Juan F M Leal

Verónica García-Hernández

Victoria Moneo

Alberto Domingo

Juan Antonio Bueren-Calabuig

Ana Negri

María Federico Gago

José Guillén-Navarro

Pablo Avilés

Carmen Cuevas

Luis Francisco García-Fernández

MD Carlos María Galmarini
email: cgalmarini@pharmamar.com

Francisco Luis

García-Fernández

Molecular pharmacology and antitumor activity of in several human cancer cell lines

Keywords: Antineoplastic agents, Tetrahydroisoquinolines, DNA breaks, apoptosis

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This compound is structurally related to jorumycin, a natural compound isolated from the skin and mucus of the Pacific nudibranch Jorunna funebris [START_REF] Fontana | A New Antitumor Isoquinoline Alkaloid from the Marine Nudibranch Jorunna funebris[END_REF], and also to renieramycins isolated from sponges and tunicates [START_REF] Frinke | Antimicrobial Metabolites of the Sponge Reniera sp[END_REF][START_REF] Oku | a highly cytotoxic tetrahydroisoquinoline alkaloid, from a marine sponge Neopetrosia sp[END_REF], safracins and saframycins isolated from bacteria and marine sponges [START_REF] Kishi | Structure-activity relationships of saframycins[END_REF], and ecteinascidins isolated from marine tunicates [START_REF] Rinehart | Ecteinascidins 729, 743, 745, 759A, 759B and 770: potent antitumor agents from the Caribean tunicate Ecteinascidia turbinata[END_REF]. These carbinolaminecontaining compounds react, through an iminium intermediate that is generated by dehydration [START_REF] Kishi | Structure-activity relationships of saframycins[END_REF][START_REF] Guan | Molecular and Crystal Structures of Ecteinascidins: Potent Antitumor Compounds from the Caribbean Tunicate Ecteinascidia turbinata[END_REF][START_REF] Pommier | DNA sequence-and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata[END_REF][START_REF] Moore | NMR-based model of an Ecteinascidin 743-DNA adduct[END_REF], with the exocyclic amino group of selected guanines in the minor groove of DNA. The resulting adduct is additionally stabilized through the establishment of van der Waals interactions and one or more hydrogen bonds with neighboring nucleotides in the opposite strand of the DNA double helix [START_REF] Moore | NMR-based model of an Ecteinascidin 743-DNA adduct[END_REF][START_REF] Seaman | Molecular basis for the DNA sequence selectivity of ecteinascidin 736 and 743: evidence for a dominant role of direct readout via hydrogen bonding[END_REF], thus creating the equivalent to a functional interstrand crosslink [START_REF] Casado | Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin[END_REF] that can lead to strong inhibition of the early phases of transcription [START_REF] Magro | Characterization of tumor cell response to the novel anti-tumor agent Zalypsis®[END_REF][START_REF] Feuerhahn | Mechanistic insights into the effect of oxidative lesions and anticancer drugs on RNA pol II transcription and transcription coupled repair[END_REF].

In this study we characterized both molecular interactions with the DNA and the cytotoxic activity of Zalypsis ® in an attempt to shed light into its mechanism of antitumor action. The DNA binding characteristics were studied using a combination of electrophoretic mobility shift assays in polyacrylamide gels and a fluorescence-based method that employs tailor-made oligonucleotides. We also looked at the type of DNA damage subsequently generated in living cells as a consequence of Zalypsis ® -DNA adduct formation and whether cell cycle arrest and cell death induced by this compound mechanistically depend on caspase activation. Then, in vitro cytotoxic activity was evaluated in a panel of 24 different cancer cell lines. Finally, the in vivo antitumor activity of Zalypsis ® was investigated using several murine xenograft models of human prostate, stomach, bladder and pancreas carcinomas.

Materials and Methods

A c c e p t e d M a n u s c r i p t 4 2.1. Drugs. Zalypsis ® is a Pharmamar SAU proprietary drug synthesized at our facilities. ET-745 is a synthetic isoquinoline derived from trabectedin (ET-743) that lacks the carbinolamine group (Supplementary Material, Figure 1) and does not alkylate DNA [START_REF] David-Cordonnier | DNA and non-DNA targets in the mechanism of action of the antitumor drug trabectedin[END_REF]. Z-Vad-fmk is a pancaspase inhibitor purchased from Sigma (St Louis, MO, USA).

DNA electrophoretic mobility shift assay.

The binding assays were performed either with a 250-bp PCR product from the human adiponectin gene or with a double-stranded oligonucleotide (ds-oligo) synthesized by Sigma (St Louis, MO, USA). Briefly, appropriate concentrations of the compound were added at 25ºC during 1 hour to 250-bp DNA or ds-oligo.

Then, DNA was subjected to electrophoresis in a 2 % (w/v) agarose/TAE gel, stained with 1 µg/mL ethidium bromide and photographed. The sequences of the oligonucleotides used in the assay are the following: NonMeth-Fw 5'-TAAATATTTCCGGATATAATTAT-3'; NonMeth-Rev 5'-ATAATTATATCCGGAAATATTTA-3'; Meth-Fw 5'-TAAATATTTCMeCGGATATAATTAT-3'; Meth-Rev 5'-ATAATTATATCMeCGGAAATATTTA-3'.

DNA melting assay.

Synthetic oligodeoxynucleotides with one strand 5'-end-labeled with the fluorophore 6-carboxyfluorescein (6-FAM, F) and the complementary strand 3'-end-labeled with the quencher tetramethylrhodamine (TAMRA, Q) were synthesized at Bonsai Technologies (Madrid, Spain) (Supplementary Table 1). For the experiments, we followed the methodology previously described in a 7500 Fast Real-Time PCR System (ABI Prism, Applied Biosystems, Foster City, CA, USA) [START_REF] Casado | Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin[END_REF][START_REF] Negri | Antitumor activity, X-ray crystal structure, and DNA binding properties of thiocoraline A, a natural bisintercalating thiodepsipeptide[END_REF]. The raw data obtained were analyzed to estimate the increases in melting temperatures (∆T m ) brought about by drug binding as well as the ligand concentration that produces half the maximal change in melting temperature (C 50 ). The inverse of this value (1/C 50 ) was taken as a measure of the relative DNA binding affinity. We additionally used the parameter ∆T m (max) to reflect the relative stability of the DNA-ligand complexes [START_REF] Negri | Antitumor activity, X-ray crystal structure, and DNA binding properties of thiocoraline A, a natural bisintercalating thiodepsipeptide[END_REF]. The complete analysis was carried out using an in-house developed Visual Basic Application running on Microsoft Excel (Microsoft, Redmond, WA, USA).

A c c e p t e d M a n u s c r i p t 5 2.4. Computational methods. We studied in atomic detail the feasibility of achieving the geometries required to activate dehydration of the carbinolamine prior to the nucleophilic attack that leads to covalent bond formation. To this end, the initial structures of the precovalent complexes between Zalypsis ® and a series of oligonucleotides of general sequence 5'd(ATAATAXYZATAATA)/5'-d(TATTATZ'Y'X'TATTAT), where XYZ/Z'Y'X' stands for AGA/TCT, AGG/CCT and AGC/GCT, were built as previously reported for trabectedin [START_REF] Marco | Further insight into the DNA recognition mechanism of trabectedin from the differential affinity of its demethylated analogue ecteinascidin ET729 for the triplet DNA binding site CGA[END_REF]. The complexes were then refined using energy minimization techniques in the AMBER force field and their stability was studied by means of 5 ns of unrestrained molecular dynamics simulations in an explicit water box using the same conditions as described previously.

Cell culture and cytotoxicity.

All the tumor cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). For the cytotoxicity experiments, cells were seeded in 96-well trays. Serial dilutions of the compound dissolved in dimethyl sulfoxide (DMSO) were prepared and added to the cells in fresh medium, in triplicates.

Exposure to the compounds was maintained during 72 hours. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, MO, USA) was added to the cells and formazan crystals dissolved in DMSO. Absorbance at 540 nM was measured with a POLARStar Omega Reader (BMG Labtech, Offenburg, Germany). Determination of IC 50 values was performed by iterative non-linear curve fitting with the Prism 5.0 statistical software (GraphPad, La Jolla, CA, USA). The data presented are the average of three independent experiments performed in triplicate.

Fluorescent microscopy. Cells were treated with the appropriate concentration of

Zalypsis ® during 6 hours, washed out and cultured for 18 additional hours. Cells were then fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100) and incubated with the primary antiγ-H2AX monoclonal antibody (Upstate, Temecula, CA) for 1 h at 37 ºC. Then cells were washed and incubated with the secondary anti-mouse AlexaFluor 594 (Invitrogen, Carlsbad, CA, USA) A c c e p t e d M a n u s c r i p t 6 for 30 min at 37ºC. Finally the slides were incubated with Hoesch 33342 (Sigma, St Louis, MO, USA) and mounted with Mowiol mounting medium. Pictures were taken with a Leica DM IRM fluorescence microscope equipped with a 100x oil immersion objective and a DFC 340 FX digital camera (Leica, Wetzlar, Germany).

Comet Assay.

For the determination of double-strand breaks (DSBs) a single cell gel electrophoresis assay was used (Trevigen's CometAssay TM ), following the manufacturer's instructions after treatment of cells for 12 hours with the appropriate concentration of Zalypsis ® .

Pictures were taken with a Leica DM IRM fluorescence microscope equipped with a DFC 340 FX digital camera (Leica, Wetzlar, Germany). Quantitation of the DNA in the tails of the comets was performed with Adobe Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA, USA). For each condition 30 cells were analyzed, and the experiments were repeated several times.

Cell cycle analysis.

For the cell cycle experiments, cells were treated with the appropriate amount of the compound for 24 h, and then stained with 0.4 µg/ml propidium iodide. Samples were analyzed with a FACScalibur flow cytometer (Beckton and Dickinson, Franklin Lakes, NJ, USA) and the FlowJo7 cytometry analysis software.

2.9. Apoptosis. For the chromatin condensation assay, cells were treated with the appropriate amount of the compound for 24 hours and stained with DAPI. Early apoptotic cells show chromatin condensation that was assessed with a DM IRM microscope (Leica, Wetzlar, Germany). For the M30-Apoptosense solid-phase sandwich enzyme immunoassay (Peviva, Bromma, Sweden), the kit's instructions were followed. Briefly, cells were exposed to the appropriate concentration of the compound for 24 hours. Cells lysates were obtained and transferred to an assay well, M30 HRP conjugate added and incubated for 4 hours. Once the antigen-M30 was bound to the M5-coated surface of the wells, they were extensively washed and incubated with TMB substrate in the dark for 20 minutes. Finally, the reaction was stopped A c c e p t e d M a n u s c r i p t 7 with the addition of a stop solution and plates were read at 450 nM within 30 minutes with a Victor3 platform (Perkin Elmer).

Western Blot assays.

For immunoblotting, cells were treated with the appropriate concentration of the compound for 24 hours and lysed with RIPA lysis buffer. Protein content was determined by the modified Bradford method. Samples were separated in 7.5 % SDS-PAGE, transferred onto an Immobilon-P membrane. Then, membranes were incubated with the appropriate primary antibody 1h, washed and incubated with the secondary antibody. Finally protein was visualized using the ECL System (GE Healthcare, Fairfield, CT, USA). We used anti-PARP rabbit polyclonal, anti-p53FL rabbit polyclonal and anti-p21/WAF1 rabbit polyclonal antibodies from Santa Cruz (Santa Cruz, CA, USA), and an anti-α-tubulin monoclonal antibody from Sigma (St Louis, MO, USA). Secondary antibodies were HRP-conjugated goat anti-rabbit secondary antibody (R&D Minneapolis, MN, USA) and HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz, CA, USA).

Antitumor activity in

Results

Zalypsis ® binds covalently to DNA.

The mechanism of action of Zalypsis ® is expected to rely on the alkylation of selected guanines in the DNA double helix. Thus, we investigated its binding to DNA by means of band shift assays that allow the qualitative assessment of the molecular weight increase of Zalypsis ® -DNA adducts. Figure 1B shows a typical band shift experiment using increasing amounts of Zalypsis ® in order to saturate the 250-bp DNA probe with the compound. ET-745, which lacks the crucial hydroxyl group required for activation and covalent DNA modification, was used as a negative control (supplementary Figure 1). As expected, Zalypsis ® bound to naked DNA and delayed its electrophoretic migration in the gel.

Although 1 µM of Zalypsis ® was enough to produce a slight delay in band migration, DNA saturation required ~15 µM of the compound.

We further assessed whether Zalypsis ® could alkylate methylated DNA, using a ds-oligo containing a methylated cytosine in the sequence meCGG. Figure 1C shows the result of a typical experiment. Zalypsis ® bound to methylated and unmethylated CGG sequences with similar efficiencies, suggesting that methylation of the cytosine in a CpG sequence context in the major groove does not interfere with guanine modification in the DNA minor groove. 1). Figure 2A summarizes the results of the DNA melting experiments.

Zalypsis

The average 1/C 50 and ∆T m (max) values for Zalypsis ® were ~0.6 µM -1 and ~10 ºC, respectively. There were differences in the affinity of Zalypsis ® for the different triplets assayed, with the highest affinity being observed for GGC, AGC, AGG and TGG triplets, and the lowest, almost negligible, being displayed by the CGA triplet. Nonetheless, some of the selected triplets (e.g. AGC or AGG) do not provide a good binding site if embedded in an alternative sequence context (e.g. AGCA vs. AGCG and AGGA vs. AGGG). On the other hand, the highest stabilities were observed for adducts formed with the oligos containing the triplets AGA, TGT, AGG, GGC and TGG. Thus, according the affinity indicator 1/C 50 , the most favorable triplets for Zalypsis ® bonding to DNA appear to be AGG, GGC, AGC, CGG and TGG whereas the thermal stability of the DNA-adduct involving an AGA or TGT site is slightly above average and those involving CGC, CGT, GGT and CGA sites are well below average.

Molecular modeling data.

The molecular models of the equilibrated Zalypsis ® -DNA precovalent complexes in aqueous solution provided a rationale for the distinct binding of the drug to three representative central triplets that provide good (AGC), intermediate (AGG) or poor (AGA) binding sites for this drug, the only difference among them being the nature of the base pair on the 3'-side of the guanine that will undergo alkylation. As shown in Table 2, the geometry required for nucleophilic attack of the exocyclic amino group of the central guanine (G8) at the carbinolamine of the drug (C21) was rapidly attained in all cases, as assessed by an interatomic distance of less than 4 Å between the two reactive atoms. Likewise, the carbinol OH was seen to act as a hydrogen bond acceptor from N2(G8) and as a hydrogen bond donor to O2 in C21, that is, the cytosine pairing with G8 (Figure 2B). These findings are consistent with the fact that Zalypsis ® is able to react with these triplet sites, and the geometry that we find in these precovalent complexes lends strong support to the proposal that the latter hydrogen bond weakens the N12-H12 covalent bond, thus facilitating transfer of the H12 proton to the carbinol A c c e p t e d M a n u s c r i p t 10 OH, whereas the first hydrogen bond debilitates the C21-OH bond, thus favoring the exit of the water molecule. On the other hand, the protonated N12 of Zalypsis ® remained hydrogenbonded to the O2 acceptor atom of T22 in the AGG and AGC triplets but not in the AGA triplet (Figure 2C). This finding, together with the fact that AGA cannot provide a hydrogen-bonding partner to the methylenedioxy oxygen facing the minor groove may account for the fact that AGA is a poorer binding site even though the DNA in the resulting complex is stabilized to a greater extent than those containing the other sequence contexts studied (Figure 2A). When this hydrogen-bonding partner is found, as is the case for the AGC and AGG sequences which possess the amino group of either G20 or G9, respectively, the additional hydrogen bond formed (Figures 2B and2C) facilitates the full insertion of the drug into the minor groove and leads to enhanced reactivity. As regards the small stabilization brought about by Zalypsis ® on the CGA-containing oligonucleotide, this can be rationalized by proposing that it arises only from the precovalent complex, which cannot evolve into a covalent adduct because of an incorrect geometry for attack [START_REF] Negri | Antitumor activity, X-ray crystal structure, and DNA binding properties of thiocoraline A, a natural bisintercalating thiodepsipeptide[END_REF].

The initial Zalypsis ® -DNA adducts eventually give rise to double-strand breaks

(DSBs) in living cells. We analyzed the formation of γ-H2AX foci as a surrogate indicator of DSB formation [START_REF] Redon | Histone H2A variants H2AX and H2AZ[END_REF][START_REF] Guirouilh-Barbat | Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex[END_REF]. Figure 3A shows typical results of MCF7 and A549 cells treated with Zalypsis ® for 6 h at the indicated concentrations. At 5 nM, only a few MCF7 or A549 cells showed γ-H2AX staining and, in the positive cells, few foci were visible (data not shown). In contrast, at 25 nM, most of the cells were stained for γ-H2AX and each of the positive cells carried a large number of foci, indicating that several DSBs per cell were induced at this drug concentration (Figure 3A). At 100 nM, nearly every single cell stained positive for γ-H2AX, with a large number of foci per nucleus, again an indication of extensive DNA damage and induction of numerous DSBs (data not shown).

A c c e p t e d M a n u s c r i p t 11 Zalypsis ® -dependent formation of DSBs was additionally evaluated using the comet assay, which provides a direct measurement of damaged DNA. Both A549 and MCF7 cell lines were treated with Zalypsis ® at 10 and 100 nM for 24 hours and the percentage of the total DNA that was in the tail of the comets was measured. A typical comet assay result (Figure 3B) shows a clear concentration-dependent increase in DSBs in both cell lines following treatment with Zalypsis ® . The use of Mann-Whitney U test demonstrated that differences between each treated-measure against the baseline were statistically significant. 2 summarizes the results of the assessment of possible cell cycle perturbations after 24 h of exposure to Zalypsis ® . At both low (15 nM) and high (150 nM) drug concentrations there was a clear increase of the S-phase population in A549 (lung), HT29 (colon) and MDA-MB-231 (breast) cancer cells. Of note, the Sphase accumulation was higher at 15 nM, probably due to a higher cell survival. In IGROV-1 cells we did not observe any S-phase accumulation, most likely due to the extensive cell death induced by the compound at both concentrations after 24 h treatment (data not shown).

Zalypsis ® -treated cells accumulate in S-phase. Table

Zalypsis ® induces apoptosis in tumor cells. Since the antitumor mechanism of action of

DNA-damaging agents is known to involve induction of apoptosis [START_REF] Norbury | DNA damage-induced apoptosis[END_REF], we investigated whether Zalypsis ® treatment was able to induce a canonical apoptosis response in tumor cells. Firstly, we determined whether the drug was able to generate late apoptosis by detecting the flowcytometric subG 1 cell population (Table 2). A549 and HT-29 cell treatment resulted in an increase in the subG 1 population. On the other hand, MDA-MB-231 cells died with a slower kinetics while IGROV-1 cells died faster (Table 2). These data indicate that the percentage of cells entering the sub-G 1 peak after 24 hours of treatment with Zalypsis ® was cell type-and concentration-dependent. After treatment of MCF7 and A549 cells with 100 nM Zalypsis ® for 24 We further analyzed the onset of apoptosis using a commercially available solid-phase sandwich enzyme immunoassay that detects the caspase cleavage of cytokeratin-18 as a sensitive and specific marker of early canonical apoptosis [START_REF] Kramer | Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18[END_REF]. Treatment with 100 nM Zalypsis ® induced a marked increase of the apoptosis-related CK-18 Asp396 epitope in A549 cells. Moreover, simultaneous treatment with the pan-caspase inhibitor Z-VAD-fmk strongly reversed this effect (Figure 3C), which clearly indicates that the pro-apoptotic effect of Zalypsis ® was dependent on caspase activity.

Finally, we studied the caspase-dependent cleavage of the nuclear protein PARP as a marker of canonical apoptosis. The p85 band, generated through proteolytic cleavage of p116 PARP by activated caspases, was clearly visible as early as 6 hours after the treatment of MCF7 and A549 cells with 100 nM Zalypsis ® (Figure 3D). After 48 hours of treatment a considerable proportion of the PARP protein had already been cleaved by caspases, indicating that most of the cells were undergoing canonical apoptosis.

Zalypsis ® shows a potent cytotoxic activity.

The in vitro cytotoxicity of Zalypsis ® was determined using a panel of 24 tumor cell lines that represent eleven relevant types of human cancer (Table 1). Most of the cell lines were very sensitive to Zalypsis ® treatment, with IC 50 values in the low nanomolar range. The panel average IC 50 was ~7 nM, with MOLT-4 (acute lymphoblastic leukemia) cells showing the lowest IC 50 and CAKI-1 (renal carcinoma) and HCT116 (colon carcinoma) cells presenting the highest IC 50 s in the mid-nanomolar range. Control animals received an equal volume of vehicle. At the drug doses used in the experiment, no significant toxicity or body weight loss was observed in the treated animals. Tumors in mice treated with Zalypsis ® as a single agent strongly reduced their proliferation over the assay period, which finished when paired vehicle-treated animals had to be sacrificed (Figure 4).

Zalypsis ® exhibits in vivo

Tumor growth reduction ranged from 69% in the Hs746t gastric cancer cell line to 42% in the MiaPaCa-2 pancreas cancer cell line. Tumor growth reductions in the 22RV1 (human prostate cancer), and SW780 (human bladder cancer) xenografts treated with Zalypsis ® were 63% and 55%, respectively. The differences in tumor volumes between the vehicle-and the Zalypsis ®treated cohorts were statistically significant (p<0.05) from Day 16 (22RV1, prostate), Day 24 (SW780, bladder), Day 19 (Hs746t, gastric) and Day 22 (Mia PaCa-2, pancreas) until the end of the experiments (Figure 4).

Discussion

In the present work we introduce Zalypsis ® , a new synthetic tetrahydroisoquinoline alkaloid that induces DNA damage and apoptosis in a variety of cancer cell lines in the low nanomolar range and shows a potent antitumor activity in several mouse xenograft models.

We first investigated whether Zalypsis ® was able to bind either naked or methylated DNA through band shift assays, a methodology that has been previously used for similar analysis with other alkylating agents [START_REF] David-Cordonnier | DNA and non-DNA targets in the mechanism of action of the antitumor drug trabectedin[END_REF][START_REF] Marco | Further insight into the DNA recognition mechanism of trabectedin from the differential affinity of its demethylated analogue ecteinascidin ET729 for the triplet DNA binding site CGA[END_REF][START_REF] David-Cordonnier | Covalent binding of antitumor benzoacronycines to double-stranded DNA induces helix opening and the formation of single-stranded DNA: unique consequences of a novel DNA-bonding mechanism[END_REF]. Zalypsis ® was able to bind to both a naked PCR product of 250 bp with multiple guanines spread over its whole length and an oligonucleotide with a single binding site. This binding was dependent on the presence of the reactive carbinolamine moiety in the drug since ET-745, a related compound without this reactive group, A c c e p t e d M a n u s c r i p t 14 did not produce any band shifts at any of the concentrations tested. Furthermore, Zalypsis ® was able to bind to guanines that are preceded by a methylated cytosine (CpG natural methylation sites), demonstrating that methylation in the major groove does not affect binding of the drug to the minor groove.

Structurally, Zalypsis ® belongs to a family of compounds that bind to the exocyclic amino group of guanines in the DNA double helix through an iminium intermediate generated in situ by dehydration of the carbinolamine moiety present in the A-ring [START_REF] Kishi | Structure-activity relationships of saframycins[END_REF][START_REF] Guan | Molecular and Crystal Structures of Ecteinascidins: Potent Antitumor Compounds from the Caribbean Tunicate Ecteinascidia turbinata[END_REF][START_REF] Pommier | DNA sequence-and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata[END_REF][START_REF] Moore | NMR-based model of an Ecteinascidin 743-DNA adduct[END_REF]. The resulting adduct is additionally stabilized through van der Waals interactions and the establishment of one or more hydrogen bonds with surrounding nucleotides [START_REF] Moore | NMR-based model of an Ecteinascidin 743-DNA adduct[END_REF][START_REF] Seaman | Molecular basis for the DNA sequence selectivity of ecteinascidin 736 and 743: evidence for a dominant role of direct readout via hydrogen bonding[END_REF][START_REF] Garcia-Nieto | Bending of DNA upon Binding of Ecteinascidin 743 and Phthalascidin 650 Studied by Unrestrained Molecular Dynamics Simulations[END_REF]. In fact, hydrogen bonding rules seem to determine the binding sequence specificity of the tetrahydroisoquinoline family of drugs [START_REF] Seaman | Molecular basis for the DNA sequence selectivity of ecteinascidin 736 and 743: evidence for a dominant role of direct readout via hydrogen bonding[END_REF][START_REF] Marco | Further insight into the DNA recognition mechanism of trabectedin from the differential affinity of its demethylated analogue ecteinascidin ET729 for the triplet DNA binding site CGA[END_REF].

Considering the diversity of regulatory mechanisms that rely on specific DNA sequences, it can be speculated that sequence selectivity may drive therapeutic specificity for the different DNAbinding compounds. Thus, while guanines are the target of many agents that alkylate DNA, anthramycin selects AGA and AGG triplets [START_REF] Pierce | Comparison of sequence preference of tomaymycin-and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis[END_REF]; saframycins prefer GGG or GGC [START_REF] Rao | DNA sequence selectivities in the covalent bonding of antibiotic saframycins Mx1, Mx3, A, and S deduced from MPE.Fe(II) footprinting and exonuclease III stop assays[END_REF]; trabectedin favors TGG, CGG, AGC and GGC (while CGA is completely refractory, unlike the N12-demethylated analogue ET-729 [START_REF] Marco | Further insight into the DNA recognition mechanism of trabectedin from the differential affinity of its demethylated analogue ecteinascidin ET729 for the triplet DNA binding site CGA[END_REF]), and Zalypsis ® is shown here to have a preference for, GGC, AGC, AGG, TGG and CGG. Whether these subtle differences in sequence selectivity may eventually generate differences in activity against diverse tumor types, is presently not known. On the other hand, the average DNA stabilization brought about on the double helix by drug binding (∆T m (max)) was different for Zalypsis ® (~10 ºC) compared to Yondelis ® (~20 ºC) (supplementary Figure 1) [START_REF] Casado | Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin[END_REF] although it is still as high as that caused by a tight-binding bisintercalating agent such as echinomycin or thiocoraline [START_REF] Negri | Antitumor activity, X-ray crystal structure, and DNA binding properties of thiocoraline A, a natural bisintercalating thiodepsipeptide[END_REF]. The differences in reactivity and stabilization found for the rather comprehensive collection of alternative DNA triplets containing a central guanine could be rationalized on the basis of molecular models that showed the importance of specific hydrogen-bonding interactions involving drug and DNA atoms in the We then verified that Zalypsis ® induces DSBs in human cancer cells other than those from multiple myeloma for which γ-H2AX foci formation following exposure to this compound has been recently reported [START_REF] Ocio | Zalypsis: A novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double strand breaks[END_REF]. Indeed, our data show that drug treatment at nanomolar concentrations for 6 hours gives rise to a high proportion of positive cells with abundant γ-H2AX foci per cell, as well as comets with a considerable percentage of DNA in the tail. Taken together, these findings most likely indicate that the Zalypsis ® -DNA adducts are eventually transformed into DSBs in replicating cells. This is not surprising in light of similar findings recently reported for Yondelis ® [START_REF] Casado | Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin[END_REF] but there still might be some differences in the way both compounds induce these DSBs . In fact, it has already been demonstrated that in Yondelis ®treated cells, stalling of the Transcription-Coupled NER (TC-NER) machinery at the adduct position is largely responsible for the toxicity of the compound [START_REF] Erba | Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action[END_REF][START_REF] Takebayashi | Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair[END_REF]. In this action, the C-ring present in Yondelis ® , which protrudes from the DNA minor groove, has been proposed to be directly involved in a hydrogen-bonding interaction with a key basic residue of the NER endonuclease XPG [START_REF] Herrero | Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin[END_REF]. Since the ring in Zalypsis ® that sticks out of the DNA minor groove is very different, this type of interaction may not take place and the repair complex can be stalled at a different step or even proceed further allowing in this case the effective repair of the DNA adducts. Preliminary data support this view as fission yeast mutants defective in the XPG orthologue Rad13 have been shown to be partially resistant to Yondelis ® but not to Zalypsis ® [START_REF] Herrero | Resilience of the cytotoxic effects of Zalypsis® (pm00104) to the lack of a functional nucleotide excision repair system[END_REF]. We can then envisage the possibility that Zalypsis ® behaves as other alkylating/crosslinking agents giving rise to DNA adducts that escape repair by the NER machinery and eventually lead to replication fork stalls in the S-phase, followed by translesion synthesis (TLS) and generation of DSBs (reviewed in [START_REF] Andreassen | DNA damage responses and their many interactions with the replication fork[END_REF]). Moreover, the recent finding that Zalypsis ® inhibits transcription with a higher potency than Yondelis ® [START_REF] Magro | Characterization of tumor cell response to the novel anti-tumor agent Zalypsis®[END_REF][START_REF] Feuerhahn | Mechanistic insights into the effect of oxidative lesions and anticancer drugs on RNA pol II transcription and transcription coupled repair[END_REF] In this regard, the data presented here show that Zalypsis ® induces an S-phase arrest at both low and high nanomolar concentrations. During S-phase, cells are continuously checking the integrity of their DNA to ensure the accuracy of the copying process. If any alteration is found, there are two safety mechanisms (DNA damage and DNA replication checkpoints) that stop Sphase progression and coordinate the repair of damaged DNA [START_REF] Andreassen | DNA damage responses and their many interactions with the replication fork[END_REF][START_REF] Zhou | The DNA damage response: putting checkpoints in perspective[END_REF][START_REF] Hakem | DNA-damage repair; the good, the bad, and the ugly[END_REF]. Thus, our data suggest that DNA damage induced by this drug arrests cells in S-phase and activates the DNA replication checkpoints. This scenario is different from that obtained in multiple myeloma cells where Zalypsis ® treatment was described to induce an increased in the G 0 /G 1 phase [START_REF] Ocio | Zalypsis: A novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double strand breaks[END_REF].

Whether these effects are cell line-or disease-dependent is presently not known. In our experiments, the S-phase accumulation was higher at 15 nM than at 150 nM. Probably, this is due to a higher cell survival rate at the lower concentration. In fact, although some of the cells treated with high concentrations of Zalypsis ® would still accumulate in S-phase, most of them would stop in the cell cycle phase in which they originally were at the onset of drug treatment, due to the high amount of DNA damage that they endure, and they would die with a slower kinetics. Our data also show that Zalypsis ® -induced cell death presents the characteristics of apoptosis, with chromatin condensation, dependence on caspase activity and cleavage of the caspase targets PARP-1 and cytokeratin 18. This is in accordance with data obtained in multiple myeloma cell lines on which Zalypsis ® treatment induced a strong apoptotic response that was partially dependent on caspase activation [START_REF] Ocio | Zalypsis: A novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double strand breaks[END_REF].

When assayed in a panel of 24 cancer cell lines, Zalypsis ® demonstrated a potent cytotoxic activity, with an average IC 50 of 7 nM in most cell lines, with the notable exception of HCT116 and CAKI-1, both of which are substantially less sensitive to the drug. Myeloma cell lines have also been recently shown to be highly sensitive to Zalypsis ® , with IC 50 values ranging from A c c e p t e d M a n u s c r i p t picomolar to low nanomolar concentrations [START_REF] Ocio | Zalypsis: A novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double strand breaks[END_REF]. Other alkaloids of the tetrahydroisoquinoline family, including jorumycin, renieramycins and saframycins, have been previously shown to possess strong cytotoxic activity, with IC 50 s also in the low nanomolar range [START_REF] Oku | a highly cytotoxic tetrahydroisoquinoline alkaloid, from a marine sponge Neopetrosia sp[END_REF][START_REF] Arai | The structure of a novel antitumor antibiotic, saframycin A[END_REF][START_REF] Lane | Antitumor activity of tetrahydroisoquinoline analogues 3-epi-jorumycin and 3-epi-renieramycin G[END_REF].

According to our data, Zalypsis ® exhibits higher cytotoxic activity against leukemia and stomach tumor cell lines, with breast cancer cell lines being less sensitive than the average. This tissue/cell-type sensitivity pattern is clearly different from that observed for Yondelis ® as this ecteinascidin shows selectivity against sarcoma, breast, ovary and lung tumor cell lines, with head-and-neck and colon tumor cell lines being slightly less sensitive [START_REF] Poindessous | Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin[END_REF][START_REF] Koeppel | Irofulven cytotoxicity depends on transcription-coupled nucleotide excision repair and is correlated with XPG expression in solid tumor cells[END_REF].

Finally, the in vitro cytotoxicity of Zalypsis ® translated well into antitumor activity in vivo in four xenograft models of human cancer. The models analyzed corresponded to cell lines ranging from slightly more sensitive (Hs746T, gastric cancer) to substantially less sensitive (SW780, bladder cancer) than the average in the in vitro cytotoxicity assays. Zalypsis ® demonstrated a clear antitumor activity that was highest for the Hs746t gastric cancer model, with a tumor growth reduction of 69%. The pancreatic cancer model MiaPaCa was the least sensitive to Zalypsis ® but, even in this case, the compound achieved a 42% tumor growth reduction. When we assayed the maximum tolerated dose of the drug in two different administration regimes no significant differences among them and no toxic effects to the animals were observed.

In summary, we have demonstrated highly potent in vitro and in vivo anticancer activities of Zalypsis ® and gained some insight into its molecular mechanism of action. This agent exerts its anticancer effects through the covalent modification of guanines in the DNA minor groove that eventually give rise to DNA double-strand breaks, S-phase arrest and apoptosis in cancer cells.

These encouraging results strongly support its development as a novel anticancer agent with a view to a wide spectrum of clinical settings.

-

  antitumor activity of Zalypsis® in several human cancer cell lines. (Leal et al. BCP-D-09-00162Formation of DNA adducts -Detection of most favorable DNA triplets for covalent adduct formation -Rationalization of these triplets by molecular modeling -Induction of double-strand DNA breaks -Triggering of S-phase accumulation -Induction of apoptotic cell death A c c e p t e d M a n u s c r i p t 1. Introduction Zalypsis ® (Figure 1A) is a new synthetic dimeric isoquinoline alkaloid that is currently in Phase I clinical development for the treatment of solid tumors and hematological malignancies.

  xenograft murine models. Four to six weeks-old athymic nu/nu mice (Harlan Sprague Dawley, Madison, WI, USA) were s.c. xenografted into their right flank with ca. 0.5-1 x 10 7 cells in 0.2 mL of a mixture (50:50; v:v) of Matrigel basement membrane matrix (Beckton Dickinson, Frankin Lakes, NJ, USA) and serum-free medium. When tumors reached ca. 150 mm 3 , mice were randomly assigned into treatment or control groups. Zalypsis ® was intravenously administered either in 3 consecutive weekly doses (0.9 mg/kg/day) or in 2 cycles of 5 consecutive daily doses (0.3 mg/kg/day) in pancreas or in breast and prostate xenograft studies, respectively. Control animals received an equal volume of vehicle. Caliper measurements of the tumor diameters were done twice weekly and tumor volumes calculated according to the following formula: (a•b) 2 /2, where a and b were the longest and shortest diameters, respectively. Animals were humanely euthanized, according to Institutional Animal Care and Use Committee of PharmaMar, Inc. (Cambridge, MA, USA) guidelines, when their A c c e p t e d M a n u s c r i p t 8 tumors reached 3,000 mm 3 or if significant toxicity (e.g., severe body weight reduction) was observed. Differences in tumor volumes between treated and control groups were evaluated using the unpaired t-Test. Statistical significance was defined as p<0.05. Statistical analyses were performed by LabCat ® v8.0 SP1 (Innovative Programming Associates, Inc. NJ, USA). All animal studies were conducted under approval from an IACUC in an AAALAC accredited animal facility.

9 (

 9 ® binds to guanines in selected DNA triplets. To investigate the ability of Zalypsis ® to stabilize the double helix, we designed several fluorophore-labeled oligonucleotides encompassing most of the possible DNA triplet combinations involving a central guanine A c c e p t e d M a n u s c r i p t Supplementary Table

A

  c c e p t e d M a n u s c r i p t 12 hours, an apoptotic nuclear morphology with strong chromatin condensation was clearly visible in fluorescent microscopy in both cell types (data not shown).

  antitumor activity in xenograft murine models. We performed xenograft studies to test whether the cytotoxicity of Zalypsis ® translates into in vivo antitumor activity. 22RV1 (prostate), SW780 (bladder), Hs746t (gastric), and Mia PaCa-2 (pancreas) cells were xenografted into the right flank of athymic nu/nu mice. Once the tumors A c c e p t e d M a n u s c r i p t 13 reached ca. 150 mm 3 , the mice were randomized into groups of 10 and Zalypsis ® was intravenously administered either in 3 consecutive weekly doses (0.9 mg/kg/day) (prostate and bladder) or in 2 cycles of 5 consecutive daily doses (0.3 mg/kg/day) (gastric and pancreas).

  A c c e p t e d M a n u s c r i p t 15 minor groove for attaining a suitable geometry for nucleophilic attack and covalent adduct formation.

  may indicate that A c c e p t e d M a n u s c r i p t 16 this latter drug principally targets TC-NER-dependent DNA repair whereas Zalypsis ® blocks transcription through a stalled initiation or early elongation complex.

Figure 1 .

 1 Figure 1. Cytotoxic effect and DNA Binding properties of Zalypsis ® . A: Structure of Zalypsis ® showing the positions of C21 and N12 atoms discussed on the main text. B: Binding

Figure 2 .

 2 Figure 2. Characteristics and modeling of DNA-Zalypsis ® complexes. A: Relative binding affinities for different DNA triplet sequences. Zalypsis ® was incubated with the labeled ds-oligos

Table 1 .

 1 Cytotoxicity of Zalypsis® in a panel of 24 human cancer cell lines*.

		Cell line	IC 50 (nM)	Tissue Average (nM)
	Prostate	PC3	2.4 ± 1.1	2.1 ± 0.4
		22RV1	1.7 ± 0.8
	Pancreas	PANC-1	4.1 ± 0.8	2.7 ± 1.9
		MiaPaCa-2	1.3 ± 0.4
	Ovary	IGROV-1	3.5 ± 0.1	2.8 ± 0.9
		A2780	2.1 ± 1.3
	Lung	NCI-H460	3 ± 1.2	2.5 ± 0.4
		NCI-H23	2.2 ± 0.3
		A549	2.4 ± 0.8
	Liver	SK-HEP-1	2.5 ± 0.8	2.5 ± 0.1
		HEPG2	2.6 ± 1.5
	Leukemia	MOLT4	0.5 ± 0.2	1 ± 0.7
		K562	1.6 ± 1.5
	Kidney	RXF393	2.1 ± 0.5	21.9 ± 27
		CAKI-1	41 ± 5
	Stomach	HS746T	1.5 ± 0.1	1.6 ± 0.07
		HGC-27	1.6 ± 0.5
	Colon	LoVo	2.4 ± 0.8	23 ± 34
		HT29	3.8 ± 0.2
		HCT-116	62 ± 30
	Bladder	SW780	10 ± 0	10
	Breast	MDA-MB-231	4.8 ± 0.8	4.1 ± 0.9
		MCF-7	4.5 ± 0.1
		BT-474	3 ± 1.2
	* Values represent mean ± SD of three different experiments

Caspase activation and PARP-1 processing. MCF7 and A549 cells were treated with Zalypsis ® 100 nM during 24 hours and then washed and lysed with RIPA buffer supplemented with a protease inhibitor cocktail. Western blot was then performed using a primary anti-PARP-1 polyclonal antibody.


Antonello A Romani

Silvia Desenzani

Marina M Morganti

Silvia La Monica

MD Angelo F Borghetti
email: angelo.borghetti@unipr.it

Paolo Soliani

Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells 1*

Keywords: Cholangiocarcinoma, Zoledronic acid, Cyclins, Cell cycle regulators, Bcl-xL, GGOH

ABSTRACT

Cholangiocarcinoma is the second most common primary hepatic neoplasia and the only curative therapy is surgical resection or liver transplantation. Biphosphonates (BPs) are an emerging class of drugs widely used to treat bone diseases and also appear to possess direct antitumor activity. In two human cholangiocarcinoma cell lines (TFK-1 and EGI-1) we investigated, for the first time, the activity of zoledronic acid by determining proliferation, cell-cycle analysis and apoptosis.

The results obtained indicate that zoledronic acid induces cell-narrowing and growth inhibition, both reversed by 25 µM GGOH, and significantly affects the colony-forming ability of these cells. The inhibition by zoledronic acid of Rap1A prenylation was reversed in cell co-treated with GGOH. At 10-50 µM zoledronic acid exerted an S-phase cell cycle arrest which was confirmed by changes in the level of cyclins and of regulators p27 KIP1 and pRb. Interestingly, the expression level of cyclin A (putative S-phase marker) shows a dose-dependent increment in contrast to the decrement of cyclin D1 (putative G1-phase marker). However, neither hypodiploid cells nor cleaved PARP or caspase-3 were detected. The lack of TP53 or loss of its function, the large constitutive expressions of antiapoptotic proteins Bcl-xL and HSP27 together with the low level of the proapoptotic Bax are the likely factors which protect cells from apoptosis.

In conclusion, our study indicates that zoledronic acid induces S-phase arrest and cell-narrowing, both reversed by GGOH and, by changing the delicate balance between pro-apoptotic and antiapoptotic proteins, allows survival of cholangiocarcinoma cells.

Running title: S-phase arrest by zoledronate in cholangiocarcinoma

Introduction

Cholangiocarcinoma are malignant tumors that derive from bile duct epithelium and are classified anatomically into intra-and extra-hepatic cholangiocarcinoma. The incidence and the mortality rates of cholangiocarcinoma are increasing worldwide [START_REF] Khan | Cholangiocarcinoma[END_REF]. At present, complete resection is the only way to cure this disease, but at the time of diagnoses more than 60% of patients have occult metastases or an advanced local disease which preclude any curative resection. Conventional anticancer treatments, such as chemotherapy or radiotherapy have minimal impact on patients survival with unresectable tumor [START_REF] Blechacz | Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment[END_REF][START_REF] Poplin | Leucovorin, 5-fluorouracil, and gemcitabine: a phase I study[END_REF][START_REF] Pazdur | Phase II trial of docetaxel for cholangiocarcinoma[END_REF][START_REF] Thongprasert | Phase II study of gemcitabine and cisplatin as first-line chemotherapy in inoperable biliary tract carcinoma[END_REF]. Up to now, the factors responsible for this relative unresponsiveness have not been clearly understood. In general, the efficacy of conventional anticancer therapies is strongly dependent on their ability to initiate programmed cell death (apoptosis) in cancer cells [START_REF] Dive | Avoidance of apoptosis as a mechanism of drug resistance[END_REF][START_REF] Eastman | Apoptosis: a product of programmed and unprogrammed cell death[END_REF][START_REF] Huether | Sorafenib alone or as combination therapy for growth control of cholangiocarcinoma[END_REF]. Therefore, cancer cells that have evolved ways to circumvent apoptosis become resistant, providing an obstacle to effective treatments [START_REF] Hannun | Apoptosis and the dilemma of cancer chemotherapy[END_REF][START_REF] Romani | The associated expression of Maspin and Bax proteins as a potential prognostic factor in intrahepatic cholangiocarcinoma[END_REF].

Previous researchers have shown that zoledronic acid (ZOL), third generation of biphosphonates (BPs), exerts powerful antitumoral activity [START_REF] Clezardin | Anti-tumour activity of zoledronic acid[END_REF] in several human neoplasms [START_REF] Clezardin | Anti-tumour activity of zoledronic acid[END_REF] such as human myeloma [START_REF] Aviles | Antitumor effect of zoledronic acid in previously untreated patients with multiple myeloma[END_REF], breast [START_REF] Gralow | Evolving role of bisphosphonates in women undergoing treatment for localized and advanced breast cancer[END_REF] and prostate [START_REF] Hoesl | Biphosphonates in advanced prostate and renal cell cancer-current status and potential applications[END_REF][START_REF] Melisi | Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer[END_REF] cancers. BPs are currently the most important class of inhibitors of osteoclast-mediated bone resorption and are used extensively for the treatment of skeletal diseases, such as Paget's disease, postmenopausal osteoporosis, and tumor-induced osteolysis [START_REF] Rogers | Cellular and molecular mechanisms of action of bisphosphonates[END_REF][START_REF] Rodan | Bone mass homeostasis and bisphosphonate action[END_REF]. The antiproliferative effect of pamidronate in human and rat osteosarcoma cells has recently been demonstrated in vitro [START_REF] Sonnemann | The bisphosphonate pamidronate is a potent inhibitor of Ewing's sarcoma cell growth in vitro[END_REF][START_REF] Mackie | Bisphosphonates regulate cell growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line[END_REF]. BPs can be divided into two distinct pharmacological classes based on their molecular mechanisms of action [START_REF] Russell | Bisphosphonates: mode of action and pharmacology[END_REF]. Non-nitrogencontaining BPs, such as clodronate and etidronate, are metabolized intracellularly as cytotoxic analogues of ATP and thus inhibit osteoclast activity [START_REF] Toyras | Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro[END_REF]. In contrast, the newer nitrogen-A c c e p t e d M a n u s c r i p t 4 containing BPs such as alendronate, residronate, pamidronate, and ZOL inhibit cell proliferation and induce apoptosis in osteoclasts [START_REF] Luckman | Nitrogencontaining bisphosphonates inhibit the mevalonate pathway and prevent posttranslational prenylation of GTP-binding proteins, including Ras[END_REF] by inhibiting farnesyl-PP-synthase of the mevalonate synthesis, a biosynthetic pathway responsible for the production of cholesterol and isoprenoid lipids, particularly farnesyl-and geranylpyrophosphates. These are required for the posttranslational modification (prenylation) of small GTP proteins (such as Rho, Ras, Rac) that play crucial roles in signaling pathways controlling cell growth. The anti-proliferative effect of ZOL was described in several tumor cell lines and it was likely due to cell cycle distribution change by accumulating and arresting cells in the S-phase [START_REF] Forsea | Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells[END_REF][START_REF] Iguchi | Zoledronate-induced S phase arrest and apoptosis accompanied by DNA damage A c c e p t e d M a n u s c r i p t 25 and activation of the ATM/Chk1/cdc25 pathway in human osteosarcoma cells[END_REF][START_REF] Kubista | Anticancer effects of zoledronic acid against human osteosarcoma cells[END_REF][START_REF] Kuroda | p53independent anti-tumor effects of the nitrogen-containing bisphosphonate zoledronic acid[END_REF][START_REF] Ory | Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status[END_REF]. This arrest was coupled to changing levels of cyclins and cell cycle regulators, and the S-phase arrest was associated with typical (caspase-dependent) [START_REF] Kubista | Anticancer effects of zoledronic acid against human osteosarcoma cells[END_REF][START_REF] Ory | Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status[END_REF][START_REF] Sewing | The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis[END_REF] or atypical (caspase-independent) apoptotic pathway. To date, however, the detailed molecular mechanism of cell cycle arrest involved remains not fully understood.

Since most of patients with cholangiocarcinoma present at diagnoses with unresectable disease or the occurrence, in this cancer, of high-rate of post-resection relapse, the current adjuvant or palliative option is of limited benefit. It is mandatory, therefore, to evaluate new potential anticancer drugs. ZOL, due to its mechanism of action so far highlighted, could represent a reliable chemotherapeutic agent able to sensitize cells to cytotoxic agents and/or radiations.

Here we investigated, for the first time on two cholangiocarcinoma cell lines, the role of ZOL on cell morphology, proliferation, cycle phases, and apoptosis to regard its potential use in cholangiocarcinoma therapy. from Gibco-BRL (Grand Island, NY, USA). Antibodies against Bcl-xL (H-5), Bax (P-19), TP53 (DO-1), Rap1A (SC-1482, unprenylated form) were obtained from Santa Cruz Biotechnology (Santa Cruz, Temecula, CA, USA). Monoclonal antibody against HSP27 (G3.1) was from StressGene (Victoria, BC Canada); caspase-3, PARP, cyclin A, cyclin B1, cyclin D1, pRb1, Rb, and p27 antibodies were from Cell Signaling Technologies (Beverly, MA, USA); monoclonal anti-actin (AC-40) antibody was from Sigma Aldrich. HRP-conjugated secondary antibodies (Pierce, Rockford, IL, USA) and the enhanced chemiluminescence system (ECL) were from Millipore (Millipore Co, Billerica, MA, USA). Reagents for electrophoresis and blotting analysis were obtained from BIO-RAD Laboratories (Richmond, CA, USA).

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

Cell culture

The cholangiocarcinoma cell lines EGI-1 and TFK-1 were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, GERMANY) and maintained in RPMI-1640 medium, containing antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) and supplemented with 10% FCS and with 2 mmol/L glutamine. Cultures were kept in an incubator at 37°C in a water-saturated 5% CO 2 atmosphere in air. Routine sub-cultivations were carried out every week. Cell morphology was assessed on May-Grünwald stained cells.

MTT assay

Viability of EGI-1 and TFK-1 cells was evaluated by [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium] (MTT) (Sigma-Aldrich) assay. 5000 cells/well were seeded into 96-well plates and left to adhere overnight. The cells were then incubated for 3 days in the absence or in the presence of 1 to 1000µM ZOL. Then 100 µl of 1 mg/ml MTT were added for 1 h. After removal of the MTT, cells were treated with 100 µl DMSO and formazan product was solved.

The absorbance was determined at 595 nm in a multiwell plate reader (BIO-RAD, Microplate Reader 555). Drug-induced proliferation changes were calculated as percentage of cell growth inhibition using the formula: (1-extinction of treated sample x 100) / (extinction of control sample). The half-maximal inhibition constant (IC 50 ) was determined using the nonlinear regression program CalcuSyn (Biosoft, Cambridge, UK).

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

Clonogenic assay

Colony formation was determined by seeding cells into a six-well culture plates at a density of 50 cells/cm 2 well and left to adhere overnight. The cells were then incubated in the absence or in the presence of 2-32µM ZOL. After 10 day-incubation, the wells were washed twice with PBS, and fixed with 1% formaldehyde. They were then stained with 0.1% crystal violet. The colony is defined to consist of at least 40-50 cells. For evaluation of cell growth, colonies were examined under a light microscope.

Western blotting

Total proteins were extracted by lysing the cells with a buffer containing 50mM Tris-HCL, pH 7.4, 150mM NaCl, 5mM MgCl 2 , 0.5mM EDTA, 0.1% SDS and protease inhibitor cocktail followed by ultracentrifugation at 13,000 rpm for 10 min. Protein concentration was determined using a protein assay from BIO-RAD according to the manufacturer's protocol, using BSA as standard. Proteins (50µg) were resolved by SDS/PAGE (12%) and blotted onto PVDF membranes (Millipore). Immunodetection was carried out in 50mM Tris-HCl, pH 7.4, 150mM

NaCl, 0.1% Tween 20, 5% non-fat dry milk, followed by HRP conjugated secondary antibodies at 1:20000 dilution. Detection was performed using an ECL kit.

Flow cytometric cell cycle and morphological analysis

Cholangiocarcinoma cells were incubated with 10 and 50µM ZOL for 72 h. For the cell cycle assay EGI-1 and TFK-1 cells were harvested and subjected centrifugation, 5x10 5 cells were

Growth inhibitory effects

In order to test the effect of ZOL on cholangiocarcinoma cell growth, EGI-1 and TFK-1 cell lines were treated with increasing concentration of ZOL (1 to 1000µM) for 72h, followed by an MTT assay (see Material and Methods section). As revealed in Figure 1, after 72h of ZOL treatment cholangiocarcinoma cells significantly reduced their growth in a dose dependent manner. In fact, the tumor cell proliferation was reduced to about 21% and 50% for TFK-1 and EGI-1, respectively, at ZOL concentration of 10µM. Then inhibition increased to 59% in TFK-1 and 83% in EG1-1 at 33µM. Higher concentrations of drug further inhibited proliferation of both cell lines. The mean IC 50 estimated from three independent experiments gave a concentration of 37µM (C.I 95%: 28.2-47.9) for TFK-1 cells and of 17µM (C.I 95%: 10.5-29.5µM) for EGI-1 cells.

Rescue by GGOH of morphological and proliferation changes induced by ZOL

Besides the significant inhibition of proliferation induced by ZOL at relatively high concentrations, concomitant alterations of cell morphology occurred. Control cultures (Figure 2A) show typical morphological variations of cholangiocarcinoma cells from cuboidal to-lowcolumnar epithelial cells with rounded to polygonal nuclei. Subsequent to 50 µM ZOL treatment long and narrowed cells resembling fibroblasts coupled with a marked reduction of cell number are observed in EGI-1 cells. Similar changes, even if less marked were observed also in TFK-1 cells. To substantiate the mechanism of ZOL activity GGOH, an acyclic isoprenoid substrate of and EGI-1 cells, respectively. Moreover, GGOH counteracted the ZOL-induced unprenylation of Rap1A in EGI-1 and TFK-1 cells (Figure 2C). This ability of GGOH to alleviate/rescue ZOL toxicity could be ascribed to cell replenishing of substrates for protein prenylation.

Loss of clonogenic survival

Because of the pronounced cytostatic effects of ZOL on cell growth observed on monolayer cultures, we then verified if ZOL could affect the ability of EGI-1 and TFK-1 cells to form colonies in vitro. Clonogenic assay, based on the ability of a single cell to grow into a colony, is the method of choice to determine the effectiveness of cytotoxic agents. After 10 days in culture both EGI-1 and TFK-1 cells, seeded at 50 cells/cm 2 , formed a large number of colonies clearly visible at naked eye (Figure 3). However, when cells were grown for ten days in the presence of 8µM ZOL, number and size of TFK-1 and EGI-1 colonies were markedly reduced. When treated with 16µM ZOL, both cell lines did not form visible colonies. These results clearly indicate that the ZOL treatment determines a long-lasting cell cycle arrest of cholangiocarcinoma cells suppressing their colony-forming ability in a dose-dependent manner.

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

Cell cycle phase distribution

We then investigated the effect of ZOL on the cell cycle phase distribution of cholangiocarcinoma cells following treatment with this drug over a 72h period. The analysis (Figure 4) revealed the cell-cycle distribution of untreated control cells showing an average of 70 and 62% cells in G1, 9 and 11% in G2/M, and 21 and 28% in S phase for EGI-1 and TFK-1, respectively. It's interesting to note that when EGI-1 cells were treated with 10 µM ZOL for 72 h, the proportion of cells in G1 was reduced to 34% and 55% of the cells occurred in S phase, whereas only at 50 µM ZOL a marked accumulation of TFK-1 cells in S phase (71%) occurred.

As evident from this figure, the proportion of cells in the G2/M phase did not change significantly indicating no additional arrest in this phase. In EGI-1 cells a distinct loss of cell in G2/M phase at the highest drug concentration (50 µM) was detectable. Taken together, these results suggest that ZOL treatment for 72h induced the entrapment of cholangiocarcinoma cells in the S phase, resulting, therefore, in an effective reduction of cells capable to complete cell-cycle, and thus leading to a diminished cell proliferation. Interestingly, in both cholangiocarcinoma cell lines, ZOL treatment did not induce any hypodiploid sub-G1 peak, a feature indicative of apoptotic cell death (Figure 4, upper panel). It's worth to note that 48 h co-treatment with GGOH partially rescues the cell-cycle phase distribution seen in ZOL-exposed cholangiocarcinoma cells (data not presented).

Expression of putative phase markers and cell cycle regulators

We then examined the mechanisms of ZOL induced S-phase arrest by focusing on the expression level of phase-specific cyclins and the cell cycle regulators such as tumor suppressor pRb and The first checkpoint at G1/S is often compromised in malignant cells due to deletion or loss of function of TP53, guardian of the genome, and/or mutation of pRb, main regulator of the cell cycle. As shown in Figure 5, while we confirmed the absence of TP53 in TFK-1 cells as known from literature data, its overexpression in EGI-1 cells is likely to be ineffective due to the presence of one deleterious transition mutation (G>A, R273H) in the DNA binding domain that prevents its binding to DNA [START_REF] Olivier | The IARC TP53 database: new online mutation analysis and recommendations to users[END_REF]. pRb protein hinders the cell from replicating damaged DNA by preventing its progression along the cell cycle through G1 into S. In the hypophosphorylated state, pRb protein is active and carries out its role as tumor suppressor by inhibiting cell cycle progression. Phosphorylation inactivates pRb. As shown in Figure 5, in both untreated cell lines the hyperphosphorylation status of pRb (SER 780) occurred and drug treatment did not affect the status of phosphorylation, except only in EGI-1 cells in the presence of 50µM ZOL. The hyperphosphorylation status of pRb (SER 780) in both treated cell lines associated with a deletion (TFK-1) or deleterious mutation (EGI-1) of TP53 greatly suggests that the first checkpoint G1/S is compromised allowing unrestrained cells freely entering in the next S-phase.

We then examined the cyclins, putative markers of cell cycle phases and regulatory subunits of the cyclin-dependent kinases (cdks), whose levels peak the specific stages of the cell cycle. The first cyclin level examined was cyclin D1, which is maximally expressed in mid-to-late G1 phase and its kinase activity, elicited by binding with cdk4 (predominantly cyclin D1/cdk4 complex), is up-regulated in cancer cells [START_REF] Sherr | Cancer cell cycles[END_REF]. As shown in Figure 6, treating cells for up to 72 h with 10-100

A c c e p t e d M a n u s c r i p t 13 µM ZOL resulted in a marked decrease of cyclin D1 in EGI-1 cells and only at higher concentrations in TFK-1. We further verified the status of p27 KIP1 whose function is to bind to cyclin D either alone, or when complexed to its catalytic subunit cdk4. In this way, p27 Kip1 inhibits the catalytic activity of cdk4, preventing cdk4 from adding phosphate residues to pRb protein. Increased levels of the p27 Kip1 protein typically cause cells to arrest in the G1 phase of the cell cycle. Upon ZOL treatment a dose-dependent decrease in the level of p27 inhibitor was observed in both cell lines, more evident in EGI-1 cells. The p16 INK4A , which shares with pRb and p27 a regulatory role at G1/S checkpoint, is frequently mutated or deleted in a wide variety of tumors, in particular in bile duct cancers, and not expressed in the cholangiocarcinoma cell lines used in this work [START_REF] Caca | Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct A c c e p t e d M a n u s c r i p t 26 cancers and bile tract cancer cell lines[END_REF][START_REF] Tannapfel | Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver[END_REF]. In several human malignancies, the loss, mutation or silencing of the gene encoding the CDK4 inhibitor p16 INK4a hyperactivates the kinase activity of CDK4 [START_REF] Borg | High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families[END_REF][START_REF] Nobori | Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers[END_REF][START_REF] Spruck | p16 gene in uncultured tumours[END_REF]. Next, cyclin A expression is important for progression through the S phase and G2/M transition [START_REF] Pagano | Cyclin A is required at two points in the human cell cycle[END_REF]. Cyclin A, in contrast to cyclin D1, showed significant increases in both cell lines following cell exposed to ZOL and thus indicating a slow-down and/or an arrest in the S-phase.

Moreover, cyclin B1, in complex with cdk1, functions as an M-phase kinase [START_REF] Dunphy | The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis[END_REF]. No changes of cyclin B1 level were observed in both cell lines except a distinct drop at 50µM ZOL in EGI-1 cells. Finally, we analyzed the phospho-histone H3, a marker of cromatin condensation. The Western blotting analysis indicated that ZOL increases the level of this M-phase marker. This increment was in agreement with the increased level of cyclins A.

These findings on the significant changes in the levels of specific cell cycle cyclins and inhibitors confirm the results observed with cell-cycle analysis and further indicate a ZOL interference with the cell cycle progression machinery at the putative S-phase check point.

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

Apoptosis assessment

As shown in Figure 4, in both cholangiocarcinoma cell lines, ZOL treatment did not induce any apoptotic sub-G1 peak. In order to confirm the absence of apoptotic death, we examined the morphological and biochemical features of apoptosis. The cells were stained with Hoechst 33342 and propidium iodide. Condensed chromatin or apoptotic cells were rarely seen in all the cell populations studied: only 8-12% of control cells and of tested cells treated with the drug up to 25µM were homogeneously and intensively stained by propidium iodide indicating cell death.

None evident characteristics of apoptosis (such as cell shrinkage, apoptotic bodies, cytoplasmic blebs, chromatin collapse into patches and dense spheres) were observed. Only when EGI-1 cells were treated with 50µM, approximately 10% of cells showed some apoptotic characteristics (not shown).

We then investigated the occurrence of apoptosis by biochemical criteria such as activation of caspases or presence of cleavage products of their substrates. Figure 6A shows that none effect on caspase-3 activation occurred after cells exposure to ZOL. Moreover, the drug did not induce cleavage of PARP, a key enzyme in the apoptotic cascade and substrate of active caspase-3.

To investigate if the cell-death resistance is specific to zoledronic acid, we evaluate the effect of staurosporine, gemcitabine and nitrogen (pamidronate) and a non-nitrogen (clodronate) containing bisphosphonate on caspase-3 and PARP activation. Experiments using the abovementioned drugs indicated that staurosporine on EGI-1 cells and gemcitabine on both cell lines were able to induce caspase-3 and PARP activation. In contrast and similarly to ZOL, pamidronate and clodronate did not activate caspase-3 nor PARP (Figure 6B).

The analysis of the expression of anti-versus pro-apoptotic proteins indicated that untreated TFK-1 cells markedly express the anti-apoptotic Bcl-xL and at greater extent the pro-apoptotic Bax protein, accordingly to literature data [START_REF] Okaro | The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium[END_REF][START_REF] Okaro | Pk11195, a mitochondrial benzodiazepine receptor antagonist, reduces apoptosis threshold in Bcl-X(L) and Mcl-1 expressing human cholangiocarcinoma cells[END_REF]. As shown in Figure 6C, in TFK-1 cells ZOL treatment significantly affected the balance of anti-apoptotic versus pro-apoptotic proteins; it is also interesting to note the marked decrease of Bax coupled to the increasing concentrations of ZOL. Moreover, in EGI-1 cells ZOL treatment did not affect the ratio of Bcl-xL/Bax, already shifted versus the anti-apoptotic Bcl-xL protein.

We then addressed the question whether the observed apoptosis resistance of S-phase arrested cells were associated to high expression of HSP27. HSP27, a powerful chaperone, is a stress protein induced in response to a variety of insults including oncogenic transformation and chemotherapic agents. Its expression allows/helps tumor cells to survive otherwise lethal conditions by counteracting at multiple control points of the apoptotic pathway and therefore increasing the tumorigenic potential of cancer cells [START_REF] Garrido | Heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties[END_REF]. As shown in Figure 6C, constitutive expression of HSP27 was clearly evident in untreated TFK-1 cells; however, ZOL treatment did not affect its expression. In contrast, in EGI-1 cells HSP27 was not present and ZOL treatment did not induce its expression.

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

Discussion

Despite recent improvements in surgery and the development of different regimens of multidrug chemotherapy over the past 25 years, survival of patients suffering from cholangiocarcinoma remains around 5 to 20% after 5 years [41,[START_REF] Malhi | Cholangiocarcinoma: modern advances in understanding a deadly old disease[END_REF]. The poor prognosis of cholangiocarcinoma [START_REF] Blechacz | Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment[END_REF] warrants new therapeutic strategies to improve the overall rate of survival, especially in high-risk subgroups.

In the present study, we examined the commonly used biphosphonate molecule ZOL, because of its high biological activity in inducing inhibition of proliferation and apoptosis in several cancer cell lines [START_REF] Russell | Bisphosphonates: mode of action and pharmacology[END_REF]. We here reported that ZOL, the most potent nitrogen containing BP, causes antiproliferative effects in EGI-1 and TFK-1 cholangiocarcinoma cells. ZOL inhibited cell growth in dose-dependent manner in both cell lines tested. The IC 50 range from 17 to 37 µM for EGI-1 and TFK-1 cells, respectively. The co-treatment with GGOH prevented morphological changes and growth inhibition induced by zoledronate suggesting that a replenishing of protein geranygeranylation could overcome the FPP synthase-block. Furthemore, ZOL-induced inhibition of Rap1A prenylation was reversed by GGOH, suggesting that ZOL-induced inhibition of proliferation is mediated via inhibition of the mevalonate pathway also in cholangiocarcinoma cells.

The ability to form a colony from a single cell, may be seen as a surrogate of the invasive ability of the tumor cell [START_REF] Reddig | Clinging to life: cell to matrix adhesion and cell survival[END_REF]. Interestingly, in cholangiocarcinoma cells this ability is markedly impaired at 8µM ZOL, a concentration 2-4 fold lower than those seen in the MTT assay. It should be noted that cells, exposed up to IC 50 ZOL for 72h, following drug removal later on resume their growth thus indicating the reversibility of the inhibition. In our opinion, this property of ZOL suggests its The anti-proliferative effect of ZOL was associated with a change of cell distribution in the cell cycle phases as indicated by the accumulation and arrest of cancer cells in S-phase. This observation is in accordance with previously studies on different types of tumor cells, all suggesting arrest of ZOL-treated cells in the S-phase [START_REF] Forsea | Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells[END_REF][START_REF] Iguchi | Zoledronate-induced S phase arrest and apoptosis accompanied by DNA damage A c c e p t e d M a n u s c r i p t 25 and activation of the ATM/Chk1/cdc25 pathway in human osteosarcoma cells[END_REF][START_REF] Kubista | Anticancer effects of zoledronic acid against human osteosarcoma cells[END_REF][START_REF] Kuroda | p53independent anti-tumor effects of the nitrogen-containing bisphosphonate zoledronic acid[END_REF][START_REF] Ory | Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status[END_REF]. To date, however, the details of the molecular mechanism of cell cycle arrest involved remain obscure. The G1/S checkpoint is often compromised in many tumor cells due to mutation or deletion of tumor suppressor genes including pRb and TP53 [START_REF] Bucher | G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer[END_REF]. Several studies conducted so far have assessed the deregulation of the pRb-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. The concomitant high level of p27 KIP1 is required for the assembly of active cyclin D1-CDK4 [START_REF] Stacey | Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells[END_REF]. In the presence of ZOL in both cholangiocarcinoma cell lines a reduced expression of the inhibitor p27 Kip1 and a concomitant suppression of the cell cycle promoter D1 were observed. The dual suppression of cyclin D1 and p27 KIP1 , two critical control molecules, and the persistence of pRb in the hyperphosphorylated status should allow the cells to freely enter in the S-phase and to accelerate through the cell cycle [START_REF] Stacey | Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells[END_REF]. The dose-dependent accumulation of cells in the S-phase, however, suggests an arrest at the S-phase checkpoint. Moreover, the observed ZOL dose-dependent increase of cyclin A expression is consistent with a large fraction of cells accumulated in the S-phase. Massive DNA repair should presumably allow slow down or arrest of S-phase progression. The concomitant high level of cyclin B1, putative marker of M phase, was not significantly affected by ZOL, thus indicating that this phase has not been passed by the cells.

Taken together our results on phase distribution of cells, expression of cyclin proteins and their regulators pRb and p27 KIP1 suggest that ZOL treatment on cholangiocarcinoma cells do not A c c e p t e d M a n u s c r i p t 18 induce G1/S arrest, as already seen in other tumor cells [START_REF] Kuroda | p53independent anti-tumor effects of the nitrogen-containing bisphosphonate zoledronic acid[END_REF][START_REF] Oades | Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway, impairing Ras membrane localization in prostate cancer cells[END_REF]. It determines an arrest of cells in the S-phase without an accumulation of cells in the G2-phase and absence of apoptosis induction.

Several studies have described induction of apoptosis (caspase-dependent and -independent) by the administration of ZOL to cancer cells. For example, in breast cancer ZOL decreased the cell growth in a dose-dependent manner, this effect being ascribed to the concomitant BP-induced increase in cell apoptosis [START_REF] Croucher | The anti-tumor potential of zoledronic acid[END_REF]. Similarly, it was reported that ZOL was able to induce apoptotic death in several pancreatic cells achieving 35-90% of apoptotic cells in the treated groups.

Independently of the TP53 and pRb status, in osteosarcoma cells, it was recently described a ZOL induction of atypical apoptosis independent of caspase activation and involving the mitochondrial pathway, in particular the translocation to the nucleus of the apoptosis-inducing factor (AIF) [START_REF] Ory | Zoledronic acid activates the DNA S-phase checkpoint and induces osteosarcoma cell death characterized by apoptosis-inducing factor and endonuclease-G translocation independently of p53 and retinoblastoma status[END_REF]. Our data support the notion that ZOL could inhibit cholangiocarcinoma cell proliferation by inducing cell accumulation/arrest in the S-phase but without inducing apoptosis.

We have found that, subsequent to ZOL treatment both TFK-1 and EGI-1 cells are prevented to activate the apoptotic pathway as shown by the lack of sub-G1 peak, and the absence of caspase 3 cleaved fragment. This outcome was confirmed by lack of morphological features of apoptosis.

This lack of apoptosis was not specific to ZOL treatment since similar results were obtained by using pamidronate (N-BP) or clodronate (BP). It worth to note that these cells are quite resistant to cell death as shown by moderate caspase-3 activation observed in the presence of a high concentration of staurosporine (a classical inducer of apoptosis in several cells). Interestingly, in staurosporine-exposed PC-3 cells the overexpression of anti-apoptotic protein Bcl-xL was found to be associated with resistance because of abrogation of cytochrome c release to the cytosol [START_REF] Li | Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells[END_REF].

Discrepant observations about the ability of biphosphonates to induce apoptosis was reported by Lee et al. [START_REF] Lee | Bisphosphonate treatment inhibits the growth of prostate cancer cells[END_REF]. These authors showed that reduction in prostate cell growth was mainly mediated by cell cycle prolongation. Other two papers related to breast and lung cancer [START_REF] Li | Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival for nonsmall-cell lung cancers[END_REF][START_REF] Fromigue | Bisphosphonates induce breast cancer cell death in vitro[END_REF] showed family. Interestingly, some authors have hypothesised that high concentrations of antiapoptotic proteins, such as Bcl-2 proteins, within the biliary tree could play a key role in the development of cholangiocarcinoma [START_REF] Okaro | The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium[END_REF][START_REF] Okaro | Pk11195, a mitochondrial benzodiazepine receptor antagonist, reduces apoptosis threshold in Bcl-X(L) and Mcl-1 expressing human cholangiocarcinoma cells[END_REF]. A few years ago it was reported that the enhanced Bcl-2 expression alters the threshold for apoptosis in a cholangiocarcinoma cell line [START_REF] Harnois | Bcl-2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line[END_REF]. Moreover, Bcl-2 over-expression could protect myeloma cells against ZOL-induced apoptosis but not against cytostasis [START_REF] Aparicio | In vitro cytoreductive effects on multiple myeloma cells induced by bisphosphonates[END_REF]. In agreement with this report, our results show that Bcl-xL, an antiapoptotic member of the Bcl-2 family proteins, is significantly expressed in both cholangiocarcinoma cell lines, while the pro-apoptotic Bax protein is largely expressed only in TFK-1 cells. ZOL treatment reverted the ratio between pro-apoptotic Bax versus anti-apoptotic Bcl-xL in TFK-1 cells, meanwhile in EGI-1 cells this ratio, already shifted versus the antiapoptotic component, was unaffected by the drug.

Our results support the plausible anti-apoptotic role of the Bcl-xL protein to contrast the cytotoxic effect of ZOL as observed by other authors [START_REF] Okaro | The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium[END_REF]. As already indicated above, Bcl-xL overexpression is one of the mediator of resistance to staurosporine-induced apoptosis [START_REF] Li | Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells[END_REF].

Recently, it was shown that Bcl-xL overexpression protects from apoptosis induced by HMG-CoA reductase inhibitors (simvastatin) in murine tubular cell line [START_REF] Blanco-Colio | Bcl-xL overexpression protects from apoptosis induced by HMG-CoA reductase inhibitors in murine tubular cells[END_REF]. Moreover, antisense Bcl-xL down-regulation has been reported to induce apoptosis in colorectal cancer cells [START_REF] Hayward | Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence to apoptosis in colorectal cancer cells, enhancing global cytotoxicity[END_REF]. Taken together, these reports and our results suggest that the expression of Bcl-xL protein might prevent cellular apoptosis, and confirm, at least in vitro, the resistance of TFK-1 and EGI-1 cells to the protein. This remark is suggested by the known role of HSP27 in conferring chemotherapy resistance, in inhibiting apoptotic cell death, and facilitating tumor progression [START_REF] Richards | Effect of overexpression of the small heat shock protein HSP27 on the heat and drug sensitivities of human testis tumor cells[END_REF][START_REF] Mori-Iwamoto | Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine[END_REF]. Our previous results suggest that HSP27 could be also a prognostic factor in cholangiocarcinoma [START_REF] Romani | The expression of HSP27 is associated with poor clinical outcome in intrahepatic cholangiocarcinoma[END_REF] In conclusion, our study provides the first evidence that ZOL elicits a direct inhibitory action on the proliferation of cholangiocarcinoma cells. The cell-cycle alteration induced by ZOL, i.è. cell accumulation/arrest in S-phase, was confirmed by changes in the level of cyclins D1 and A, and of regulators p27 KIP1 and pRb. ZOL therefore, could represent a reliable chemotherapeutic agent able to sensitize cells to cytotoxic agents and/or radiations.

Our data indicate that a dose of 10-50 µM ZOL is required for a significant cell cycle arrest in cholangiocarcinoma cells. This range of concentrations, however, may preclude ZOL to be used as monotherapy in cholangiocarcinoma, thus making mandatory the need to investigate differently active molecules to associate with ZOL, in order to obtain a synergistic therapeutic improvement and minimizing, in the same time, the side effects.

  Zoledronic acid (ZOL, ZOMETA®, Novartis Europharm, UK ) and gemcitabine (GEM, GEMZAR®, Eli Lilly, Italy) were kindly provided by Drs. Vittorio Rizzoli and Maria Cristina Baroni, Dipartimento di Medicina Interna e Scienze Biomediche, Università degli Studi di Parma. ZOL was dissolved in phosphate buffer saline (PBS) as a 5 mM stock solution while GEM as 100mM stock solution and both stored at -20°C . RPMI 1640 medium, staurosporine, geranylgeraniol (GGOH), pamidronate, clodronate, and phosphorylated histone H3 (pHH3, 8656-R), were purchased from Sigma-Aldrich (St. Louis, MO, USA), and fetal bovine serum (FBS)

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

 8 overnight at 4° C in 1ml of hypotonic fluorochrome solution. DNA content was determined by flow cytometry using a Coulter EPICS XL-MCL cytometer (Coulter Co., Miami, FL, USA) and the cell-cycle-phase distribution was analysed by ModFit 2.0 software (Verity Software House, Topsham, Me. USA). Respective to their phase-specific DNA-content cells were divided into G1, S, G2/M phases and, when present, sub-G1 phase counted as apoptotic cells. Cell death was also assessed by morphological criteria on cells stained with Hoechst 33342 (a fluorescent cell-permeant DNA stain) and propidium iodide (PI, a fluorescent permeant stain for membrane damaged cells) using fluorescence microscopy.

  A c c e p t e d M a n u s c r i p t 10 protein prenylation, was added during drug treatment. The addition of 25 µM GGOH determines a rescue of zoledronate-induced morphological changes in both cell lines. The ability of GGOH to attenuate/rescue ZOL cytotoxicity was assessed by plating cells and then treating them with various ZOL concentrations or in combination with GGOH as described in the legend to Figure 2B. By using Western blotting analysis in the presence of 50µM of ZOL the band for the unprenylated form of Rap1A (a small GTP-protein of Ras family) in both cell lines became visible, indicating high level of the unprenylated form. Co-treatment with GGOH significantly attenuated ZOL cytotoxicity, increasing the IC 50 by 4.5-fold and 10-fold for TFK-1

A c c e p t e d M a n u s c r i p t 12 p27

 12 KIP1 . Modified expression and/or phosphorylation of these molecules are indicative of which checkpoints may be involved. Checkpoints are important sensors that ensure the proper sequence of cell cycle events allowing cells to repair DNA damage. There are at least three checkpoints at G1/S, S and G2/M phases that can allow DNA repair and promote cell death in unrepaired cells.

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

 15

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

 17 use, in cholangiocarcinoma affected patients, as effective adjuvant therapy for metastasis and disease relapse.

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

 19 ZOL administration inhibited cancer cell proliferation, although only a minor fraction of cells (less than 20% in the case of lung cancer) entered in the apoptotic process. Some mechanisms have been proposed to explain the potential refractory of tumor cells to activate the apoptotic program following ZOL treatment. For instance, the failure of ZOL to induce apoptosis is linked to the high levels of anti-apoptotic signals such as Bcl-2 proteins

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

 20 -inducing effects of ZOL. An additional mechanism that emerges from this study and might explain the higher resistance to ZOL exhibited by TFK-1 cells, in comparison to EGI-1 cells (IC 50 = 37 vs 17), could be linked to the constitutive expression of the antiapoptotic HSP27

A c c e p t e d M a n u s c r i p t 21 5 .

 5 AcknowledgementsThis study was supported by RE.GA.STR project (Programma di Ricerca Regione-Università 2007-2009, Emilia-Romagna, Italy) and FIL grants from MIUR (Rome, Italy). Silvia Desenzani was supported by a fellowship from Fondazione Cariparma, Parma, Italy. We thank Simona Stefàno for her precious technical assistance.

Figure 1 .

 1 Figure 1. ZOL inhibits EGI-1 (□) and TFK-1 (■) cell proliferation. Cells were exposed to ZOL

Figure 2 .

 2 Figure 2. Cholangiocarcinoma cell changes induced after ZOL treatment in the absence and in the presence of 25 µM GGOH. (A) Morphology: EGI-1 cells were plated and then treated for

Figure 3 .

 3 Figure 3. Colony formation assay. Influence of 2-32µM ZOL treatment for ten days on the

Figure 4 .

 4 Figure 4. Flow cytometric analysis of DNA content in EGI-1 and TFK-1 cells, in the absence or

Figure 5 .

 5 Figure 5. Cell cycle regulators and inhibitors. Western blotting detection of TP53, pRb, phospho-pRb, p27 KIP1 , cyclin D1, cyclin A, cyclin B1, phospho-HH3 in EGI-1 and TFK-1 cells after 72h

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