suggested that mouse liver TP contains more than one allosteric (i.e. thymine binding) site, and that when thymine is bound to one of these sites, the enzyme is inhibited [START_REF] Iltzsch | Kinetic studies of thymidine phosphorylase from mouse liver[END_REF]. We found thymine (100 µM) inhibitory to the human TP-catalysed hydrolysis of 100 µM dThd, but thymine had no effect on the inhibitory activity of 5'-O-tritylinosine (200 µM and 100 µM) (data not shown), making it unlikely that 5'-O-tritylinosine may bind to one of such potential thymine-binding sites.

However, we previously showed that 5'-O-tritylinosine inhibits TP in a noncompetitive fashion [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF]. If Asp-203 is part of a putative allosteric site to which this inhibitor binds or contributes to its formation, one would expect that mutation of this amino acid residue to alanine would affect the inhibitory activity of this compound against TP. To test this hypothesis, wild-type and mutant TP enzymes were incubated for 15 minutes with 100 In sharp contrast, the competitive inhibitors 6AT and 6A5BU, which have been shown to interact with the dThd-binding site of TP [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF], were able to dose-dependently decrease the enzymatic activity of wild-type and mutant D203A TP to a similar extent (Fig. 7, panels B and C) (6AT and 6A5BU inhibitor concentrations used: 50, 25 and 5 µM for wild-type TP and 1,250, 625 and 125 µM for mutant D203A TP in the presence of 100 µM dThd for wild-type TP and 2,500 µM dThd for mutant D203A TP). These findings are compatible with the view that binding of dThd, 6AT and 6A5BU to the active site is similarly compromised in the mutant enzyme relative to the wild-type TP whereas the putative allosteric site for 5'-Otritylinosine binding is no longer present on the mutant D203A TP due to loop disruption.

Concluding remarks

Earlier site-directed mutagenesis studies on TP have confirmed the importance for catalysis of Lys-115, Leu-148, Arg-202, Ser-217, and Lys-221 [START_REF] Miyadera | Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase[END_REF][START_REF] Moghaddam | Thymidine phosphorylase is angiogenic and promotes tumor growth[END_REF]. All of these are active-site residues directly involved in the stabilization of the substrates (dThd and phosphate) and the TS, as explained above. Asp-203, on the other hand, is positioned at the end of an α-helix and extends its side chain towards a solvent region comprised between the glycine-rich 144-153 loop and the more mobile arginine-rich 406-415 loop. In this location, the Asp-203 carboxylate can accept two hydrogen bonds: one from the backbone NH of Gly-149 in the former loop (Fig. 2 and5) and a second one from a water molecule that is, in turn, hydrogen bonded to the guanidinium of Arg-408 in the latter loop (Fig. 2). In addition, the Asp-203 carbonyl can also make a water-mediated hydrogen bond with the carbonyl of Arg-408 (Fig. 5'-O-Tritylinosine has been described previously as an inhibitor of both human and bacterial TP enzymes [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF][START_REF] Liekens | 5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis[END_REF]. Given the non-competitive nature of this inhibition, this purine riboside derivative was not expected to interact directly with either the pyrimidine nucleosidebinding or phosphate-binding site of the enzyme. On the contrary, this novel inhibitory mechanism was indicative of the possible existence of a yet unidentified allosteric site in the enzyme. Nonetheless, a crystal structure of human TP produced in the presence of 5'-Otritylinosine revealed no traces of this inhibitor neither in the active site, which was occupied by the reaction product, thymine, nor anywhere else.

By using a combination of NMA, docking and MD simulations methods, a location for this binding site is now proposed that relies on the loop stabilization brought about by Asp-203. The site-directed mutant D203A TP, which was constructed to assess the importance of this residue, displayed a ~60-fold reduction in phosphorolytic capacity (V max /K m ) relative to the wild-type enzyme. Importantly, both wild-type and mutant TP were inhibited to a similar extent by the competitive inhibitors 6AT and 6A5BU whereas the non-competitive inhibitor KIN59 lost its inhibitory activity against the mutant D203A enzyme. These data thus support a role for Asp-203 in loop stabilization, which is important for catalytic activity, as well as for creating a rather unique binding site for 5'-O-tritylinosine. A multiple sequence alignment of human, mouse, rat and E. coli TP (Figure 8) shows that Asp-203 is highly conserved during evolution, which reinforces the idea that this amino acid may indeed be crucial for the biological activities of TP. We previously hypothesized that this putative allosteric site might be involved in the angiogenic activity of TP [START_REF] Liekens | The nucleoside derivative 5'-O-trityl-inosine (KIN59) suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive mechanism of action[END_REF][START_REF] Liekens | 5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis[END_REF]. Therefore, further investigations on the angiogenic and anti-apoptotic properties of the mutant D203A enzyme would be warranted. V max is the maximum velocity of the enzyme reaction. c V max /K m ratio or catalytic phosphorolytic capacity of the enzyme. Values are presented as means ± standard deviation of at least three independent experiments.

Acknowledgements

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

 7 by calculating the 10 lowest-frequency normal modes, which are those with the highest amplitudes and those most often related to large-scale structural rearrangements in proteins. Each mode was explored in its two opposite directions, thus resulting in a series of conformations different from the crystallographic structure within a maximum root-meansquare deviation (rmsd) value of 2 Å.

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

 9 -type protein (TP) and the transition state of the reaction (TS), calculated over a series of snapshots (i) taken from the MD trajectory:

  A c c e p t e d M a n u s c r i p t 11 The pMOAL-10T vector containing the wild-type TP or the mutant D203A TP was transfected into E. coli BL21(DE3)pLysS. Bacteria were grown overnight at 37 °C in 2YT medium containing ampicillin (Bristol Myers Squibb, Brussels, Belgium) (100 μg/mL) and chloramphenicol (Sigma) (100 μg/mL), diluted 1:10 in fresh medium and further incubated for 5 h at 27 °C. Then isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma) was added to a final concentration of 0.1 mM to induce the expression of the GST-TP fusion protein. After a further incubation for 15 h at 27 °C, the cells were pelleted (10,000×g for 10 min at 4 °C) and resuspended in lysis buffer (50 mM Tris, pH 7.5, 1 mM dithiothreitol, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 0.1 mM phenylmethylsulfonylfluoride [PMSF] and 0.15 mg/ml lysozyme). The bacterial suspensions were passed through the SLM Aminco French Pressure Cell Press (Beun de Ronde, La Abcoude, The Netherlands) and centrifuged at 20,000×g for 15 min at 4 °C. The GST-TP fusion protein was further purified from the supernatant using Glutathione Sepharose 4B (GE Healthcare, Little Chalfont, Buckinghamshire, UK), following the instructions of the manufacturer. Briefly, a 50% glutathione-agarose slurry was added to the bacterial supernatant, incubated for 30 min at 4 °C, and then washed three times with 10 bed volumes (7.5 mL) of lysis buffer without lysozyme and PMSF. Bound proteins were eluted in 50 mM Tris (pH 8.0) containing 0.1% Triton X-100 and 10 mM glutathione. Protein content of the purified fractions was determined using Bradford reagent (Sigma, St-Louis, MO).

A c c e p t e d M a n u s c r i p t 13 3. Results and discussion 3 . 1

 1331 5 and 0.2 mM dThd, while the mutant D203A TP was tested at a concentration of 10 µg/500 µL reaction mixture in the presence of 40, 30, 20, 15 and 10 mM dThd. Incubation was performed for 15 min. At this time point, the reaction still proceeded linearly.To evaluate the inhibitory activity of 5'-O-tritylinosine, 6AT and 6A5BU several concentrations of these TP inhibitors were added to the reaction mixtures containing 100 µM (for wild-type TP) or 2.5 mM (for mutant D203A TP) of dThd. Wild-type TP and mutant D203A TP were used at a concentration of 0.5 and 10 µg/500 µL reaction mixture, respectively. The mixtures were incubated for 15 minutes. The amount of thymine formed was determined by HPLC analysis as described above. Proposal of a binding mode for KIN59 on human TP Inspection of the Gly-405-Val-419 loop (which was clipped in the structure of Norman et al.[START_REF] Norman | Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor[END_REF]), in the crystallographic structure of El Omari et al.[START_REF] El Omari | Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design[END_REF], revealed an intricate hydrogen-bonding network, the most relevant part of which is schematically represented in Figure2:(i) the backbone NH of Arg-408 is hydrogen-bonded to a welldefined water molecule (W98 in subunit A, designated WAT1) that is fixed in place by two additional hydrogen bonds: one with the backbone carbonyl of Gly-149 and another with one carboxylate oxygen from Asp-203, a residue that is situated at the terminus of -helix 9, (ii) the same carboxylate oxygen of Asp-203 forms a good hydrogen bond with the backbone NH of Gly-149, which is next to Leu-148, a residue whose side-chain provides a hydrophobic surface for interaction with the pyrimidine base of both the dThd substrate and competitive inhibitors such as TPI, (iii) the guanidinium group of Arg-408 establishes two strong hydrogen bonds with the carboxylate oxygens of Asp-156 (not shown) and also a watermediated (W80 in subunit B, designated WAT2) hydrogen bond with the other Asp-203 carboxylate oxygen, (iv) the carbonyl groups of the backbones of Asp-203 and Arg-408 are both accepting a hydrogen bond from a common water molecule (designated WAT3); (v) the guanidinium group of Arg-410 is partly exposed to the solvent but also engaged in an intersubunit hydrogen bond with the carbonyl group of Ser-65 (not shown); and (vi) the backbone CO and NH groups of Ala-411 in one protomer establish two good hydrogen bonds with the side-chain carboxamide of Gln-67 in the neighboring subunit (not shown). Thus, it appears that the Gly-405-Val-419 loop consists of closely interacting amino acids that contribute to the stabilization of the closed conformation of the enzyme (the only state that presents the integral active site) by helping to hold the α and α/β domains together.

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

 14

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

 15 partially sits on top of the side-chain methyl of Ala-200 (not shown), N3 is close to Nε of Arg-146 and N1 is oriented towards the carboxylate of Asp-203 and the isopropyl of Val-204

  (i) the pyrimidine ring gets sandwiched between the side chains of Tyr-199 and Leu-148, on one side, and that of Ile-218 on the other side, and places the methyl group in the vicinity of Val-241 (not shown); (ii) the O2 and O4 of thymine accept hydrogen bonds from the amino group of Lys-221 and the guanidinium group of Arg-202, respectively, whereas the hydroxyl of Ser-217 accepts a hydrogen bond from N3 and the imidazole ring of His-116 makes a bifurcated hydrogen bond with N1 and O2; (iii) the O5' of the deoxyribose accepts a H-bond from the hydroxyl of Thr-151 and donates one to the carbonyl of the backbone of Gly-119 while the backbone NH of Thr-154 hydrogen bonds to O3' of the deoxyribose, which in turn donates another H-bond to the phosphate, precisely to the same oxygen (OP1) that accepts a second H-bond from the hydroxyl of Thr-154; and (iv) the phosphate OP3 accepts two hydrogen bonds, one from the amino group of Lys-115 and another from the hydroxyl of Ser-117 (not shown). This elaborate hydrogen-bonding pattern, which is maintained during the whole length of the MD simulation, stabilizes the TS geometry and facilitates the nucleophilic attack of the phosphate O2P onto the sugar ring atom C1'.

A c c e p t e d M a n u s c r i p t 19 µM

 19 dThd in the presence of various concentrations of either the non-competitive inhibitor 5'-O-tritylinosine or the known competitive inhibitors 6AT and 6A5BU. As shown in Figure7, 5'-O-tritylinosine was completely unable to inhibit the enzymatic activity of the D203A mutant TP regardless of the concentrations used (100, 50 and 25 µM) whereas a concentration-dependent inhibition of the wild-type enzyme was observed (Fig.7, panel A).

A c c e p t e d M a n u s c r i p t 20 2

 20 ). It thus appears that Asp-203 plays a crucial role in loop stabilization, and this action is important for both catalysis and inhibition by the non-competitive inhibitor 5'-O-tritylinosine.

Figure 1 .

 1 Figure 1. Chemical structures of relevant TP inhibitors.

Figure 2 .

 2 Figure 2. Scheme depicting the most relevant hydrogen bonds that contribute to loop

Figure 3 .

 3 Figure 3. Proposed binding site for 5'-O-tritylinosine (previously designated KIN59 [20]) in

Figure 4 .

 4 Figure 4. Conversion of thymidine to thymine by wild-type and mutant D203A TP as a

Figure 5 .

 5 Figure 5. Detail of the active site of wild-type TP containing the transition state of the reaction

A c c e p t e d M a n u s c r i p t 30 Figure 6 .

 306 Figure 6. Time evolution of the overall interaction energy between the transition state of the

Figure 7 .

 7 Figure 7. Effect of the inhibitors 5'-O-tritylinosine (panel A), 6AT (panel B) and 6A5BU

Figure 8 .

 8 Figure 8. ClustalW multiple sequence alignment of the amino acid sequences of mouse, rat,

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

Figure 1 Page

 1 Figure 1

Figure 2 A

 2 Figure 2

Figure 3 A

 3 Figure 3

Figure 4 A

 4 Figure 4

Figure 5 A

 5 Figure 5

Figure 6 A

 6 Figure 6

Figure 8

 8 Figure 8

Table 1 .

 1 Kinetic parameters of human wild-type and mutant D203A TP Enzyme activity is expressed as the amount of dThd converted to thymine [µmol/mg protein/min]. a K m is the Michaelis-Menten constant of the enzyme for dThd.

	TABLES
		K m a (µM)	V max b (µmol/mg	V max /K m c
			protein/min)
	Wild-type TP	414 ± 96	1026 ± 460	2.48
	D203A mutant TP	9828 ± 3200	400 ± 136	0.041

b

We thank Mrs. Kristien Minner for her dedicated technical help and Mrs. Christiane Callebaut for fine editorial assistance. This research was supported by grants to J.B. from the Flemish FWO [Krediet nr. G. 0486.08], the Concerted Actions [GOA nr. 05/19], the Centers of Excellence [Krediet nr. 05/15] (to J.B. and S.L.), and from Comisión Interministerial de Ciencia y Tecnología [SAF2006-12713-C02] and Comunidad de Madrid [S-BIO/0214/2006] (to M.J.C, M.-J.P.-P. and F.G.). Annelies Bronckaers benefits from a PhD scholarship of the FWO and Sandra Liekens is a postdoctoral fellow of the FWO. Leire Aguado thanks the Spanish Ministerio de Educación y Ciencia for a FPU predoctoral fellowship.

The search for the binding site of the allosteric inhibitor 5'-O-trylinosine has allowed the identification of Asp203 as an important residue for catalysis in human thymidine phosphorylase.

Graphical Abstract


Mahmoud Youns
email: m.youns@dkfz.de

Thomas Efferth

Jürgen Reichling

Kurt Fellenberg

Andrea Bauer

Jörg D Hoheisel

Youns Mahmoud

Gene expression profiling identifies novel key players involved in the cytotoxic effect of Artesunate on pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies, with an extremely poor prognosis. The paucity of curative therapies has translated into an overall 5year survival rate of less than 5%, underscoring a desperate need for new therapeutic options. Artesunate (ART), clinically used as anti-malarial agent, has recently revealed remarkable anti-tumor activity. However, the mechanisms underlying those activities in pancreatic cancer are not yet known. Here we evaluated the antitumor activity of Artesunate and the possible underlying mechanisms in pancreatic cancer. MiaPaCa-2 (poorly differentiated) and BxPC-3 (moderately differentiated) pancreatic cancer cell lines were treated with Artesunate

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

Gene expression profiling identified Artesunate as a novel topoisomerase

IIα inhibitor that inhibits pancreatic cancer cell growth through modulation of multiple signalling pathways.

Graphical Abstract

and the effect was monitored by a tetrazolium-based assay (MTT) for evaluating cell viability and by flow cytometry and caspase 3/7 activation for apoptosis evaluation. In addition cDNA arrays were used to identify differentially expressed genes. The microarray data were then validated by RT-PCR and western blotting. Moreover, pathways associated with these expression changes were identified using the Ingenuity Pathway Analysis. The expression analysis identified a common set of genes that were regulated by Artesunate in pancreatic cancer. Our results provide the first in vitro evidence for the therapeutic utility of Artesunate in pancreatic cancer. Moreover, we identified Artesunate as a novel topoisomerase IIα inhibitor that inhibits pancreatic cancer growth through modulation of multiple signaling pathways. The present analysis is a starting point for the generation of hypotheses on candidate genes and for a more detailed dissection of the functional role of individual genes for the activity of Artesunate in tumor cells.

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

Introduction

Pancreatic cancer remains a devastating and poorly understood malignancy with increasing incidence worldwide. Cancer of the exocrine pancreas is the fourth most common malignancy in the United States. The annual incidence rate is almost identical to the mortality rate. Currently, surgery is the only treatment, although due to its late presentation, only 9% to 15% of patients are suitable for surgery [START_REF] Jemal | Cancer statistics[END_REF].

Gemcitabine, the standard of care since 1997 [START_REF] Burris | Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial[END_REF], is the current drug of choice for treatment of pancreatic cancer. It has been shown to improve the clinical outcome and survival compared with 5-fluorouracil [START_REF] Burris | Assessing clinical benefit in the treatment of pancreas cancer: gemcitabine compared to 5-fluorouracil[END_REF], however, the median survival for all pancreatic cancer stages is still ~ 3 to 5 months from diagnosis [START_REF] Shore | Cancer in the elderly: pancreatic cancer[END_REF] and the five-year survival rate remains less than 5% [START_REF] Saif | Controversies in the adjuvant treatment of pancreatic adenocarcinoma[END_REF]. Therefore, new therapeutic strategies are necessary to combat this deadly disease. To obtain an effective regimen, a careful and well-designed combination of multi-therapeutic agents with different modes of action will be required and that was a goal in our study.

Artemisinin, a sesquiterpene isolated from Artemisia annua L., is used in traditional Chinese medicine for the treatment of fever and chills [START_REF] Klayman | Qinghaosu (artemisinin): an antimalarial drug from China[END_REF]. Artesunate (ART) is a semisynthetic derivative of artemisinin. ART and other artemisinin derivatives are promising novel drugs in the treatment of malaria [START_REF] Price | Artemisinin drugs: novel antimalarial agents[END_REF]. Large clinical studies with malaria patients have shown that ART is well tolerated, with a few and insignificant side effects [START_REF] Hien | An open randomized comparison of intravenous and intramuscular artesunate in severe falciparum malaria[END_REF][START_REF] Hien | Qinghaosu[END_REF][START_REF] Fishwick | The toxicity of artemisinin and related compounds on neuronal and glial cells in culture[END_REF]. In addition to the well known anti-malarial activity of ART, recently a cytotoxic action of ART against cancer cell lines of different tumor types is identified [START_REF] Efferth | The anti-malarial artesunate is also active against cancer[END_REF][START_REF] Singh | Selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells[END_REF][START_REF] Dell'eva | Inhibition of angiogenesis in vivo and growth of Kaposi's sarcoma xenograft tumors by the antimalarial artesunate[END_REF][START_REF] Chen | Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro[END_REF][START_REF] Li | Artesunate attenuates the growth of human colorectal carcinoma and inhibits hyperactive Wnt/beta-catenin pathway[END_REF][START_REF] Efferth | Molecular modes of action of artesunate in tumor cell lines[END_REF]. Until now, the accepted antitumor mechanism is similar to the antimalarial mechanism; the artemisinin structure contains an endoperoxide bridge that reacts with an iron atom to form free radicals [START_REF] Zhang | Hemin-catalyzed decomposition of artemisinin (qinghaosu)[END_REF][START_REF] Efferth | Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron[END_REF][START_REF] Kelter | Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate[END_REF], which causes macromolecular damage and cell death [START_REF] Anderson | Free radicals and reactive oxygen species in programmed cell death[END_REF].

A c c e p t e d M a n u s c r i p t 4 Previous studies showed that ART induces ROS-mediated apoptosis in doxorubicinresistant T leukemia cells [START_REF] Efferth | Artesunate induces ROSmediated apoptosis in doxorubicin-resistant T leukemia cells[END_REF], attenuates the growth of human colorectal carcinoma, inhibits hyperactive Wnt/β-catenin pathway [START_REF] Li | Artesunate attenuates the growth of human colorectal carcinoma and inhibits hyperactive Wnt/beta-catenin pathway[END_REF], exhibits anticancer growth activities in human ovarian cancer cells [START_REF] Jiao | Dihydroartemisinin is an inhibitor of ovarian cancer cell growth[END_REF], inhibits nitric oxide [START_REF] Konkimalla | Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells[END_REF] and induces DNA damage and repair [START_REF] Li | Artesunate derived from traditional Chinese medicine induces DNA damage and repair[END_REF].

Despite the great efforts that have been done, the effect of ART on pancreatic cancer and its possible molecular mechanisms are not yet known.

In the present study, we established that ART induces growth arrest and apoptosis in pancreatic cancer cell lines and its effect depends on the differentiation stage, being more effective against the poorly differentiated cells. In addition, our results suggest that ART potentiates the antitumor effects of gemcitabine in pancreatic cancer. Moreover, to the best of our knowledge, this is the first study demonstrating ART as a novel topoisomerase IIα containing 100 units /ml penicillin and 100 µg /ml streptomycin and supplemented with heatinactivated 10% fetal bovine serum (FBS). MiaPaCa-2 cells were maintained in DMEM containing 100 units /ml penicillin, 100 µg/ml streptomycin and 10% FBS (Invitrogen, Carlsbad, CA).Cultured cells were maintained in a humidified environment at 37°C with 5% CO 2 . Artesunate (purity ≥99%) was purchased from Saokim Pharma (Hanoi,Vietnam) and a stock solution in DMSO at 1 M was prepared. Gemcitabine was kindly provided by Eli Lilly (Gemzar®, Bad Homburg, Germany). The concentration of DMSO was kept at or below 0.1 % in all experiments.

MTS cell proliferation assay.

To assess cell proliferation, CellTiter 96® Aqueous Nonradioactive Cell Proliferation Assay (Promega, Mannheim, Germany) was used according to the manufacturer's instructions. The assay tests cellular viability and mitochondrial function.

Briefly, cells were grown in tissue culture flasks, and then harvested by treating the flasks with 0.025% trypsin and 0.25mM EDTA for 5 min. Once detached, cells were washed, counted and an aliquot (5x10 3 cells) was placed in each well of a 96-well cell culture plate in a total volume of 100 μl. Cells were allowed to attach overnight and then treated with or without increasing concentrations of ART. After 24, 48, and 72 h, 20 μl MTS solution was added to each well and the plates were incubated at 37°C for 3h. The absorbance of the product formazan, which is considered to be directly proportional to the number of living cells in the culture, was measured at 490 nm and at 650 nm using a Precision Microplate Reader (Molecular Devices, Sunnyvale, CA). The same routine was done after treating cells

Flow Cytometry analysis of apoptotic cells. Cells treated with gradient concentrations

of ART were harvested and washed with ice-cold phosphate-buffered saline (PBS; Invitrogen) and resuspended in 150 µl hypotonic fluorochrome solution [50 µg/ml propidium iodide, 0.1% (w/v) sodium citrate (pH 7.4) and 0.1% (v/v) Triton X-100]. The cells were incubated in the dark at 4°C overnight before performing FACS analysis. The propidium iodide fluorescence of individual nuclei was measured using a FACS-Calibur cytometer (BD Biosciences, Heidelberg, Germany). Data were analysed with the CellQuess Pro V5.2.1 software (BD Biosciences). For each condition, at least three independent experiments were performed.

2.4.

Caspase-Glo 3/7 assays. Caspase-Glo TM 3/7 Assay (Promega) was used to detect Caspase 3/7 activities of MiaPaCa-2 cancer cell lines triggered by ART. This test provides a proluminescent caspase-3/7 substrate, which contains the caspase-3 specific tetrapeptide sequence DEVD in a reagent optimized for cell lysis and determination of caspases and luciferase activity. MiaPaCa-2 cells cultured in DMEM were seeded in 96-well plates and treated with ART. Six hours after treatment, cellular caspase 3/7 activity was determined according to the manufacturer's protocol. Luminescence was measured using Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). Cellular apoptosis was expressed as percentage of the untreated medium control.

Microarray gene expression profiling.

MiaPaCa-2 and BxPC-3 cell lines were treated with 25 µM ART or with DMSO alone (control) for 48 h. Total-RNA from each sample was isolated with the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The integrity of the isolated RNA was checked on an Agilent Bioanalyser 2100 using the RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, USA). Fluorescently A c c e p t e d M a n u s c r i p t 7 labelled cDNA samples were prepared from 15 µg total-RNA. Cy3-or Cy5-labelled dCTP was directly incorporated during first-strand synthesis [START_REF] Beier | Fluorescent sample labeling for DNA microarray analyses[END_REF] Microarrays were produced and processed as described in detail previously [START_REF] Esposito | Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?[END_REF], representing a well-defined subset of some 7,000 genes that are highly associated with the occurrence of pancreatic cancer including apoptotic and oncogenic genes, growth factors, angiogenic, cell cycle, metastasis-associated, and housekeeping genes. Hybridization was done in TeleChem Chambers at 62°C overnight.

After washing, fluorescence signals were detected with a confocal ScanArray 5000 scanner (Packard Bioscience, USA) and analyzed with GenePix Pro 6 (Axon Instruments, Union City, USA).

Real-time Reverse Transcription-PCR.

Total RNA was extracted and purified from cells treated with 0 or 25 µM ART for 48 h using RNeasy kit (Qiagen) according to the manufacturer's instructions. Total RNA was converted to cDNA by reverse transcriptase (Invitrogen) with random hexamer primers. The cDNAs were quantified by real-time PCR using the QuantiTect SYBR Green PCR Kit (Qiagen) and the Light Cycler 480 instrument (Roche Diagnostics), PCR was done by initial denaturation at 95°C for 15 min and 40 cycles of strand separation at 94°C for 15 s, annealing at 56°C for 20 s and extension at 72°C for 20 s. Expression levels were normalised relative to the transcription level of β-actin. All samples were run in triplicate.

Western blotting.

Cells were seeded as noted above. 24 h after incubation, the cells were treated with 0 or 25 µM ART for 48 h. Total protein was extracted using Qproteome mammalian protein preparation kit (Qiagen) according to the manufacturer's instructions.

Protein samples were resolved on SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). After blocking, membranes were incubated with primary antibodies against the following proteins: TOP2A (Millipore, Schwalbach, Germany), PCNA (Biozol, Eching, Germany), AREG, A c c e p t e d M a n u s c r i p t 8 NAG-1 and DDIT3 (Sigma-Aldrich, Munich, Germany) at 4°C overnight. After incubation with secondary antibody, bands were detected by chemiluminescence using the ECL Western Blotting Detection System (Amersham) and images were acquired with western blot documentation instruments LAS-3000 (Fuji Film, Tokyo, Japan) and the signal intensities were quantified using the Image Gauge software (ver. 4.23; Fujifilm). Data are presented as the mean ± S.E.

Topoisomerase II Assays. Inhibition of topoisomerase II activity by ART was

measured by a supercoiled DNA relaxation assay using a topoisomerase II drug screening kit (TopoGEN, FL, USA). The kit allows detection of two kinds of topoisomerase inhibitors: those that antagonize topoisomerase II action on the DNA (relaxation assay) and those that stimulate formation of cleavable complexes (stabilization assay). Briefly, 0.25 µg supercoiled DNA (pRYG) was suspended in a reaction buffer. ART, etoposide or a solvent control was added to the mixture before the reaction was started by topoisomerase II enzyme addition. After 30 minutes incubation at 37 º C, the reaction was stopped by adding 0.1 volumes of 10% SDS. The DNA-bound protein (topoisomerase IIα) was digested by proteinase K (50 µg/mL) at 37 º C for 15 min. The proteinase K was removed by chloroform/isoamyl alcohol (24:1, v/v) treatment. DNA samples were then analyzed by 1% agarose gel electrophoresis. The gel did not contain ethidium bromide and was stained by 0.5 µg/mL ethidium bromide before UV photography. The stabilization of topoisomerase IIα cleavage complex was studied by using pRYG plasmid, and 1% agarose gel containing 0.5 µg/mL ethidium bromide. The other procedures were the same as the relaxation assay. The highly stringent 'min-max separation' is calculated by talking the minimum distance between all data points of two conditions. The less stringent criterion 'standard deviation separation' is defined as the difference of the means of the two data sets diminished by one standard deviation. Only variations with a P-value of less than 5% were taken into account.

Cluster analysis was performed using correspondence analysis [START_REF] Esposito | Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?[END_REF].

Artesunate inhibits growth and proliferation of pancreatic cancer cell lines.

Recently, it was shown that ART has profound cytotoxic activities against cancer of different tumor types. To determine its effect on pancreatic cancer cells, MiaPaCa-2 and BxPC-3 cells were cultured in a monolayer and treated with ART for 24, 48, and 72 h. The MTS assay was performed to assess the rate of proliferation, and the resulting growth curves showed that ART has a concentration and time-dependent inhibitory effect (Fig. 1). In comparison of differentiation stage with the decrease in cell viability, the moderately differentiated BxPC-3 cell line (Fig. 1B) were less sensitive to ART treatment than the poorly differentiated MiaPaCa-2 cells (Fig. 1A)

Artesunate induces apoptosis in a dose-dependant manner.

To analyze the mechanism of cell death induced by ART, flow cytometry analysis following 48 hr treatment with ART (0-100 µM) was performed. ART significantly increased the percentage of cells with hypo-diploid or sub-G1 peaks, the hallmark of apoptosis [START_REF] Darzynkiewicz | Features of apoptotic cells measured by flow cytometry[END_REF], in a concentration dependant manner (Fig. 2A) and the apoptotic effect was more prominent in the poorly differentiated MiaPaCa-2 cells compared to the moderately differentiated BxPC-3 cells (Fig. 2B).

Artesunate induces caspase 3/7 activation.

It is well established that the induction of the apoptotic cascade is one of the main mechanisms of chemotherapy-induced cell death [START_REF] Igney | Death and anti-death: tumour resistance to apoptosis[END_REF]. To determine whether the chemosensitizing effect of Artesunate demonstrated above is secondary to its ability to activate the apoptotic cascade, MiaPaCa-2 cells were treated with ART. Six hours after treatment, the activity of caspase 3/7 were measured using the Caspase-Glo 3/7 assay. Fig. 2C shows that ART caused significant increase in activation of caspase-3/7 in a dose dependent manner. These results suggest that ART-induced apoptosis is, in part, due to activation of caspases 3/7. normalization and subsequent analysis were performed as described before [START_REF] Esposito | Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?[END_REF][START_REF] Fellenberg | Systematic interpretation of microarray data using experiment annotations[END_REF] by procedures that meet or exceed the MIAME-criteria of microarray analysis [START_REF] Brazma | Minimum information about a microarray experiment (MIAME)-toward standards for microarray data[END_REF]. In MiaPaCa-2 and BxPC-3 cells the expression of 1161 genes, was found to be significantly regulated (p<0.05) (for a detailed list see supplemental table 1).

For further interpretation, the results were subjected to correspondence cluster analysis [START_REF] Esposito | Microcystic tubulopapillary carcinoma of the pancreas: a new tumor entity?[END_REF][START_REF] Fellenberg | Correspondence analysis applied to microarray data[END_REF]. It was apparent that the two cell lines exhibited markedly different expression profiles and form distinct clusters (data not shown). Replicate experiments of the two cell lines always fell in the same respective cluster, demonstrating the high degree of experimental reproducibility. The analysis documented clearly, that the principle difference (distance between clusters along the vertical axis) in expression between the two cell lines exhibits correlation with the differentiation status of the respective cells.

Verification of Microarray Results.

For independent verification of the expression variations, RT-PCR and/or western blotting were performed on some genes selected during the analysis process (e.g. TOP2A, COX-2, NAG-1, PCNA, DDIT3, RRM2, AREG and FOS). All results were in accordance with the array results (Fig. 3 and4) 3.6. Functional classification of microarray regulated genes. In addition to the purely statistical analysis of the correspondence analysis, we employed the Ingenuity Pathway Analysis Knowledge database (www.ingenuity.com) to improve further the understanding of the biological consequences of ART treatment. Among the differentially expressed genes, 1161 genes were in the Ingenuity Pathway Analysis (version 6.5) database, and 1155 genes mapped to genetic networks as defined by the IPA tool. 42 and 12 networks were found to be A c c e p t e d M a n u s c r i p t 12 highly significant in that they had more of the identified genes present than would be expected by chance in MiaPaCa-2 and BxPC-3 cell lines respectively (Supplemental table 2 A, 2 B, 2 C and 2 D). These networks were associated with cancer, cell cycle, cell death, cellular growth and proliferation. The tool then associates these networks with known biological pathways (Fig. 5). Moreover the gene ontology analysis showed that 51 and 56 relevant biological functions and diseases were identified as high-level functions (data not shown), of which, the top functions were cell death, cell cycle, and cellular growth and proliferation, and the top diseases were cancer, gastrointestinal diseases, immunological and inflammatory diseases in MiaPaCa-2 and BxPC-3 cell lines, respectively. Moreover we carried out a biomarker analysis, which allows identifying and prioritizing the most relevant and promising molecular biomarker candidates from datasets from nearly any step of the drug discovery process or any type of disease research. Using the Biomarker Comparison Analysis, Moreover, we identified 48 common biomarkers between the 2 cell lines that are common to ART response (Fig. 6) and 133 unique potential specific biomarkers to MiaPaCa-2 cells and 21 unique potential biomarkers to BxPC-3 cells that discriminate between ART responses (Fig. 7).

Artesunate Targets Topoisomerase

IIα. Agents that target Top2, involving etoposide and doxorubicin, are among the most effective anticancer drugs used in the clinic. Top2A is essential for cell proliferation and is highly expressed in vigorously growing cells [START_REF] Toyoda | NK314, a topoisomerase II inhibitor that specifically targets the alpha isoform[END_REF]. Here, our microarray gene expression results (Supplemental table 1) showed that ART significantly down-regulates Top2A expression which was confirmed also by RT-PCR (Fig. 3) and by western blotting (Fig. 4 A andB). Down-regulation was higher in case of Miapaca-2 cells. In addition, to test whether ART could antagonize topoisomerase IIα action on the DNA, using etoposide as a drug positive control, pRYG and topoisomerase IIα as a non solvent control and pRYG, topoisomerase IIα plus DMSO as a solvent control, in a cell-free topoisomerase A c c e p t e d M a n u s c r i p t 13 IIα relaxation assay, we found that 50 -100 µM ART to 50-100 µM etoposide indeed inhibited the relaxation of supercoiled plasmid DNA by topoisomerase IIα (Fig. 8A), and the inhibition was dose dependent, indicating that ART is a as potent topoisomerase IIα inhibitor as etoposide.

Effect of Artesunate on the Topoisomerase II-mediated DNA cleavage-religaion

reaction. Several potent and clinically relevant anti-neoplastic agents stabilize the topoisomerase II-DNA cleavage complex by inhibiting the topoisomerase II-mediated religation reaction [START_REF] Hutt | Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene[END_REF]. When this stabilization occurs, the DNA fragments resulting from the double-strand breaks appear in the gel as linear species [START_REF] Gantchev | The ortho-quinone metabolite of the anticancer drug etoposide (VP-16) is a potent inhibitor of the topoisomerase II/DNA cleavable complex[END_REF]. To test if ART traps topoisomerase IIα in its DNA cleavage complex form, we used the pRYG plasmid as supercoiled DNA substrate and detected linear DNA formation after incubation with topoisomerase enzyme in presence of either DMSO (solvent control), ART or etoposide. and also we used linear DNA as a marker. As can be seen in (Fig. 8B), when topoisomerase II was incubated in the cleavage assay in the presence of ART (25 -100 µM), no linear pRYG DNA was produced. We also found that ART at 100 times the concentration that inhibits topoisomerase II activity in the relaxation assay was also unable to stabilize the topoisomerase II-mediated DNA cleavage-religation complex (data not shown). Linear DNA was produced clearly from pRYG DNA by topoisomerase II enzyme when the incubation was carried out in the presence of 150 µM etoposide (drug control), an agent that stabilizes the cleavage complex (Fig. 8B). These results suggest that topoisomerase IIα is a major intracellular target of Artesunate.

Artesunate potentiates growth inhibition induced by gemcitabine.

Ribonucleotide reductase 2 (RRM2) was among the significantly down-regulated genes in our microarray data. Previous studies showed that over-expression of RRM2 was associated with resistance to gemcitabine in patients with pancreatic cancer [START_REF] Goan | Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line[END_REF]. Also, it was demonstrated that A c c e p t e d M a n u s c r i p t 14 systemic delivery of siRNA-based therapy can enhance the efficacy of gemcitabine [START_REF] Duxbury | RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine[END_REF].

Accordingly, we hypothesized that Artesunate could potentiate the growth inhibitory effects of gemcitabine on pancreatic cancer cells. MiaPaCa-2 (Fig. 9A) and BxPc-3cells (Fig. 9B) were either treated with gemcitabine alone or in combination with Artesunate (10 µM), and the number of viable cells were evaluated 48 h post-treatment by MTS assay. Data presented here showed that BxPc-3 cells were more sensitive to gemcitabine compared to MiaPaCa-2 cells and that Artesunate potentiates the growth inhibitory effect of gemcitabine on both cell lines in a dose-dependent manner and the effect was sometimes additive or synergistic depending on gemcitabine doses.

A c c e p t e d

M a n u s c r i p t

Discussion

Natural products have been a continuous source of novel compounds for the treatment of numerous diseases, with natural products and their synthetic derivatives comprising over 60% of the approved anticancer drug candidates developed between 1981 and 2002 [START_REF] Newman | Natural products as sources of new drugs over the period 1981-2002[END_REF]. In order to observe an effect using compounds like Artesunate (ART) in clinical trials, the identification of potential responders would greatly increase the power of such trials.

Potential responders are those patients whose tumors express molecular characteristics that match the molecular effects of Artesunate, and this was our motivation to understand the effects of Artesunate on pancreatic cancer cells.

Artemisinin and its derivative Artesunate, distinguish themselves as a new generation of anti-malarial drugs with a few and insignificant side effects [START_REF] Hien | An open randomized comparison of intravenous and intramuscular artesunate in severe falciparum malaria[END_REF][START_REF] Hien | Qinghaosu[END_REF][START_REF] Fishwick | The toxicity of artemisinin and related compounds on neuronal and glial cells in culture[END_REF]. Recently it has been reported that they also possess antitumor activity. Although the anti-tumor effects of Artesunate have been previously investigated in vitro and in vivo, the effect of Artesunate and its possible mechanisms of action have not been studied with regard to pancreatic cancer. Gene expression profiling using cDNA microarray has been widely used in screening drug targets. In order to investigate the antitumor potential of Artesunate, we treated MiaPaCa-2 (poorly differentiated) and BxPC-3 (moderately differentiated) pancreatic cancer cells with Artesunate and the expression of cancer related-genes were monitored using microarray technology. Differentially expressed genes were then organized into functionally annotated networks.

Pharmacokinetic studies indicate that the concentration of Artesunate applied clinically for the treatment of malaria (e.g. 2 mg/kg intravenously) results in peak plasma drug concentration of 2640 ± 1800 µg/l (6.88 ± 4.69 mM) [START_REF] Efferth | The anti-malarial artesunate is also active against cancer[END_REF][START_REF] Batty | Selective highperformance liquid chromatographic determination of artesunate and alpha-and beta-dihydroartemisinin in patients with falciparum malaria[END_REF].The doses used in our investigation were approximately two orders of magnitude lower than those used clinically. At the cellular level, our data shows that Artesunate inhibited growth and induced apoptosis of human pancreatic cancer cells and the effect was more prominent with the poorly differentiated MiaPaCa-2 cells.

At the molecular level, a network analysis was done on the basis of expression profiling data, in order to discover relevant connections and pathways among the regulated genes.

Our gene expression analysis identified many significantly up-and down-regulated genes, some of them are already established to be involved directly in the apoptotic pathway and others recently identified either as a potential pre-apoptotic and apoptotic genes, cancer prognostic agents, oncogenes, cancer drug targets or genes responsible for drug resistance mechanisms but their actual rules remains to be elucidated. DNA topoisomerase II is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes through a transient DNA double-strand break (DSB) and subsequent religation of the DSB. The enzyme has been implicated in many aspects of DNA metabolism, including DNA replication, repair, transcription, and chromosome condensation /segregation. Topoisomerase II-targeting agents, including etoposide, doxorubicin, and mitoxantrone, are among the most effective and widely used anticancer drugs in cancer chemotherapy [START_REF] Toyoda | NK314, a topoisomerase II inhibitor that specifically targets the alpha isoform[END_REF][START_REF] Berger | Structure and mechanism of DNA topoisomerase II[END_REF]. Topoisomerase-targeting anticancer drugs can be divided into two broad classes that vary widely in their mechanisms of action. Class I drugs include acridines, anthracyclines, actinomycins and quinolones act by stabilizing covalent topoisomerase-DNA complexes that are the intermediates during the catalytic cycle of the enzyme. They are also referred to as `topoisomerase poisons' because they transform the enzyme into a potent cellular toxin. Class II drugs; by contrast, interfere with the catalytic function of the enzyme without trapping the covalent complex. The drugs in this class are referred to as `topoisomerase inhibitors'. The main topoisomerase inhibitors are coumarin antibiotics and fostriecin analogues [START_REF] Topcu | DNA topoisomerases as targets for anticancer drugs[END_REF].

The results presented here clearly demonstrate for the first time that Artesunate downregulates expression TOP2A at both mRNA and protein levels, Moreover, we showed that 100 µM Artesunate completely inhibited the activity of topoisomerase IIα. However, ART was unable to trap topoisomerase IIα in its cleavage complexes, demonstrating that Artesunate is a novel topoisomerase inhibitor but not a topoisomerase poison. The concentrations required for Artesunate (100 µM) and etoposide (100 µM) to maximally inhibit topoisomerase IIα activity in our cell-free topoisomerase II relaxation assay could be higher than those needed actually in vivo. One reason may be that the DNA damage threshold to evoke checkpoint response is far less than which can be detected by relaxation of supercoiled DNA by topoisomerase IIα in this cell-free assay. In addition, the cell-free assay generates nearly total conversion of the plasmid substrate, whereas fewer lesions may be detected by the checkpoint sensors.

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. Killing of tumor cells by most anticancer strategies currently used in clinical oncology, such as chemotherapy or immunotherapy, has been linked to activation of apoptosis signal transduction pathways in cancer cells such as the intrinsic and/or extrinsic pathway [START_REF] Fulda | Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy[END_REF]. Our results demonstrate that apoptosis induction was among the mechanisms triggering Artesunate inhibitory effects on pancreatic cancer cell lines, Where Artesunate induces sub-G1 phase (Fig. 2A), Caspase 3/7 activation (Fig. 2C) and upregulates a variety of important and well-known apoptotic and proapoptotic molecules including APAF1, BAX, BAK and caspases 2, 3, 4, 5, 6, 8, 9 and 10 [41] as shown in the gene expression results (Supplemental table 1).

A c c e p t e d M a n u s c r i p t 18 GADD153, also known as DDIT3 (DNA-damage-inducible transcript 3) has been shown to be involved in growth arrest and apoptosis following DNA damage and a variety of stress conditions, such as nutrient deprivation and treatment with anticancer agents [START_REF] Kim | GADD153-mediated anticancer effects of N-(4-hydroxyphenyl)retinamide on human hepatoma cells[END_REF].

Here, we demonstrate that DDIT3 is up-regulated in MiaPaCa-2 and BxPC-3 cell lines upon treatment with Artesunate which could explain, in part, the cytotoxic activities of Artesunate.

Proliferating cell nuclear antigen (PCNA) plays important roles in nucleic acid metabolism. The protein is essential for DNA replication and has been shown to be involved in RNA transcription [START_REF] Kelman | PCNA: structure, functions and interactions[END_REF]. Recent work has shown that PCNA expression is significantly higher in pancreatic cancer [START_REF] Hu | Expression of sonic hedgehog, EGFR and PCNA proteins in pancreatic cancer and their correlations to cell proliferation[END_REF][START_REF] Makinen | Tumour suppressor protein (p53), apoptosis inhibiting protein (Bcl-2) and proliferating cell nuclear antigen (PCNA) expressions in a rat pancreatic tumour model[END_REF]. Down-regulation of PCNA by Artesunate at mRNA and protein level in pancreatic cancer cell lines implies that Artesunate inhibits their growth, in part, by decreasing the PCNA level.

Non steroidal anti-inflammatory drug-activated gene (NAG-1) was another key gene among the up-regulated genes. NAG-1 is a distant member of the transforming growth factor super family. NAG-1 has been identified as an anti-tumorigenic and pro-apoptotic protein and its expression is able to be induced by NSAIDs and several other anti-cancer compounds [START_REF] Baek | Expression of NAG-1, a transforming growth factor-beta superfamily member, by troglitazone requires the early growth response gene EGR-1[END_REF][START_REF] Lee | Indole-3-carbinol and 3,3'diindolylmethane induce expression of NAG-1 in a p53-independent manner[END_REF]. Furthermore, over-expression of NAG-1 is reported to induce caspase-dependent apoptosis in prostate cancer cell line DU-145 [START_REF] Song | Cyclooxygenase-2, player or spectator in cyclooxygenase-2 inhibitor-induced apoptosis in prostate cancer cells[END_REF] which is consistent with our results.

Ribonucleotide reductase (RRM) mediates the rate-limiting step in DNA-synthesis because it is the only known enzyme that converts ribonucleotides to deoxynucleotides. The enzymatic activity of ribonucleotide reductase is modulated by the levels of RRM type 2 (RRM2) [START_REF] Nutter | Nature and properties of mammalian ribonucleoside diphosphate reductase[END_REF]. Previous studies showed that over-expression of RRM2 was associated with resistance to gemcitabine in patients with pancreatic cancer [START_REF] Goan | Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line[END_REF], and demonstrated that systemic delivery of siRNA-based therapy can enhance the efficacy of gemcitabine [START_REF] Duxbury | RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine[END_REF].

Here, we demonstrate that RRM2 is significantly down-regulated in both pancreatic cancer cells by Artesunate treatment, which is to our knowledge the first study to identify RRM2 as

(

  TOP2A) inhibitor, and indicating that Proliferating cell nuclear antigen (PCNA), DNAdamage-inducible transcript 3 (DDIT3), Non steroidal anti-inflammatory drug-activated gene (NAG-1) and Ribonucleotide reductase 2 (RRM2) are among the novel markers modulating ART effect on pancreatic cancer cells.

1 .

 1 Cell lines and treatment. The human pancreatic cancer cell lines, MiaPaCa-2 (poorly differentiated) and BxPC-3 (moderately differentiated), were obtained from the American Type Culture Collection (Rockville, USA). BxPC-3 cells maintained in RPMI 1640

  with increasing concentrations of gemcitabine in absence or presence of 10 µM ART, respectively.

2. 9 .

 9 Identification of signalling pathways. The Ingenuity Pathway Analysis software (IPA) (Ingenuity Systems, Mountain View, USA) was utilized to identify networks of interacting genes and other functional groups. A cut-off ratio of 1.5 was used to define genes. Using the A c c e p t e d M a n u s c r i p t 9 IPA Functional Analysis tool, we were able to associate biological functions and diseases to the experimental results. Moreover we used a biomarker filter tool and the Network Explorer for visualizing molecular relationships. 2.10. Data analysis. Microarray data quality assessment, normalisation and correspondence cluster analysis were performed with the MIAME-compatible analysis and data warehouse software package M-CHiPS [27, 28] (www.mchips.org). Signal intensities of repeated hybridizations were normalised and significance levels assessed by two stringency criteria.

A c c e p t e d M a n u s c r i p t 11 3. 4 .

 114 Microarray Analysis identifies novel Artesunate targets significantly up-and downregulated in their expressions. For the identification of possible targets and mechanisms of action of ART; MiaPaCa-2 and BxPC-3 pancreatic cancer cell lines were treated with 0 or 25 µM ART for 48 h. RNA preparation, hybridization, data quality assessment, filtering,

  selected concentrations that we used for gene expression profiling and for other experiments were much lower than the clinically relevant molar concentrations.

A c c e p t e d M a n u s c r i p t 19 a

 19 marker for the cytotoxic effect of Artesunate on pancreatic cancer. Moreover, we proved that Artesunate at lower molecular concentration significantly potentiates the growth inhibitory effects of gemcitabine on both pancreatic cancer cell lines.In combination, our data demonstrate that Artesunate inhibits cell growth and promotes apoptosis in pancreatic cell lines through the modulation of multiple signaling pathways. The mechanisms driving Artesunate-induced growth arrest of human pancreatic cancer cells were dependent, in part, on the differentiation stage of the cells. At the molecular level, many genes related to apoptosis, cell cycle, angiogenesis, metastasis and differentiation are significantly regulated upon Artesunate treatment. Moreover, we showed for the first time that the common mechanisms modulating Artesunate effect may be mediated through down-regulation of TOP2A, RRM2 and PCNA and up-regulation of NAG-1 in both cell lines. In addition we introduced Artesunate for the first time to be a novel topoisomerase IIα inhibitor. The present analysis is a starting point for the generation of hypotheses on candidate genes and for a more detailed dissection of the functional role of individual genes for the activity of Artesunate in tumor cells. Moreover, our results provide new insights into Artesunate-related signalling activities, which may facilitate the development of Artesunatebased anticancer strategies and/or combination therapies. Further preclinical and clinical investigations are required to elucidate the full potential of Artesunate as a powerful cytotoxic agent for treatment of pancreatic cancer.

Figure and Table legends

  Figure and Table legends

Fig. 1 .

 1 Fig.1. Growth inhibition of human pancreatic cancer cells by ART. Cultures of exponentially

Fig. 2 .

 2 Fig.2. ART induces apoptosis in pancreatic cancer cells. MiaPaCa-2 and BxPc-3 cells were

Fig. 3 .

 3 Fig.3. Results of real-time reverse transcriptase PCR analysis. BxPc-3 cells were treated with

Fig. 4 .

 4 Fig.4.Western blot analysis. (A) In order to confirm changes in Top2A, AREG, NAG-1,

Fig. 5 .

 5 Fig.5. Functional assignment of genes significantly transcribed in MiaPaCa-2 (A) and BxPc-

Fig. 6 .

 6 Fig.6. Biomarker analysis displaying common genes that were significantly regulated after

Fig. 7 .

 7 Fig.7. Specific biomarker genes significantly regulated after 48 h treatment with Artesunate

Fig. 8 .

 8 Fig.8. Action of ART on topoisomerase IIα: (A) Supercoiled plasmid DNA (pRYG) was

Fig. 9 .

 9 Fig.9. Potentiating effect of Artesunate on the growth inhibition of gemcitabine. The viability

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