R Dornetshuber

M R Kamyar

P Rawnduzi

I Baburin

K Kouri

T Hornbogen

R Zocher

W Berger

Rosa Lemmens-Gruber

Effects of the anthelmintic drug PF1022A on mammalian tissue and cells

Keywords: PF1022A, anthelmintics, ionophore, cytotoxicity, apoptosis

Nematode infections cause human morbidity and enormous economic loss in livestock. Since resistance against currently available anthelmintics is a worldwide problem, there is a continuous need for new compounds. The cyclooctadepsipeptide PF1022A is a novel anthelmintic that binds to the latrophilin-like transmembrane receptor important for pharyngeal pumping in nematodes. Furthermore, PF1022A binds to GABA receptors, which might contribute to the anthelmintic effect. Like other cyclodepsipeptides, PF1022A acts as an ionophore. However, no correlation between ionophoric activity and anthelmintic properties was found. This is the first study describing the effect of PF1022A on mammalian cells and tissues. While channel-forming activity was observed already at very low concentrations, changes in intracellular ion concentrations and reduction of contractility in isolated guinea-pig ileum occurred at multiples of anthelmintically active concentrations.

PF1022A did not induce necrotic cell death indicated by complete lack of cellular lactate dehydrogenase release. In contrast, apoptosis induction via the mitochondrial pathway was suggested for long-term drug treatment at high concentrations due to numerous apoptotic morphological changes as well as mitochondrial membrane depolarisation. Short time effects were based on cell cycle blockade in G 0 /G 1 phase. Additionally, the cell cycle and apoptosis regulating proteins p53, p21 and bax, but not Bcl-2 were shown to impact on PF1022Ainduced cytotoxicity. However, since PF1022A-induced cytotoxicity was found at drug concentrations higher than those used in anthelmintic treatment, it can be suggested that PF1022A intake might not impair human or animal health. Thus, PF1022A seems to be a safe alternative to other anthelmintic drugs.

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

Introduction

Nematode infections are a major cause of human morbidity and mortality in tropic and temperate climates [START_REF] Bundy | Can we deworm this wormy world?[END_REF]. Moreover, these pathogenes cause enormous economic loss in cattle, horses, pigs and sheep [START_REF] Waller | Anthelmintic resistance[END_REF][START_REF] Von Samson-Himmelstjerna | Efficacy or two cyclooctadepsipeptides, PF1022A and emodepsidde, against anthelmintic-resistant nematodes in sheep and cattle[END_REF]. However, resistance against the major currently available anthelmintics has increasingly become a worldwide problem [START_REF] Geary | Frontiers in anthelmintic pharmacology[END_REF]. Therefore, new compounds with different modes of action are needed to manage the problem of resistance. The 24membered N-methylated cyclooctadepsipeptide PF1022A belongs to a novel class of anthelmintic agents with potent broadspectrum anthelmintic activity and low toxicity in animals [START_REF] Sasaki | A new anthelmintic cyclodepsipeptide, PF1022A[END_REF][START_REF] Conder | Anthelmintic profile of the cyclodepsipeptide PF1022A in in vitro and in vivo models[END_REF][START_REF] Kachi | Effect of PF1022A on adult Angiostrongylus cantonensisin the pulmonary arteries and larvae migrating into the central nervous system in rats[END_REF][START_REF] Von Samson-Himmelstjerna | In vivo activities of the new anthelmintic depsipeptide PF1022A[END_REF][START_REF] Zahner | Filaricidal efficacy of anthelmintically active cyclodepsipeptides[END_REF]. Additionally this drug was found to be a good candidate for the treatment of human angiostrongyliasis [START_REF] Mentz | Drug trials for treatment of human angiostrongliasis[END_REF].

PF1022A is a secondary metabolite of the fungus imperfectus Mycelia sterilia (Rosellinia sp.) isolated from the plant Camellia japonica [START_REF] Sasaki | A new anthelmintic cyclodepsipeptide, PF1022A[END_REF]. So far, the nonribosomal N-methylcyclooctadepsipeptide synthetase responsible for PF1022A biosynthesis has been isolated and characterized [START_REF] Weckwerth | Biosynthesis of PF1022A and related cyclooctadepsipeptides[END_REF]. Consequently, this discovery allows the development of new N-methylcyclooctadepsipeptide-derived compounds [START_REF] Krause | Untersuchungen zur Biosynthese des Cyclooctadepsipeptids PF1022A[END_REF]. PF1022A serves as starting material for the synthesis of semi-synthetic cyclooctadepsipeptide derivatives with improved anthelmintic potency and broadspectrum activity [START_REF] Yanai | Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes[END_REF][START_REF] Jeschke | Influence of the cyclooctadepsipeptide PF1022A and PF1022E as natural products on the design of the semi-synthetic anthelmintics such as emodepside[END_REF][START_REF] Jeschke | Synthesis and anthelmintic activity of thioamide analogues of cyclic octadepsipeptides such as PF1022A[END_REF][START_REF] Jeschke | Synthesis of anthelmintically active N-methylated amidoxime analogues of the cyclic octadepsipeptide PF1022A[END_REF]. Meanwhile, also total synthesis of cyclooctadepsipeptides has been accomplished [START_REF] Dyker | Chimeric cyclodepsipeptides as mimetics for the anthelmintic PF1022A[END_REF][START_REF] Dutton | Synthesis of PF1022A, an anthelmintic cyclodepsipeptide[END_REF][START_REF] Dutton | Restricted conformation analogues of an anthelmintic cyclodepsipeptide[END_REF][START_REF] Lee | Generation of a small library of cyclodepsipeptide PF1022A analogues using a cyclization-cleavage method with oxime resin[END_REF][START_REF] Scherkenbeck | PF1022A and related cyclodepsipeptides -a novel class of anthelmintics[END_REF][START_REF] Scherkenbeck | PF1022A -a novel anthelmintic cyclooctadepsipeptide. Modification and exchange of the N-methyl leucine residues[END_REF].

With regard to the molecular mechanisms underlying its anthelmintic activity, PF1022A has been shown to bind to the amine terminus of the 110-kDa heptahelical, latrophilin-like transmembrane receptor HC-110R, isolated from Haemonchus contortus (Rudolphi). This receptor has an important regulatory function on pharyngeal pumping in nematodes. By binding to this receptor, PF1022A induces an influx of external Ca 2+ into the cell [START_REF] Saeger | Latrophilin-like receptor from the parasitic nematode Haemonchus contortus as target for the anthelmintic depsipeptide PF1022A[END_REF], consequently affecting several presynaptic signal transduction pathways [START_REF] Harder | Cyclooctadepsipeptides -an anthelmintically active class of compounds exhibiting a novel mode of action[END_REF]. Due to this effect, treatment with PF1022A as well as the semi-synthetic emodepside causes worm A c c e p t e d M a n u s c r i p t 4 paralysis [START_REF] Harder | Cyclooctadepsipeptides -an anthelmintically active class of compounds exhibiting a novel mode of action[END_REF][START_REF] Harder | Mechanisms of action of emodepside[END_REF]. These modes of action are different from the mechanisms of known anthelmintics like benzimidazoles, imidazothiazoles, tetrahydropyrimidines and macrocyclic lactones [START_REF] Harder | Cyclooctadepsipeptides -a new class of anthelmintically active compounds[END_REF]. Thus, these new drugs might solve the increasing problem of resistance development against available anthelmintics. Indeed, PF1022A and emodepside were effective in benzimidazole-, levamisol-and ivermectin-resistant populations of Haemonchus contortus in sheep when applied orally, subcutaneously or intravenously. Additionally, they showed activity in ivermectin-resistant Cooperia oncomorpha population in cattle [START_REF] Von Samson-Himmelstjerna | Efficacy or two cyclooctadepsipeptides, PF1022A and emodepsidde, against anthelmintic-resistant nematodes in sheep and cattle[END_REF].

Furthermore, in binding studies it could be demonstrated that PF1022A binds to GABA receptors in nematodes, which might contribute to the anthelmintic effect [START_REF] Chen | Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A, a new anthelmintic, on the receptor[END_REF]. PF1022A acts as an ionophore in planar lipid bilayers like other cyclodepsipeptides, however, testing structurally related derivates revealed no correlation between ionophoric activity and anthelmintic properties [START_REF] Gessner | Ionophore and anthelmintic activity of PF1022A, a cyclooctadepsipeptide, are not related[END_REF].

However, at least to our knowledge, no data describing the effect of PF1022A on mammalian cells and tissues are published so far. PF1022A is structurally related to cyclohexadepsipeptides of the enniatin-type for which synergistic effects on antifungal drug activity were described [START_REF] Zhang | High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections[END_REF][START_REF] Fukuda | New beauvericins, potentiators of antifungal miconazole activity, produced by Beauveria sp. FKI-1366. Taxonomy, fermentation, isolation and biological properties[END_REF]. Moreover, potent cytostatic and cytotoxic effects against diverse cancer cell types [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF][START_REF] Lin | Involvement of Bcl-family, cytochrome c and caspase 3 in induction of apoptosis by beauvericin in human non-small cell lung cancer cells[END_REF][START_REF] Jow | Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium[END_REF], antiangiogenic activity [START_REF] Zhan | Search for cell motility and angiogenesis inhibitors with potential anticancer activity: beauvericin and other constituents of two endophytic strains of Fusarium oxysporum[END_REF] as well as anthelmintic properties [START_REF] Jeschke | Synthesis and anthelmintic activity of substituted (R)-phenyllactic acid containing cyclohexadepsipeptides[END_REF] could be demonstrated for these structurally related cyclohexadepsipeptides.

Aim of this study was to investigate the effect of PF1022A on force of contraction in isolated guinea-pig ileum, its electrophysiological properties and the resulting impacts on cell homeostasis. Moreover, PF1022A-induced cytotoxic modes of action were elucidated in mammalian cells.

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

Materials and Methods

Test compound

PF1022A was kindly supplied by Bayer AG, Germany. The compound is poorly soluble in water, therefore a stock solution with Tween 80 : MeOH = 1:2 (Sigma-Aldrich, Vienna, Austria) was prepared. This solution was further diluted to the nutrient solution at the final concentration.

Cell lines

The following human cell lines were used in this study: the epidermal carcinoma-derived cell line KB-3-1 (generously donated by Dr. Shen, Bethesda, USA) [START_REF] Shen | Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins[END_REF], and the colon carcinoma cell line CaCo-2 (American Tissue Culture Collection), the colon carcinoma cell model HCT116 and respective sublines with deleted p53, p21, or bax genes were generously donated by Dr. Vogelstein, John Hopkins University, Baltimore [START_REF] Bunz | Targeted inactivation of p53 in human cells does not result in aneuploidy[END_REF][START_REF] Waldman | p21 is necessary for the p53-mediated G1 arrest in human cancer cells[END_REF][START_REF] Zhang | Role of BAX in the apoptotic response to anticancer agents[END_REF], and the Bcl-2-negative non-small cell lung cancer cell line A549 and its bcl-2 pBabe/Puro-transfected subline [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF].

HCT116 cell lines were grown in McCoy´s culture medium, Caco-2 cells in MEME, and KB-3-1 as well as A549 cells and the transfected subline in RPMI 1640 medium. All culture media were purchased from Sigma-Aldrich GmbH (St. Louis, MO) and supplemented with 10% fetal calf serum (PAA, Linz, Austria). Cultures were periodically checked for Mycoplasma contamination.

Contractility measurement

The effect of PF1022A on force of contraction (f c ) of isolated terminal ilea of the guinea pig was studied as previously described [START_REF] Kamyar | Investigation of the electrophysiological properties of enniatin[END_REF]. All chemicals were purchased by Sigma-Aldrich A c c e p t e d M a n u s c r i p t 6 (Vienna, Austria). The test compound was added cumulatively to the bathing solution in rising concentrations every 30 min when a steady-state effect has been reached.

Ionophoric activity -single channel current

Electrical activity was studied on CaCo-2 cells at room temperature (19-22°C) as previously described [START_REF] Kamyar | Investigation of the electrophysiological properties of enniatin[END_REF][START_REF] Kouri | Beauvericin induced channels in ventricular myocytes and artificial membranes[END_REF]. Chemicals for pipette and bathing solutions were supplied by Sigma-Aldrich (Vienna, Austria). After a control period of 15 min during which no electrical activity could be observed, PF1022A was added to the bathing solution. The wash-in phase lasted about 1 to 2 min until single channel current could be detected. The same experimental protocol (E h = -60, -40, -20, 0, +20, +40, +60 mV) was applied as during control recordings.

Electrophysiological measurements were carried out with an Axopatch-1D patch clamp amplifier (Axon Instruments, CA) at a cut-off frequency (-3 dB) of 2 kHz. Currents were filtered at 5 kHz with a dual variable filter (VBF 8, Kemo Ltd, Beckenham, Kent, U.K.), digitized via an AD converter (TL-1 interface, Axon Instruments, CA) and sampled at 5-10 kHz. Data acquisition and storage were processed directly to a PC equipped with pCLAMP 6 software (Axon Instruments, CA). Single channel analysis was performed with the ASCD software (G. Droogmans, Leuven, Belgium).

Two-microelectrode voltage clamp studies -GABA A receptor

Stage V-VI oocytes from Xenopus laevis were prepared and cRNA injected as previously described [START_REF] Khom | Pharmacological properties of GABA A receptors containing gamma1 subunits[END_REF][START_REF] Methfessel | Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels[END_REF]. On the 2 nd day after cRNA injection, electrophysiological experiments were performed by the two-microelectrode voltage clamp method using of a TURBO TEC 01C amplifier (npi electronic GmbH, Tamm, Germany) at a holding potential of -70 mV. The bath solution contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 and 5 mM HEPES (pH 7.4). PF1022A was co-applied with GABA EC 3-10 (effective concentration of A c c e p t e d M a n u s c r i p t 7 GABA that induces 3-10 % of maximal GABA-evoked current) by means of an automated fast perfusion system according to [START_REF] Baburin | Automated fast perfusion of Xenopus oocytes for drug screening[END_REF]. Percent potentiation of I GABA by the PF1022A was calculated using formula: (R/C)*100%, where R is the amplitude of the chloride current evoked by co-application of control GABA EC 3-10 and the indicated concentration of the compound, and C is the amplitude of the chloride current evoked by application of control GABA EC 3-10 alone.

Fluorescence imaging

Caco-2 cells were loaded with 1 μM FURA 2AM and an equivalent concentration of Pluronic 20% in DMSO at room temperature (18-23C) for 30-45 min. Fluorescent dye and Pluronic were procured from Molecular Probes (Leiden, The Netherlands). After washout cells were allowed to hydrolyze the dye for 30 minutes to one hour, and experiments were performed as previously described [START_REF] Kamyar | Effects of moniliformin in presence of cyclohexadepsipeptides on isolated mammalian tissue and cells[END_REF]. Ratiometric measurements were realized following background subtraction with the Axon Imaging Workbench 2.2 software (Axon Instruments, CA) averaging 2 frames. Results are presented as the relative change (-ΔF/F o ) of the F 340 /F 380 signal.

Cell viability assays

KB-3-1 cells were seeded (2×10 4 cells/ml) in 96-well plates and were allowed to recover for 24 h. Concentrations of PF1022A ranging from 0.5 µM up to 10 µM were applied with an incubation time of 24, 48 and 72 h. The cytotoxic effect of PF1022A was determined with an MTT-based vitality assay (EZ4U, Biomedica, Vienna, Austria) as described previously [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF].

Moreover, to study the reversibility of the cytotoxic PF1022A effect, after an incubation time of 1, 2, 4, 6, 8, 24 and 48 h the test compound was removed from the wells and substituted by A c c e p t e d M a n u s c r i p t 8 fresh medium. After a total incubation period of 72 h the EZ4U assay was performed and data were evaluated by means using the GraphPadPrism 4 software.

Lactate dehydrogenase release assay

The release of lactate dehydrogenase (LDH) from the cytosol, which indicates loss of membrane integrity and is further associated with necrotic cell death, was studied with a cytotoxicity detection kit (Roche, Basel, Switzerland). Adherent KB-3-1 cells (2×10 4 cells/ml) were incubated in 96-well plates for 24 h with PF1022A (0.5 µM -10 µM). Afterwards, from each well 100 µl of the supernatant was transferred to a 96-well microplate and incubated for 20 min at 37°C with reagent solution of the kit according to the manuel. Finally, absorbance was measured with a microplate reader, set at 490 nm and 620 nm as reference.

DAPI Staining

1x10 5 KB-3-1 cells/well were treated in 6 well plates with 2.5 -10 µM PF1022A for 24 h and 48 h, respectively. Subsequently, cells were harvested, cytospins were prepared, and apoptosis was evaluated by staining with 4´,6-diamidino-2-phenylindole (DAPI) containing antifade solution (Vector Laboratories, Inc., Burlingame, CA). The nuclear morphology of cells was examined with a Leica DMRXA fluorescence microscope (Leica Mikroskopie and System, Wetzlar,Germany) equipped with appropriate epifluorescence filters and a COHU chargecoupled device camera. The rate of apoptosis was determined as the percentage of apoptotic nuclei of at least 500 nuclei/experimental group.

Cell cycle analysis

Adherent KB-3-1 (5x10 5 cells/well) were incubated in 6-well plates at 37°C for 24 or 48 h with 0.5, 1, 2.5, 5 and 10 µM PF1022A. Cell cycle progression was analysed by flow A c c e p t e d M a n u s c r i p t 9 cytometry using FACS Calibur (Becton Dickinson, Palo Alto CA) as described [START_REF] Heffeter | Multidrug-resistant cancder cells are preferential targets of the new antineoplastic lanthanum compound KP772 (FFC24)[END_REF]. Cell Quest Pro software (Becton Dickinson and Company, New York, USA) was used to analyse the resulting DNA histograms.

Detection of mitochondrial membrane potential

KB-3-1 cells (5x10 6 cells/well) were exposed to 1, 2.5, 5 and 10 µM PF1022A for 24 h before cell staining was performed with the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3',3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Mitochondrial Membrane Potential detection Kit; Stratagene, La Jolla, CA, USA) as described [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF][START_REF] Heffeter | Multidrug-resistant cancder cells are preferential targets of the new antineoplastic lanthanum compound KP772 (FFC24)[END_REF].

Statistical analysis

Values are expressed as means  S.E. where appropriate. Significance was calculated with Student's t-test for paired (f c ) and unpaired observations. Statistical significance was determined at levels of P<0.05, P<0.01 and P<0.001.

Results

Effect on force of contraction

Isometrically measured f c of isolated, pre-contracted (60 mM KCl) terminal ilea of the guinea pig was concentration-dependently (0.1 to 30 µM PF1022A) decreased with an IC 50 of 25 ± 3 µM (n=3). At solvent concentrations up to 3.5 µl methanol/Tween in 28 ml nutrient solution, which corresponds to the solvent concentration at 10 µM PF1022A, no effect on contractility of isolated smooth muscle preparations was observed. At 10 µl methanol/Tween in 28 ml nutrient solution, that is the solvent concentration at 30 µM PF1022A, f c was decreased by A c c e p t e d M a n u s c r i p t 10 4.4% (n=3). Therefore, results obtained with 30 µM PF1022A were corrected for this solvent induced decrease in contractility. Reduction of f c was only significant at 30 µM PF1022A (n=3, P<0.05). At the anthelmintically active concentration of 1 µM PF1022A [START_REF] Conder | Anthelmintic profile of the cyclodepsipeptide PF1022A in in vitro and in vivo models[END_REF], the decrease of f c from the control value of 17.8  2.6 mN to 15.9  2.2 mN was not significant (n=3, P>0.05).

Pore-forming activity

After a control period of 15 min during which no electrical activity could be observed at holding potentials between -60 and +60 mV, PF1022A was added to the bathing solution.

One to two minutes after addition of 1 µM PF1022A pore forming activity was detected in inside-out patches of Caco-2 cells with mono-and divalent cations (Na + , K + , Ca 2+ ) as the charge carriers. Higher PF1022A concentrations achieved similar effects, but faster onset of action and multiple channel openings (Fig. 1A). Washout of the substance was not effective indicating permanent incorporation into the cell membrane.

The experimental reversal potentials for potassium (n=3) and sodium (n=3) were comparable to the theoretical values for the ionic equilibrium potential calculated according to the Nernst equation. Further, current amplitudes did not vary when aspartate was substituted by chloride in the K + and Na + bathing solutions in equal cation molarity (n=3), also indicating the channel's cationic selectivity.

Current to voltage relationships for potassium, sodium and calcium currents presented a symmetrical behaviour upon hyper-and depolarization, rectifying at potentials higher than -20 mV and +20 mV. Single channel conductance was therefore estimated in the linear range of -20 mV to +20 mV and was calculated for K + at 39.3  2.8 pS (n=5), for Na + at 52. conducting channels these periods without electrical activity were almost twice the duration of those with the studied monovalent cations.

Effect on intracellular ion concentration

For studying the effect of PF1022A on intracellular calcium, sodium and potassium concentrations, the ionophores ionomycin, gramicidine and valinomycin (n=3 for each) were used as reference compounds. Although PF1022A incorporated into the cell membrane leading to conduction of calcium, sodium and potassium ions, for Na + ions no effect on the intracellular concentration was observed, while the intracellular K + and Ca 2+ concentrations were changed. The effect of PF1022A on intracellular Ca 2+ concentration, however, was weak compared to the control with ionomycin. Representative recordings are presented for Ca 2+ in Figure 1B andC. Only a small increase in the intracellular Ca 2+ concentration was caused by 100 µM PF1022A (n=7) compared to 100 µM ionomycin, while at 10 µM PF1022A (a concentration causing multiple channel openings, see Fig. 1A,n=6) no changes in [Ca 2+ ] i were observed (data not shown). The effect on intracellular K + concentration is illustrated for 100 µM PF1022A (E) and 100 µM valinomycin (D) for two representative cells each. The effect on intracellular K + was more pronounced than on intracellular Ca 2+ , but still weak at

Effect on chloride currents through GABA A receptors by PF1022A

As an anthelmintic effect via binding to the GABA receptor in nematodes was suggested [START_REF] Chen | Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A, a new anthelmintic, on the receptor[END_REF],

we also studied the electrophysiological effect of PF1022A on GABA A channels. An effect on this channel is of interest e.g. in respect to sedative side effects of the drug. At 1 and 10 µM PF1022A, the potentiation of GABA-activated chloride current (I GABA ) through 122S receptors was 20.8±11.2% (n=4) and 78.2±16.1% (n=6), respectively. However, an effect of PF1022A on GABA A can be excluded, because the solvent-induced increase in I GABA was 70.8±39.4% at a solvent concentration which corresponds to the concentration present at 10 µM PF1022A.

Effects of PF1022A on cell proliferation and viability

To investigate the impact of the anthelmintic drug PF1022A on human cell proliferation, dose-response curves (0.5-10 µM) were established in human KB-3-1 cells using different exposure times. Based on MTT assays an increased cell proliferation was observed in KB-3-1 cells after 24 and 48 h exposure to PF1022A concentrations ≤ 1 µM (Fig. 2). The maximal increase in cell growth (1.25-fold) was observed after 24 h incubation with 0.5 µM PF1022A.

At higher PF1022A concentrations the number of viable cells decreased concentration

dependently with an IC 50 of 8.1 µM (24 h), 6.6 µM (48 h) and 5.9 µM (72 h), respectively. Moreover, recovery of cells exposed for 1, 2, 4, 6, 8, 24 or 48 h to increasing PF1022A concentrations was studied after 72 h. These experiments revealed enhanced cell proliferation at concentrations ≤ 2.5 µM and short exposure times (1 to 8 h). A maximum growth stimulation (1.3-fold growth) was obvious at 2 h treatment with 2.5 µM PF1022A. However, A c c e p t e d M a n u s c r i p t 13 in experiments with expanded drug exposure time (24 and 48 h), no stimulating effect was observed (data not shown). This is in contrast to experiments without wash-out phase (Fig. 2).

The cytotoxic effect at higher drug concentrations (>5 µM) and long time exposure (24 and 48 h) did not differ significantly from data obtained without wash-out. This indicates potent cytotoxic effects of PF1022A at high concentrations (data not shown).

Apoptosis induction by PF1022A

To gain more insights into the PF1022A-induced cell death, LDH release from mitochondria to cytosol, which is indicative for necrotic cell death, was measured. Since no cytosolic LDH was detectable after 24 h PF1022A treatment at concentrations ranging from 0.5-10 µM (data not shown), we hypothesized that the decrease in the number of viable cells was due to apoptotic cell death. This was further confirmed by results obtained by Dapi stainings of control and PF1022A-treated KB-3-1 cells. By direct counting under the microscope a concentration-and time-dependent increase of apoptotic nuclei was observed indicated by characteristic apoptotic features like cell shrinkage, chromatin condensation and apoptotic body formation. In more detail, after 24 h exposure to 2.5, 5 and 10 µM PF1022A a relative minor increase in the number of apoptotic nuclei from 2% in the control to 4%, 12% and 14% was observed (Fig. 3A). In contrast, after 48 h treatment with the above mentioned PF1022A concentrations the proportion of apoptotic nuclei remarkably increased from 3% in the control to 6%, 15% and 36%, respectively (Fig. 3B).

Since programmed cell death can be initiated via the so-called mitochondrial pathway which is based on depolarisation of the outer mitochondrial membrane, FACS analyses with JC-1stained KB-3-1 cells were performed. JC-1 is a lipophilic cation fluorescent dye, which can enter into intact mitochondria selectively [START_REF] Heffeter | Multidrug-resistant cancder cells are preferential targets of the new antineoplastic lanthanum compound KP772 (FFC24)[END_REF]. While after 24 h exposure to 10 µM PF1022A a moderate mitochondrial membrane depolarisation was shown (12.4% of cells vs. 4.3% in A c c e p t e d M a n u s c r i p t 14 control) this effect was distinctly enhanced after 48 h treatment (32% of cells vs. control of 3%) (Fig. 4C). In contrast, PF1022A did not affect the mitochondrial membrane potential at concentrations ≤ 2.5 µM, independent of exposure time (data not shown).

Impact of PF1022A on cell cycle distribution

To further investigate whether PF1022A affect the cell cycle distribution flow cytometric analyses were performed in KB-3-1 cells and histograms were gated on live cells. PF1022A treatment caused a moderate concentration-dependent increase in the number of cells in G 0 /G 1 -phase after 24 h and 48 h (Fig. 4). This effect was distinct at concentrations ≥ 5 µM as shown by an increase of cells in G 0 /G 1 -phase by 23% and 19% after 24-and 48 h exposure, respectively. Additionally, after 24-and 48 h treatment with 10 µM PF1022A a further increase was observed by 43% and 34%, respectively. In accordance, the number of cells in the S-and G 2 /M-phase significantly decreased at these concentrations

Impact of apoptosis-and cell cycle-regulating proteins p53, p21, Bax, and Bcl-2 on

PF1022A-induced cell death.

To further investigate the mechanisms underlying PF1022A-induced cell death, the effect of apoptosis-and cell cycle regulating proteins was studied. For this purpose, HCT116 cells with disrupted p53, p21 or bax genes as well as A549 cells with stably overexpressing bcl-2 gene (A549/Bcl-2) were used. In general, in all tested cell lines a distinct cytotoxic effect was observed at high concentrations (10 µM). Remarkably, deletion of p53 was accompanied with loss of sensitivity to high PF1022A concentrations (Tab. 1). Comparable effects were observed in HCT116 bax (-/-) cells (Tab. 1). In contrast, the cytotoxic effect of PF1022A was more prominent in HCT116 p21 (-/-) cells than in the respective parental cell clone (Tab. 1).

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

Effects of PF1022A on cell growth of A549 with transfected Bcl2 and the A549 vector control did not differ significantly from each other.

Discussion

The anthelmintic drug PF1022A is active against diverse worms when administered orally or parenterally. Depending on the species of worms and the host, helmints are paralysed in concentrations ranging from 1 to 10 mg/kg body weight [START_REF] Von Samson-Himmelstjerna | In vivo activities of the new anthelmintic depsipeptide PF1022A[END_REF][START_REF] Harder | Cyclooctadepsipeptides -a new class of anthelmintically active compounds[END_REF], which coincides with the in vitro activity at 1 µg/ml against Trichinella spiralis and Nippostrongylus brasiliensis [START_REF] Martin | Anthelmintic actions of the cyclic depsipeptide PF1022A and its electrophysiological effects on muscle cells of Ascaris suum[END_REF] as well as the effect at 1 µM PF1022A against Haemonchus contortus [START_REF] Conder | Anthelmintic profile of the cyclodepsipeptide PF1022A in in vitro and in vivo models[END_REF]. The lack of an effect on f c at 1 µM and an estimated IC 50 of 25 µM for isolated guinea-pig ileum demonstrate that contractility is not reduced significantly at anthelmintic active concentrations. Consequently, the peristalsis which is necessary to transfer food through the gut should not be affected by the drug. Comparable to other cyclohexadepsipeptides [START_REF] Kouri | Effects of beauvericin on the metabolic state and ionic homeostasis of ventricular myocytes of the guinea pig[END_REF][START_REF] Kamyar | Effects of moniliformin in presence of cyclohexadepsipeptides on isolated mammalian tissue and cells[END_REF], PF1022A influenced the intracellular Ca 2+ concentration in Caco-2 cells only at high drug concentrations (≥ 30 µM).

Sustained Ca 2+ elevation usually results in dysfunction of the contractile mechanism, which might be the cause of the observed reduction in the force of contraction in isolated guinea pig ileum at high drug concentrations.

Results obtained from electrophysiological experiments give evidence that the cyclooctadepsipeptide PF1022A interacts with mammalian cells. The drug was shown to incorporate into cell membranes forming cation selective channels, similar to the cyclohexadepsipeptides beauvericin [START_REF] Kouri | Beauvericin induced channels in ventricular myocytes and artificial membranes[END_REF] and enniatin [START_REF] Kamyar | Investigation of the electrophysiological properties of enniatin[END_REF]. Patch clamp studies revealed that PF1022A manifests common traits of physiological ion channel behaviour such as unitary transitions in conductance levels characteristic for channels, selectivity, rectification, and A c c e p t e d M a n u s c r i p t 16 conductances in the pS range. This strongly suggests that PF1022A is a potential channelforming ionophore. Moreover, formation of a sandwich complex can be postulated similar to the cyclohexadepsipeptides beauvericin [START_REF] Ivanov | Sandwich complexes as a functional form of the enniatin ionophores[END_REF] and enniatin [START_REF] Ovchinnikov | The enniatin ionophores. Conformation and ion binding properties[END_REF]. Through such PF1022Ainduced membrane spanning pores, ions are able to diffuse at both directions following their electrochemical gradient. This conformation would allow the symmetry depicted in the current-voltage curves and the sort of electrostatic interaction that prevailed in our experiments, making the pore selective to cations. However, contrary to beauvericin and enniatin, the kinetics of the PF1022A-induced channel were not influenced by conducted cations. Although this ionophoric action was found already at very low drug concentrations, ion influx affected intracellular ion concentrations only minimally, even at high drug concentrations. Thus, these changes in intracellular ion concentrations might not contribute markedly to cellular toxicity. Apart from these findings, it has been already demonstrated that the ionophoric activity does not correlate with the anthelmintic potency of cyclooctadepsipeptides [START_REF] Gessner | Ionophore and anthelmintic activity of PF1022A, a cyclooctadepsipeptide, are not related[END_REF].

Besides the ionophoric activity, electrophysiological effects via modulation of the GABA current are discussed, as in binding studies it could be demonstrated that PF1022A binds to GABA receptors in nematodes, probably contributing to the anthelmintic effect [START_REF] Chen | Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A, a new anthelmintic, on the receptor[END_REF].

Therefore, we tested whether PF1022A exerts any effect on GABA A receptors composed of 1, 2 and 2S subunits, which might be of interest in terms of sedative side effects during anthelmintic therapy. Significant activation of the GABA current, however, was not observed.

For the reason of diverse pharmacological properties of structurally related compounds, and in order to prove low cellular toxicity of PF1022A at anthelmintic concentrations, a panel of cytotoxicity tests was performed in mammalian cells. Cytotoxic effects after different exposure times to PF1022A occurred at micromolar concentrations but higher than the active anthelmintic concentrations. Since the results obtained in experiments with and without A c c e p t e d M a n u s c r i p t washout phase did not differ significantly at PF1022A concentrations > 5µM and only a small number of cells could recover, a potent cytotoxic effect of this cyclooctadepsipeptide was suggested at high concentrations. In contrast, at low anthelmintic concentrations (0.5-1 µM) PF1022A significantly enhanced proliferation of the tumour cell line KB-3-1. Comparable effects were reported for the structurally related enniatins [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF]. This growth stimulating activity was suggested to be based on compensatory mechanisms to enniatins-induced stress signals [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF].

Regarding PF1022A-induced cytotoxicity, apoptosis induction via the mitochondrial pathway was suggested at least for long-term PF1022A treatment at high concentrations. This was based on a significant depolarisation of the mitochondrial membrane potential, which is an important step to trigger programmed cell death, after 48 h PF1022A treatment with 10 µM [START_REF] Ovchinnikov | The enniatin ionophores. Conformation and ion binding properties[END_REF]. Moreover, several characteristics of programmed cell death like cell shrinkage, chromatin condensation and apoptotic body formations were observed after 48 h drug treatment with 10 µM PF1022A. Furthermore, the complete lack of cellular lactate dehydrogenase release indicative for necrotic cell death emphasizes the apoptotic activity of the anthelmintic drug. These results are comparable to the cytototoxic effects of the structurally related cyclohexadepsipeptides enniatins [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF] and beauvericin [START_REF] Lin | Involvement of Bcl-family, cytochrome c and caspase 3 in induction of apoptosis by beauvericin in human non-small cell lung cancer cells[END_REF][START_REF] Jow | Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium[END_REF]. Moreover, since the ionophoric activity of these cyclic peptides was suggested to contribute to their potent cytotoxicity [START_REF] Jow | Beauvericin induces cytotoxic effects in human acute lymphoblastic leukemia cells through cytochrome c release, caspase 3 activation: the causative role of calcium[END_REF][START_REF] Kouri | Effects of beauvericin on the metabolic state and ionic homeostasis of ventricular myocytes of the guinea pig[END_REF][START_REF] Tang | Beauvericin activates Ca 2+ -activated Clcurrents and induces cell deaths in Xenopus oocytes via influx of extracellular Ca 2+[END_REF], comparable modes of actions might be hypothesized for PF1022A. However, our study revealed that only high concentrations (100 µM) of the cyclooctadepsipeptide PF1022A cause an increase in the intracellular Ca 2+ levels. Thus, this effect does not seem to contribute markedly to cytotoxicity.

In addition to apoptosis induction, our study revealed that long-term but also short-term PF1022A treatment concentration-dependently led to cell cycle arrest in G 0 /G 1 phase. Thus, the cytotoxic modes of actions of short-term PF1022A exposure might be based on a cell A c c e p t e d M a n u s c r i p t 18 cycle blockade. Consistently, data obtained by DAPI staining revealed a significant reduction of countable nuclei compared to control after 24 h drug treatment. Moreover, cells with deleted cyclin-dependent kinase inhibitor p21 were more sensitive to PF1022A-induced cytotoxicity. This suggests that cell cycle blockade via p21 might rescue cells from PF1022Ainduced cell death as it was also suggested for the structurally related enniatins [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF].

To further investigate the impact of PF1022A on apoptosis-and cell cycle-regulating proteins, HCT116 cells with disrupted p53, p21 or bax genes [START_REF] Bunz | Targeted inactivation of p53 in human cells does not result in aneuploidy[END_REF][START_REF] Waldman | p21 is necessary for the p53-mediated G1 arrest in human cancer cells[END_REF][START_REF] Zhang | Role of BAX in the apoptotic response to anticancer agents[END_REF] and A549 cells transfected with Bcl-2 were used. In MTT assays, a small shift of cytotoxicity to higher PF1022A concentrations was observed in p53 and bax disrupted cells, whereas the apoptosis inhibitor Bcl-2 had no significant influence on PF1022A-induced cell death. Thus, our data suggest that PF1022A-induced mitochondrial membrane depolarisation occurs via a molecular mechanism involving p53-mediated apoptosis effects. This is in contrast to enniatins, which mediated apoptosis induction independent of either of these proteins, but activated the proapototic p53-downstream target bax at relatively low enniatin concentrations [START_REF] Dornetshuber | Enniatin induces apoptosis and exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells[END_REF].

To conclude, data obtained with PF1022A in mammalian tissue and cells point to drug safety in terms of lower susceptibility of mammalian tissue compared to the paralysing effect in helmints. Moreover, due to the lack of an effect on I GABA(A) at anthelmintic concentrations no sedative side effects are to be expected. In addition, the drug PF1022A and its derivatives might be of interest to be studied in more detail in respect to anticancer activity.

PF1022A (µM)

  3  3.0 pS (n=5) and for Ca 2+ at 50.5 2.2 pS (n=3).

  A c c e p t e d M a n u s c r i p t 11 Probability density functions for open times of the PF1022A channel conducting potassium, sodium or calcium ions at potentials ranging from -60 to +60 mV could be fitted with one exponential. The kinetics of the openings at those potentials was not significantly (P>0.05)affected by voltage. Further, time constants for open times did not significantly differ for Na + , K + and Ca 2+ (P>0.05). Open state probability was independent of voltage, however, varying markedly in different patches and also within one patch with time. Openings occurred usually in bursts of fast flickerings, which alternated with periods of electrical inactivity. For Ca2+

A

  c c e p t e d M a n u s c r i p t 12 concentrations that already caused multiple channel openings (10 µM, n=6), and ineffective at 1 µM PF1022A (n=5).

Figure legends : Fig. 1 :

 :1 Figure legends:

Fig. 2 :

 2 Fig. 2: Concentration-dependent effect on cell growth is demonstrated in KB-3-1 cells after

Fig. 3 :

 3 Fig. 3: (A, B) Nuclei of KB-3-1 cells were stained with DAPI, classified (in interphase,

Fig. 4

 4 Fig.[START_REF] Geary | Frontiers in anthelmintic pharmacology[END_REF] PI staining and FACS analyses were performed on KB-3-1 cells after 24 h and 48 h

Fig. 1 Time

 1 Fig. 1

Fig. 2 *

 2 Fig. 2

  Fig. 3

  Fig. 4

Figure 4 A

 4 Figure 4

Table 1 :

 1 Impact of apoptosis-and cell cycle-regulating proteins p53, p21, Bax, and Bcl-2 on PF1022A-induced cell death. IC 50 values (µM) for the time-dependent cytotoxic effect of PF1022A are given.

	Cell line	48 h	72 h
	HCT 116 p53 (+/+)	8.4 ± 0.1	7.5 ± 0.1
	HCT 116 p53 (-/-)	9.6 ± 0.3	9.1 ± 0.1
	HCT 116 bax (+/+)	7.4 ± 0.3	7.1 ± 0.1
	HCT 116 bax (-/-)	9.5 ± 0.1	8.2 ± 0.1
	HCT 116 p21 (+/+)	9.4 ± 0.1	8.2 ± 0.1
	HCT 116 p21 (-/-)	6.0 ± 0.1	4.6 ± 0.1
	A549/Bcl-2	7.5 ± 0.1	4.3 ± 0.2
	A549/vc	7.3 ± 0.1	4.6 ± 0.1

Table 1

 1


Arpad Szanto

Eva E Hellebrand

Zita Bognar

Zsuzsanna Tucsek

Aliz Szabo

Ferenc Gallyas

Balazs Sumegi

M.D Gabor Varbiro
email: g.varbiro@keele.ac.uk

PARP inhibition promotes Taxol resistance via Akt activation 1 PARP-1 INHIBITION-INDUCED ACTIVATION OF PI-3-KINASE-AKT PATHWAY PROMOTES RESISTANCE TO TAXOL

Keywords: antitumor treatment, Taxol resistance, PARP inhibition, Akt activation, PI-3 kinase

PARP inhibition promotes Taxol resistance via Akt activation

The nonstandard abbreviations used are: PARP, Poly (ADP-ribose) polymerase; PAR, poly (ADP-ribose); PI-3K, phosphatidylinositol-3-

INTRODUCTION

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage [START_REF] Virag | The therapeutic potential of poly(ADP-ribose) polymerase inhibitors[END_REF]. Single-and/or double-strand DNA breaks induce the production of branched chain ADP-ribose polymers that are covalently attached to numerous nuclear proteins like histones or the PARP itself and this process represents an early event in DNA repair.

Although it is well-documented that inhibition of PARP-1 has cytoprotective effects against oxidative stress [START_REF] Halmosi | Effect of poly(ADP-ribose) polymerase inhibitors on the ischemia-reperfusion-induced oxidative cell damage and mitochondrial metabolism in Langendorff heart perfusion system[END_REF], there is growing evidence suggesting that inhibition of PARP-1 sensitizes cells to DNA-damaging agents [START_REF] Oliveira | Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction[END_REF]. This later effect of PARP-1 inhibition is attributed to the DNA-damage sensing function of PARP-1, namely that it responds to single-and/or doublestrand DNA breaks, and facilitates DNA repair and cell survival. Furthermore, it was shown that cells deficient in breast cancer associated gene-1 and -2 (BRCA1/2) are extremely sensitive to PARP inhibition because of defective double-strand DNA break repair [START_REF] De Soto | The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors[END_REF]. Based on these data, PARP inhibition is considered a useful therapeutic strategy not only for the treatment of BRCA mutation-associated tumours, but also for the treatment of a wider range of tumours bearing a variety of deficiencies in the homologous recombination DNA repair pathway [5]. However, it has also been shown that inhibition of PARP leads to phosphorylation, and thus activation, of Akt in various tissues [START_REF] Veres | Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADPribose) polymerase 1 inhibitor in endotoxin-induced septic shock[END_REF][START_REF] Veres | Regulation of kinase cascades and transcription factors by a poly(ADP-ribose) polymerase-1 inhibitor, 4hydroxyquinazoline, in lipopolysaccharide-induced inflammation in mice[END_REF][START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF]. It raises the possibility that application of PARP inhibitors in tumour therapy may activate the phosphatidylinositol-3 kinase (PI-3K)-Akt pathway, which initiates processes like the inactivation of glycogen synthase kinase-3, caspase-9, Bad or forkhead homolog rhabdomyosarcoma (FKHR) transcription factors [START_REF] Birkenkamp | FOXO transcription factors as regulators of immune homeostasis: molecules to die for?[END_REF] leading to cytostatic resistance.

A c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 4 Paclitaxel (taxol) interferes with the mitotic spindle during mitosis of cells, stabilizing the microtubule by inhibiting tubulin dimerisation and so inhibiting the separation of the sister chromatids [START_REF] Torres | Mechanisms of Taxol-induced cell death are concentration dependent[END_REF][START_REF] Blagosklonny | Molecular effects of paclitaxel: myths and reality (a critical review)[END_REF][START_REF] Wang | Paclitaxel-induced cell death: where the cell cycle and apoptosis come together[END_REF]. Paclitaxel can affect kinases [START_REF] Mcdaid | Enhancement of the therapeutic efficacy of taxol by the mitogen-activated protein kinase kinase inhibitor CI-1040 in nude mice bearing human heterotransplants[END_REF] that play important roles in cell death processes, and regulate the expression of tumour suppressor genes and cytokines [START_REF] Sunters | FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines[END_REF]. In addition, paclitaxel can induce cytosolic calcium oscillations [START_REF] Boehmerle | Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism[END_REF] and mitochondrial permeability transition, as well as elevated generation of reactive oxygen species predominantly at cytochrome oxidase in tumour cells [START_REF] Varbiro | Direct effect of Taxol on free radical formation and mitochondrial permeability transition[END_REF]. In the paclitaxel-induced cell death process, activation of c-Jun N-terminal kinase (JNK) plays a critical role by suppressing Akt activation and promoting the nuclear accumulation of forkhead-related transcription factor-3a (Foxo3a; [START_REF] Sunters | Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt[END_REF]). Nuclear translocation of Foxo3a can facilitate apoptosis by inducing the expression of Bim, a BH3-only proapoptotic bcl-2 homolog protein [START_REF] Van Der Heide | The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation[END_REF]. It has also been demonstrated that Akt overexpression prevented paclitaxel-induced cell death [START_REF] Vanderweele | Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin[END_REF][START_REF] Priulla | Preferential chemosensitization of PTEN-mutated prostate cells by silencing the Akt kinase[END_REF][START_REF] Asnaghi | Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt[END_REF][START_REF] Clark | Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells[END_REF][START_REF] Bevilacqua | Bcl-2 protein is required for the adenine/uridine-rich element (ARE)-dependent degradation of its own messenger[END_REF], probably by a mechanism involving Akt dependent phosphorylation of FOXOs that stabilizes their binding to cytosolic 14-3-3 protein and so prevents their translocation to the nucleus, resulting in inhibition of transcription of FOXO dependent genes such as Bim [START_REF] Luhn | Identification of FAKTS as a novel 14-3-3associated nuclear protein[END_REF].

In the present paper, we provide evidence that inhibition of PARP-1 activity can indeed cause resistance to paclitaxel induced death in tumour cells, and activation of the PI-3K-Akt pathway is significantly involved in this effect. inhibitor PJ-34, protease inhibitor cocktail, and all the chemicals for cell culture were purchased from Sigma-Aldrich Kft (Budapest, Hungary). InSolution Akt Inhibitor IV was from Calbiochem (Darmstadt, Germany) The following antibodies were used: anti-Akt, anti-phospho-Akt, antiglycogen synthase kinase-3 (GSK), anti-phospho-glycogen synthase kinase-3ß (GSK), anti-JNK, anti-phospho-c-Jun N terminal kinase (JNK), anti-p38 MAPK, anti-phospho-p38 mitogen activated protein kinase (p38 MAPK) and anti-p44/42 MAPK (Erk1/2), (Cell Signalling Technology, Beverly, MA); anti-phospho-extracellular signal regulated kinase (Erk1/2) anti-PAR and anti PARP (Alexis Biotechnology, London, UK); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPD, Delta Biolabs, Gilroy, CA); anti-mouse IgG and anti-rabbit IgG (Sigma-Aldrich Kft, Budapest, Hungary).

Cell culture.

Hela human cervical cancer and T24 human bladder carcinoma cells were from American Type Culture Collection (Wesel, Germany). The cells were maintained as monolayer adherent culture in Minimum Essential Eagle's Medium containing 1% antibioticantimycotic solution (Sigma-Aldrich Kft Budapest, Hungary) and 10% fetal calf serum (MEM/FCS) in humid 5% CO 2 atmosphere at 37 °C. 2.6. Determination of paclitaxel uptake. One million/well T24 cells were seeded to 6-well plates and incubated for 3 h in the presence of 10, 100 or 1000 nM paclitaxel only, or together with 10 µM PJ-34 and/or 40 µM verapamil. After the incubation, the cells were harvested, and homogenized by sonication. Paclitaxel content of the samples were determined by high-pressure liquid chromatography and mass spectrometry (HPLC-MS) after deproteinization by perchloric acid. For the separation, an isocratic method was applied using 5mM sodium-acetate, pH4.5acetonitrile 50:50 V/V mixture as solvent at a flow rate of 1.2 ml/min. The same HPLC system that was used for cytochrome c measurement was connected to a Bruker HCT Esquire (Bruker Daltonics, Bremen, Germany) MS instrument through a microsplitter valve (Upchurch Scientific, Oak Harbor, WA), the flow rate was 1.2 ml/min with a splitting ratio of 7 over 3. The electrospray ion source was operated in positive mode. Nitrogen was used as drying gas at 250 ºC, with a flow rate of 8 liters per minute, the pressure of the nebulizer was set at 12 psi. We used the Smart Parameter Setting (SPS) with target mass of 854 m/z (wide range). The scanning mass to charge range was 50 to 2000 m/z with a scanning speed of 8100 m/z per second. Cells were treated with paclitaxel and PJ-34 as for the cell viability assay using three replicate cultures and each experiment was repeated twice. The NAD + level was measured exactly as described previously [START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF]. Briefly, cells were cultured in a 96 well plate and treated with paclitaxel in the presence or absence of PJ-34 and PI-3K inhibitor LY-294002 as described. Cellular NAD + levels were measured by the micro plate version of the enzymatic cycling method using alcohol dehydrogenase exactly as described [START_REF] Shah | Methods for biochemical study of poly(ADP-ribose) metabolism in vitro and in vivo[END_REF]. The reaction was monitored at 550 nm and was allowed to run for 10 min. A standard curve was generated using known concentrations of NAD + for the calculation of the cellular NAD + levels.

Transdominant expression of

Western blot analysis.

The cells were seeded and treated as for the cell viability assay.

After the time indicated, the cells were harvested in a chilled lysis buffer containing 0.5 mM sodium-metavanadate, 1 mM EDTA and protease inhibitor cocktail in PBS. The proteins were precipitated with TCA, washed 3 times with -20C acetone and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. Proteins (20 g per lane) were separated on 12% gels then transferred to nitrocellulose membranes. The membranes were blocked in 5% low-fat milk for 1 hour at room temperature then exposed to the primary antibodies at 4C overnight at a dilution of 1:1000 in blocking solution. Appropriate horseradish peroxidaseconjugated secondary antibodies were used for 3 hours at room temperature at a dilution of 1:5000. Peroxidase labeling was visualized with enhanced chemiluminescence labeling using an ECL Western blotting detection system (Amersham Biosciences, Budapest, Hungary). The developed films were scanned and the pixel volumes of the bands were determined by using NIH's Image J software. All experiments were repeated 3 times [START_REF] Varbiro | Protective effect of amiodarone but not N-desethylamiodarone on postischemic hearts through the inhibition of mitochondrial permeability transition[END_REF].

2.9. Caspase-3 activity assay. Caspase-3 activity following paclitaxel administration in the presence or absence of the PARP inhibitor PJ-34 was carried out exactly as described previously ). Similar results were obtained using HeLa cells (data not shown). These data show that paclitaxel treatment resulted in a massive activation of PARP-1 activity that was effectively prevented by all the three methods used for suppression of the catalytic activity of the enzyme. Under our experimental conditions, 12 hours or longer exposure to 100 nM paclitaxel significantly decreased the viability (p < 0.01) of T24 (Fig. 2A) and HeLa (data not shown) cells.

However, when 10 µM PJ-34 was added to the medium 30 min before the application of paclitaxel, the effect of the drug on cell viabilities was significantly attenuated (p < 0.01) indicating that the PARP inhibitor provided protection against paclitaxel in the cancer cell lines studied (Fig. 2.A.). In order to reveal whether the observed paclitaxel-resistance was due to any interference with ABC transporters, we blocked P-glycoprotein pathway by 40 µM verapamil. Verapamil indeed enhanced the effect of paclitaxel in both cell lines, so in the presence of verapamil, maximal effect of paclitaxel was observed at 10 rather than 1000 nM concentration.

On the other hand, PJ-34 desensitized T24 and HeLa cells towards paclitaxel, and increased cell viability at all paclitaxel concentrations (Fig. 2.B., Fig. 2.C.). The fact that at higher paclitaxel concentrations verapamil did not interfere with the desensitizing effect of PJ-34 suggests that the PARP inhibition evoked drug resistance in tumor cells was not likely to be related to ABC transporter mechanisms.

We approached the question of the interference between the PARP inhibitor and the ABC transporter more directly by determining the amount of paclitaxel taken up by T24 cells during 3h incubation in the presence of 10, 100 and 1000 nM of paclitaxel alone or together with 10 µM PJ-34 and/or 40 µM verapamil. As shown in Table I. the PARP inhibitor slightly, although not significantly, decreased paclitaxel uptake, while verapamil very significantly increased it, regardless of the presence or absence of PJ-34. This result confirmed that the PARP inhibition induced paclitaxel resistance by an alternative mechanism, and not by interacting with ABC transporter systems.

Transdominant expression of DNA-binding domain of PARP.

To demonstrate that the inhibition of nuclear PARP-1 and not a side effect of the pharmacological PARP inhibitor was indeed responsible for the paclitaxel-resistance, we assessed the effect of non-pharmacological PARP inhibition on paclitaxel induced cell death. We transiently transfected T24 bladder carcinoma cells with a construct (pEGFP-N3) expressing a fusion protein consisting of the ). When the cells were transfected with the plasmid expressing the hybrid protein, the paclitaxel induced cytotoxicity was significantly lower (p < 0.01) when compared to non-transfected control cells (Fig. 3.A.). Similar results were detected in the HeLa cell line (data not shown).

Suppression of PARP expression by RNA-interference. PARP inhibition was also

achieved by suppressing its expression with RNA interference. T24 bladder carcinoma cells were transfected with PARP siRNA in accordance with the manufacturer's recommendations. The knock-down of PARP was verified by Western blotting (data not shown). Following 24 hours of paclitaxel treatment, no significant difference was detected between the control and siRNA transfected cells up to the paclitaxel concentration of 10 nM. However above this concentration, the viability of siRNA transfected cells was significantly higher (p < 0.01) when compared to controls (Fig. 3.B.). We obtained similar results in the HeLa cell line (data not shown).

PARP inhibition decreases the paclitaxel induced caspase-3 activation. According to

previous studies, paclitaxel administration induces mainly apoptotic cell death, so we tested caspase-3 activation and cytochrome c release in our experimental setup. In T24 bladder carcinoma cells, twelve hours of paclitaxel treatment at the concentration of 100 and 1000 nM resulted in marked activation of caspase-3 (Fig. 4.A.), and this effect was significantly reduced A c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 13 (p < 0.01) when the cells were pretreated with 10 µM of PJ-34 (Fig. 4.A.). The time-course for the activation of caspase-3 by paclitaxel was also investigated. The administration of paclitaxel at the concentration of 100 nM induced a significant increase in caspase-3 activity (p < 0.05) in T24 bladder carcinoma cells after 3 hours when compared to untreated control (Fig. 4.B.). When the cells were pretreated with 10 µM of PJ-34, the level of caspase-3 activation was significantly lower compared to the cells that were treated solely with paclitaxel. Similar results were obtained with HeLa cells (data not shown). Mitochondrial cytochrome-c release was determined by a quantitative HPCL method. In T24 cells, 12 h of 100 nM paclitaxel treatment resulted in an increased release of cytochrome-c. When the cells were pretreated with 10 µM PJ-34, this effect was significantly reduced (p < 0.01). Furthermore, 5 µM of LY294002 significantly enchanced cytochrome-c release induced by paclitaxel (p < 0.01) and diminished the reducing effect of PJ-34. Similar results were obtained in case of the HeLa cells (data not shown).

PARP inhibition leads to the activation of Akt/PKB.

To elucidate the role of the nuclear enzyme PARP-1 in regulating the proteomic signal transduction pathway, we analyzed activation of Akt/protein kinase B, Erk, JNK and p38 MAP kinases in response to paclitaxel treatment in the presence of PJ-34 in T24 bladder carcinoma cells. Previously, we observed Akt activation as soon as 15 min after PJ-34 treatment, so we assessed the levels of kinases up to 3 hours following 100 nM of paclitaxel administration in the presence or absence of 10 µM of PJ-34.

The level of total Akt was unaltered in response to either paclitaxel or PJ-34 administration (Fig.

5.

). Paclitaxel administration resulted in a slightly increased Akt (Ser 473 ) phosphorylation after only 3 hours. However, it increased within 15 min of PJ-34 administration, and the increased level was maintained throughout the observation period (Fig. 5). The total level of glycogen synthase kinase3β (GSK-3β), the downstream target of Akt, was not altered in response to either (Fig. 5). Contrary to phospho-Akt, neither paclitaxel nor PJ-34 administration influenced the level of phosphorylated p38 or Erk1/2. Paclitaxel treatment increased JNK activation, however, pretreatment with 10 µM of PJ-34 failed to modify this effect (Fig. 5). When we determined the total MAP kinase levels (Erk1/2, p-38 and JNK, respectively), no alteration was detected up to 3 hours following 100 nM of paclitaxel administration in the presence or absence of 10 µM of PJ-34. ). Since the ADP-ribose polymers are synthesized by PARP using NAD + as its substrate and resynthesis of NAD + is energetically costly, PARP inhibition could cause paclitaxel resistance by relieving this metabolic burden. To address this issue, we measured intracellular NAD + levels following paclitaxel administration either alone or in combination with PJ-34 and LY-294002 or Akt inhibitor IV. T24 cells were treated with paclitaxel at the concentration of either 100 nM or 1000 nM for 24 hours. This leads to the activation of PARP and the decrease of intracellular NAD + level. When the cells were pretreated with 10 µM of PJ-34 for 30 min prior to the administration of paclitaxel, the level of NAD + following paclitaxel treatment was significantly higher (p < 0.01) than without it. However, neither 5 µM of LY-294002 (Fig. 7.A) nor 5 µM of Akt inhibitor IV (Fig. 7.B) affected the NAD + levels when applied alone or in combination with PJ-34 and paclitaxel. Similar effect was noted in HeLa cells (data not shown). Since the inhibition of PI-3K/Akt pathway did not interfere with the intracellular level of NAD + (Fig. 7) but significantly counteracted the effect of PARP inhibition on the cell viability compromised by paclitaxel administration (Fig. 6), reduction of NAD + depletion could not account for the paclitaxel resistance caused by the PARP inhibition, rather, PARP inhibition caused paclitaxel resistance was achieved by activating the PI-3K-Akt pathway to a very significant extent.

Inhibition of the

DISCUSSION

It has been suggested that transient inhibition of DNA repair using potent PARP inhibitors could improve the efficacy of cancer treatments. Although more study is needed, recent reports demonstrated that the inhibition of poly(ADP-ribose) synthesis could selectively kill cancer cells when used for treating tumours with defective BRCA proteins [START_REF] De Soto | The inhibition and treatment of breast cancer with poly (ADP-ribose) polymerase (PARP-1) inhibitors[END_REF]5]. These reports shed some light on the DNA damage signalling and repair processes involving PARPs. Recently it has been proposed that, in addition to the effects on BRCA defective tumour cells, targeting specific DNA repair enzymes can open a new type of chemotherapeutic approach to malignant diseases. In particular, inhibitors of PARP-1 that sensitize cells to DNA-damaging agents are under extensive investigation [START_REF] Virag | The therapeutic potential of poly(ADP-ribose) polymerase inhibitors[END_REF][START_REF] Oliveira | Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction[END_REF]. It is well-documented that PARP-1 functions as a DNA damage sensor that responds to both single and/or double strand DNA breaks (SSBs, DSBs), facilitating DNA repair and cell survival. PARP-1, following binding to DNA, cleaves NAD + to ADP-ribose and nicotinamide and converts ADP-ribose into polymers of branched or linear poly(ADP-ribose) units (PAR) which can be attached to PARP-1 itself and to other nuclear acceptor proteins, including XRCC1, histones and so on [START_REF] Oliveira | Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction[END_REF][START_REF] Keil | MNNG-induced cell death is controlled by interactions between PARP-1, poly(ADP-ribose) glycohydrolase, and XRCC1[END_REF]. These processes are important in the survival of the cells after extensive DNA damage but in normal cells the complete absence of PARP-1 protein or the inhibition of PARP-1 catalytic activity produces no significant growth defect [START_REF] Tentori | Poly(ADPribose) W polymerase (PARP) inhibition or PARP-1 gene deletion reduces angiogenesis[END_REF]. This is supported by the observation that PARP-1 defective mice survive and have no obvious growth defect [START_REF] Oliver | Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice[END_REF]. However, PARP-1 defective mice are more sensitive to high levels of high energy irradiation and to alkylating agents, showing that under some condition PARP-1 inactivation can facilitate cell death [START_REF] Wesierska-Gadek | Nonapoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)[END_REF].

On the other hand, it is well documented that, in response to extensive DNA damage, PARP-1 can be hyperactivated, eliciting activation of cell death by inducing signal transduction [START_REF] Veres | Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADPribose) polymerase 1 inhibitor in endotoxin-induced septic shock[END_REF][START_REF] Veres | Regulation of kinase cascades and transcription factors by a poly(ADP-ribose) polymerase-1 inhibitor, 4hydroxyquinazoline, in lipopolysaccharide-induced inflammation in mice[END_REF][START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF], by direct mitochondrial damaging effect and can suppress the activity of the cytoprotective PI-3-kinase Akt pathway, and also can induce rapid cellular NAD + and ATP pool depletion resulting in necrotic or apoptotic cell death. PARP-1 hyperactivation has been documented in a number of pathological conditions including ischemia-reperfusion, myocardial infarction, and reactive oxygen species (ROS)-induced injury (2). In each case, inhibition of PARP-1 improved the survival of damaged cells or tissues [START_REF] Halmosi | Effect of poly(ADP-ribose) polymerase inhibitors on the ischemia-reperfusion-induced oxidative cell damage and mitochondrial metabolism in Langendorff heart perfusion system[END_REF][START_REF] Wang | Poly(ADP-ribose) polymerase inhibitor is neuroprotective in epileptic rat via apoptosis-inducing factor and Akt signaling[END_REF][START_REF] Pacher | Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents[END_REF]. In several cases, there are data showing that PARP-1 inhibition activated the PI-3-kinase -Akt pathway which can lead to cytostatic resistance [START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF][START_REF] Vanderweele | Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin[END_REF][START_REF] Priulla | Preferential chemosensitization of PTEN-mutated prostate cells by silencing the Akt kinase[END_REF][START_REF] Asnaghi | Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt[END_REF][START_REF] Clark | Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells[END_REF][START_REF] Bevilacqua | Bcl-2 protein is required for the adenine/uridine-rich element (ARE)-dependent degradation of its own messenger[END_REF], therefore PARP-1 inhibition depending on the precise conditions can facilitate, or inhibit, cell death.

In the present paper, we investigated the effect of PARP inhibition on the paclitaxel induced cell death process using two different tumour cell lines. According to our data inhibition of PARP-1 significantly compromises the cell death inducing effect of paclitaxel, resulting in cytostatic resistance to a wide range of paclitaxel concentration (Fig. 2. and 3.). This paclitaxel resistance was unlikely to be mediated by ABC transporter related mechanisms, since verapamil that blocks the P-glycoprotein pathway did not interfere with the desensitizing effect of PJ-34.

Furthermore, we determined directly the interaction of PJ-34 and verapamil on taxol uptake of T24 cells by measuring the cellular paclitaxel concentrations after incubating the cells with paclitaxel in combination with PJ-34 and/or verapamil (Table I). This experiment confirmed that ABC-transporter related mechanisms were not significantly involved in the paclitaxel resistance induced by PARP inhibition.

Although PJ-34 is a well-characterized PARP-1 inhibitor, the specificity of a small molecular weight synthetic inhibitor is always questionable because of the presence of several enzymes with poly-and mono-ADP-ribosylating activity in cells [START_REF] Virag | The therapeutic potential of poly(ADP-ribose) polymerase inhibitors[END_REF]. Knocking down of PARP-1 A c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 18 in T24 cells by siRNA technique induced paclitaxel resistance (Fig. 3B) similar to that caused by PJ-34, indicating that PARP-1 protein played a significant role in this process, although the question remains as to whether the suppression of PARP-1 catalytic activity or the absence of PARP-1 protein was responsible for the observed phenomenon. The transdominant expression of PARP-DBD inhibits ADP-ribosylation by PARP because binding to single-strand DNA breaks is essential for the activation of PARP-1, and the PARP-DBD competes with PARP-1 in binding to single-strand DNA breaks, and the former does not have catalytic activity. In a previous study, we demonstrated that PARP-DBD was localized almost exclusively to the nucleus [START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF], so it was clearly in position to compete with PARP-1. Transdominant expression of PARP-DBD induced paclitaxel resistance in tumor cells (Fig. 3A), which was similar to the effect caused by PJ-34.

Since the design of the siRNA and the PARP-DBD was based on the sequence of nuclear PARP-1, it is unequivocal that the paclitaxel resistance was the consequence of the inhibition of the single-strand DNA break-induced PARP-1 activation, and was not due to the absence of PARP-1 protein or to another mechanism that might be regulated by the pharmacological inhibitor.

However, since pharmacological PARP inhibitors are expected to be used in the clinical practice for supplementing anticancer agents, in the following experiments of our study we used a pharmacological agent in modeling the effect of PARP-1 inhibition.

In a previous report, we showed that PARP inhibition protected the mitochondrial membrane system, and this mechanism was significantly involved in its cytoprotective effect during oxidative stress [START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF]. We addressed the question of whether such a mechanism was involved in the PJ-34 induced paclitaxel resistance by comparing release of cytochrome c from the mitochondria to the cytosol and caspase-3 activation (Fig. 4) in response to paclitaxel treatment alone vs. in combination with PJ-34. We found that PJ-34 significantly decreased both A c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 19 hallmarks of apoptosis suggesting that the preservation of the mitochondrial membrane system indeed might be involved in the effects of the PARP inhibitor. We checked what kinase signaling pathways were activated by the paclitaxel treatment when applied alone or in combination with PJ-34. In agreement with the literature [START_REF] Sunters | Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt[END_REF], paclitaxel treatment induced the activation of JNK, but it was not significantly affected by PJ-34 (Fig. 5). Several previous studies demonstrated that activation of the PI-3K-Akt system was strongly involved in mediating drug resistance under various conditions [START_REF] Vanderweele | Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin[END_REF][START_REF] Priulla | Preferential chemosensitization of PTEN-mutated prostate cells by silencing the Akt kinase[END_REF][START_REF] Asnaghi | Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt[END_REF][START_REF] Clark | Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells[END_REF][START_REF] Bevilacqua | Bcl-2 protein is required for the adenine/uridine-rich element (ARE)-dependent degradation of its own messenger[END_REF]. In accord with our previous data [START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF], PARP inhibition induced the phosphorylation and thus the activation of Akt which could phosphorylate and inactivate FOXO transcription factors [START_REF] Burgering | Cell cycle and death control: long live Forkheads[END_REF] and so compromised the activation of the cell death process. In addition, Akt activation could protect mitochondrial membrane systems and could inactivate caspase-3 [START_REF] Zhang | Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion[END_REF][START_REF] Tapodi | Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADPribose)polymerase-1 inhibition in oxidative stress[END_REF] therefore it is likely that PARP-1 inhibition induced Akt activation plays a pivotal role in the resistance against taxol induced cell death.

The significance of Akt activation in PARP inhibition-induced paclitaxel resistance can be assessed by inhibiting Akt activation. When we blocked Akt activation either by inhibiting its upstream activator, the PI-3-kinase using LY-294002 or another upstream activator using Akt inhibitor IV, we observed significantly decreased PJ-34-induced paclitaxel resistance (Fig. 6).

That is, Akt activation played a pivotal role in PARP inhibitor induced paclitaxel resistance.

Although specificity and possible side-effects of a pharmacological agent is always an issue, LY 294002 has been reported to inhibit all isoforms of PI-3 kinase while not affecting other kinases such as PKC, PKA, MAP kinase, S6 kinase, EGF tyrosine kinase, c-src kinase, PI-4 kinase and diacylglycerol kinase [START_REF] Vlahos | A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)[END_REF]. Akt inhibitor IV has been less thoroughly characterized, but it was reported not to affect PI-3K, and to block Akt-mediated FOXO1a nuclear export and cell proliferation in 786-O cells [START_REF] Kau | A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells[END_REF]. Since two inhibitors of different chemical structure and It is well-documented that FOXO1 and FOXO3 have a proapoptotic function in cell death processes and that FOXOs induce the overexpression of their downstream targets such as Fas ligand and Bim [START_REF] Sunters | FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines[END_REF]. These processes and FOXO-dependent overexpression of the cell cycle inhibitor p27 (kip1) may be responsible for taxol-induced cell death [START_REF] Trotman | Identification of a tumour suppressor network opposing nuclear Akt function[END_REF][START_REF] Sunters | FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines[END_REF]. NAD + depletion and induction of mitochondrial permeability transition were implicated as intermediate steps linking PARP-1 activation to mitochondrial cytochrome c release and consequent activation of the caspase pathway. We observed significant NAD + depletion in response to paclitaxel treatment that was significantly attenuated by PJ-34 (Fig. 7). It is worth mentioning that even when 1000 nM of paclitaxel was administered, a substantial amount of NAD + remained allowing the operation of ATP dependent cell functions, such as apoptotic processes and operation of kinase signaling pathways. However, and in contrast to the viability experiments, the PI-3K and Akt inhibitors did not counteract, in fact did not affect at all (Fig. 7), the protection of NAD + pool by PARP inhibition. This suggests that PI-3K and Akt activities are not involved in the regulation of intracellular NAD + level, and prevention of NAD + depletion by the PARP inhibitor did not play significant role in the PARP inhibition-induced paclitaxel resistance. Rather, activation of the PI-3K-Akt pathway was the significant factor in the drug resistance inducing effect of PARP inhibition, as described schematically in Fig. 8.

This study demonstrates that drug-induced drug resistance can be responsible for the reduced efficacy of antitumor treatment. The data show that although PARP-1 inhibition can inhibition on the PI-3K-Akt signal transduction pathway could counteract this effect. Therefore, application of PARP inhibitors may represent a double-edged sword, which on the one hand, promotes cell death by inhibiting DNA repair while on the other hand, via activation of PI-3K/Akt pathway, promotes cell survival. This dual effect of PARP inhibition could be responsible for the contradictory data in this field [START_REF] De Soto | PARP-1 inhibitors: are they the long-sought genetically specific drugs for BRCA1/2-associated breast cancers?[END_REF][START_REF] Gallmeier | Absence of specific cell killing of the BRCA2-deficient human cancer cell line CAPAN1 by poly(ADP-ribose) polymerase inhibition[END_REF][START_REF] Bryant | Poly(ADP-ribose) polymerase inhibitors as potential chemotherapeutic agents[END_REF][START_REF] Malanga | The role of poly(ADP-ribose) in the DNA damage signaling network[END_REF]. It also suggests that to utilize the cell death promoting effect of PARP-1 inhibition in cancer therapy, the activation of PI-3k-Akt pathway should be suppressed by specific inhibitors.

1 .

 1 Materials. Taxol (paclitaxel) was from ICN Biomedicals Inc (Aurora, OH), Verapamil was from Richter Gedeon Rt. (Budapest, Hungary), PI3 kinase inhibitor LY-294002, PARP-1

  DNA-binding domain of PARP. The coding region of the N-terminal DNA-binding domain (DBD) of PARP (PARP-N214, AA residues 1-214; 25) was amplified by PCR and cloned in-frame into pEGFP-C1/N3 vectors (Clontech, Palo Alto, CA) after cutting with Hind III and EcoRI restriction enzymes (Fermentase, Vilnius, Lithuania). For

A 2 . 5 .

 25 figure-legends. After 24 hours of treatment, the medium was removed and fresh MEM/FCS

  Maximum accumulation time was 200 ms. For control of the instrument, the Esquire Control Version 5.3 Build 11, and for data evaluation the Data Analysis Version 3.3 Build 146 software was used (Bruker Daltonics, Bremen, Germany). Quantization was carried out using peak areas method. Results are expressed as pmole paclitaxel/mg protein, mean ± S.E.M of three independent experiments.

A 8 2. 7 .

 87 c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation Determination of NAD + .

A 2 . 10 .

 210 c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 9 [30]. Briefly, the cells were treated with paclitaxel in the presence or absence of PJ-34 for the time indicated. The cells were harvested, washed twice in PBS and resuspended in a cell lysis buffer. Forty μg of protein were incubated with 50 µM of fluorescent caspase substrate Ac-DEVD-AMC (Sigma-Aldrich Kft Budapest, Hungary) in four parallels for 3 hours. Fluorescence was detected by a fluorescent ELISA plate reader at the excitation and emission wavelength of 360 nm and 460 nm respectively. Determination of cytochrome c level by HPLC method. The analysis of cyt-c from the cytosol fraction of T24 or HeLa cells treated with paclitaxel in the presence or absence of PJ-34 for 18 h was performed on a non-porous 33 X 4.6 mm KOVASIL-MS C18 column (Zeochem AG, Uetikon, Switzerland). Measurements were performed on a Dionex HPLC system consisting of a Dionex P 580 low pressure gradient pump, a Dionex UVD 340S diode array detector (chromatograms were detected at 393 nm) and a Rheodyne 8125 injector equipped with a 20 μl loop. Instrument control and data acquisition was carried out using Chromeleon data management software. The following gradient was used at a 1 ml/min flow rate; eluent A consisted of 10:90 acetonitrile-water + 0.1% (v/v) trifluoroacetic acid and eluent B consisted of 90:10 acetonitrile-water + 0.1% (v/v) trifluoroacetic acid; 0 → 7 min: from 0 % B to 70 % B, 7 → min 12: from 70 % B to 100 % B, 12 → 12.5 min: from 100 % B to 0 % B, 12.5 → 14.5 min: 0 % B. Data acquisition was performed from at least three independent experiments. 2.11. Statistical Analysis. Data were presented as means ± S.E.M. For multiple comparisons of groups, ANOVA was used. Statistical difference between groups was established by paired or unpaired Student's t test, with Bonferroni's correction.

1 .

 1 PARP inhibition induces paclitaxel-resistance in tumor cell lines. Exposing wild type or T24 bladder carcinoma (Fig. 1) or HeLa cervix tumor (data not shown) cells to 100 nM of paclitaxel induced a massive increase in poly(ADP-ribosylation) of nuclear proteins that reached its maximum in about 3 hours and did not change significantly further on (Fig. 1.A.). When the wild type T24 bladder carcinoma cells were pre-treated with 10 µM of PJ-34 (31), an effective inhibitor of PARP-1, 30 min prior to the administration of paclitaxel, no ADP-ribosylation of the nuclear proteins was detected (Fig. 1.B.). When the cells were mock-transfected or transfected with a construct expressing DNA binding domain of PARP-1 (PARP-DBD; 8) or siRNA designed for the suppression of PARP protein expression at the translational level (8), paclitaxel induced ADP-ribosylation was also abolished in the cells transfected with construct expressing DNA binding domain of PARP-1 or transfected with siRNA (Fig. 1.C.) exactly as observed in the case of wild type cells treated with PJ-34 (Fig. 1.B.

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 11 Co-treating the cells with verapamil and PJ-34 significantly reduced the viability of both tumor cell lines even in the absence of paclitaxel, while verapamil by itself caused a slight, statistically non-significant decrease in the viabilities of both T24 cells (Fig. 2.B.) and Hela cells (Fig. 2.C.).

  A c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 12 nuclear localization signal and the DNA-binding domain of PARP attached to the N terminus of green fluorescent protein (GFP). Control cells were transfected with the same construct expressing only the GFP. While latter protein was localized in the cytosol, the hybrid protein with the nuclear localization signal was localized to the nucleus as detected by fluorescent microscopy (data not shown). No significant difference between the viability of cells either nontransfected or mock transfected (GFP-expressing only) was detected in response to paclitaxel administration (Fig. 3.A.

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 14 paclitaxel or PJ-34 administration (Fig. 5.). However the phosphorylation of GSK-3β presented a similar pattern to Akt, showing increased phosphorylation 30 minutes after paclitaxel and PJ-34 co-administration and slightly increased phosphorylation after 3 hours in the absence of PJ-34

  PI-3K/Akt pathway diminishes paclitaxel resistance induced by inhibition of PARP. Since PARP inhibition leads to the activation of the Akt/PKB-GSK-3β pathway and also to paclitaxel resistance, it seemed reasonable to investigate whether the paclitaxel resistance was mediated by Akt activation. To this end, we inhibited Akt by two different inhibitors, and determined the effect of PARP inhibition on paclitaxel induced cell death under these conditions. Five µM of the PI-3K inhibitor LY-294002 decreased viability of T24 cells by about 20% when applied alone, and significantly decreased paclitaxel resistance caused by PJ-34 (Fig6A). When Akt/PKB was inhibited by a different inhibitor, Akt Inhibitor IV, viability of T24 cells was reduced by about 30% when the drug was applied alone, and decreased paclitaxel resistance caused by PARP inhibition more effectively than LY-294002 did (Fig.6.B). Similar results were obtained in the case of Hela cells (data not shown). These results suggest that paclitaxel resistance induced by PARP inhibition was indeed mediated by Akt activation in a significant extent.

A 3 . 7 .

 37 c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 15 PARP inhibition but not the inhibition of Akt influences the intracellular level of NAD + . Paclitaxel treatment lead to protein poly(ADP-ribosylation) as detected by western blotting (Fig. 1.A.

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 16

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 17 pathways

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 20 targeting different upstream activators of Akt gave the same results, the effect of the aforementioned kinase inhibitors on the PARP inhibition-induced paclitaxel resistance was most likely due to their main pharmacological effect on their respective kinases rather than the result of a side-effect.

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation 21 facilitate cell death in cancer cells induced by DNA damaging agent, the effect of PARP-1

29 FIGURES LEGENDS Fig. 1 .

 29LEGENDS1 FIGURES LEGENDS

Fig. 2 .

 2 Fig. 2. Effect of paclitaxel, PJ-34 and verapamil on the viability of cell lines. In (A) the effect of

Fig. 3 .

 3 Fig. 3. Effect of non-pharmacological PARP inhibition on cell viability. In (A) wild type (filled

A 31 Fig. 4 .

 314 Fig. 4. Effect of paclitaxel and PJ-34 on the activity of caspase-3 in T24 cells. Cytosolic fraction

Fig. 5 .Fig. 6 .

 56 Fig. 5. Effect of paclitaxel and PJ-34 on the activation of various intracellular protein kinases.

Fig. 7 .

 7 Fig. 7. Effect of PI3-K/Akt pathway inhibition on the intracellular NAD + content of paclitaxel

Fig. 8 .

 8 Fig. 8. Proposed molecular mechanism of the effect of PARP inhibition on paclitaxel resistance.

A

  c c e p t e d M a n u s c r i p t PARP inhibition promotes Taxol resistance via Akt activation TABLES Table I. : Effect of PJ-34 and verapamil on paclitaxel uptake of T24 cells. Paclitaxel concentration in the cells were determined by HPLC-MS after incubating them for 3 h in the presence of 10, 100 or 1000 nM paclitaxel only, or together with 10 µM PJ-34 and/-----------------------------alone14.0 ± 5

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ACKNOWLEDGMENTS

This work was supported by grants from the Hungarian Science Foundation T 34320 as well as T 34307, from the Ministry of Health and Welfare ETT 35/2000, from PTE DD-KKK, and by grant NKFP 2001 1/026.