Johan Vande Voorde

Sandra Liekens

Christopher Mcguigan

Paola G S Murziani

Magdalena Slusarczyk

Jan Balzarini
email: jan.balzarini@rega.kuleuven.be

Johan Vande Voorde

The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2 -deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes

Keywords: nucleoside analogues, cancer, phosphoramidate prodrugs, mycoplasma, thymidine phosphorylase, 5-fluoro-2'-deoxyuridine (FdUrd)

A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.

Introduction

Current treatment of cancer using chemotherapeutics is largely based on the use of nucleoside analogues. These molecules are designed to mimic natural pyrimidine and purine nucleosides.

After uptake by the cell, they are phosphorylated by cellular enzymes such as (deoxy)cytidine kinase (dCK), thymidine kinase (TK) and/or nucleo(s)(t)ide kinases. These antimetabolites can subsequently interfere with the de novo synthesis of DNA/RNA precursors to eventually inhibit DNA/RNA synthesis resulting in cytotoxic/static activity [1;2].

Fluoropyrimidine-based antimetabolites such as fluorouracil (5-FU), capecitabine and 5fluoro-2'-deoxyuridine are mainly used in the treatment of colon, breast and ovarian carcinoma [START_REF] De Bruin | Role of platelet derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1-phosphate[END_REF][START_REF] Ishikawa | Tumor selective delivery of 5-fluorouracil by capecitabine, a new oral fluoropyrimidine carbamate, in human cancer xenografts[END_REF][START_REF] Walko | Capecitabine: a review[END_REF]. Intracellularly, these drugs are metabolised to the monophosphate 5-FdUMP, which forms a stable inhibitory complex with thymidylate synthase (TS) and the reduced co-substrate 5,10-methylenetetrahydrofolate, thereby blocking binding of the normal substrate dUMP to the enzyme [START_REF] Beck | A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil[END_REF][START_REF] Tanaka | The history, mechanism and clinical use of oral 5-fluorouracil derivative chemotherapeutic agents[END_REF][START_REF] Longley | 5-fluorouracil: mechanisms of action and clinical strategies[END_REF]. TS is the enzyme responsible for the conversion of dUMP to TMP and is therefore indispensible for cell proliferation, making it a crucial target for drug action. Among the fluoropyrimidines mentioned above, 5-FdUrd requires only one metabolic conversion, a phosphorylation catalysed by TK to generate 5-FdUMP [START_REF] Longley | 5-fluorouracil: mechanisms of action and clinical strategies[END_REF]. This obligatory phosphorylation is often the rate-limiting step in the metabolism of many anticancer drugs (including 5-FdUrd), and is therefore still one of the limiting factors for the therapeutic use of nucleoside analogues. Hence, different strategies to improve the antitumour efficacy of nucleoside analogues are being investigated [START_REF] Galmarini | Nucleoside analogues and nucleobases in cancer treatment[END_REF].

The charged nature of nucleoside monophosphates under physiological conditions results in poor, if any, penetration across the cell membrane [START_REF] Mehellou | Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells[END_REF]. They are also subject to extracellular dephosphorylation. Therefore, the direct administration of phosphorylated molecules to strategies for bypassing the rate-limiting phosphorylation using various types of nucleoside 5'-monophosphate prodrugs for more efficient drug-delivery have been explored [START_REF] Hecker | Prodrugs of phosphates and phosphonates[END_REF]. The administration of lipophilic phosphoramidate nucleotide prodrugs (ProTides) has proved successful for several molecules with anti-viral/cancer activity [START_REF] Harris | Synthesis and antiviral evaluation of phosphoramidate derivatives of (E)-5-(2-bromovinyl)-2'deoxyuridine[END_REF][START_REF] Congiatu | Novel potential anticancer naphthyl phosphoramidates of BVdU: separation of diastereoisomers and assignment of the absolute configuration of the phosphorus center[END_REF][START_REF] Derudas | The application of phosphoramidate protide technology to acyclovir confers anti-HIV inhibition[END_REF]. By masking the charges of the phosphate motif, good passive membrane diffusion of the prodrugs can be accomplished after which the prodrug is rapidly converted intracellularly into the nucleoside monophosphate by enzymatic cleavage [START_REF] Mehellou | Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells[END_REF].

Mycoplasmas are the smallest known self-replicating organisms and are characterized by the lack of a cell wall and a strongly reduced genome (600-1 200 kb). Many of these bacteria have a parasitic lifestyle and reside in the human body causing asymptomatic infections [START_REF] Razin | Molecular biology and pathogenicity of mycoplasmas[END_REF].

It was shown that these prokaryotes tend to preferentially colonize tumour tissue: Huang et al.

reported that 40-56% of human gastric, colon, oesophageal, lung and breast cancers are infected with mycoplasmas compared to 21-30% in non-tumourigenic tissue [START_REF] Huang | Mycoplasma infections and different human carcinomas[END_REF]. Pehlivan et al. found 22% of small cell lung cancer tissue samples and >80% kidney tissue samples of patients suffering renal cell carcinoma to be infected with mycoplasmas compared to 5% and 14% respectively in control tissue samples [16;17]. Chan et al. reported a 59% mycoplasma infection rate in ovarian cancer tissues [START_REF] Chan | Prevalence of mycoplasma conserved DNA in malignant ovarian cancer detected using sensitive PCR-ELISA[END_REF] and other studies also reported a high infection rate in gastric [START_REF] Sasaki | Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer[END_REF][START_REF] Yang | Mycoplasma hyorhinis infection in gastric carcinoma and its effects on the malignant phenotypes of gastric cancer cells[END_REF] and cervical condyloma tissues [START_REF] Kidder | Assessment of archived paraffinembedded cervical condyloma tissues for mycoplasma-conserved DNA using sensitive PCR-ELISA[END_REF]. Due to their reduced set of genes, mycoplasmas lack the pathway for de novo pyrimidine and purine synthesis and therefore express a wide array of salvage nucleo(s)(t)ide-metabolizing enzymes, such as thymidine phosphorylase (TP), deoxycytidine deaminase, etc. [START_REF] Razin | The mycoplasmas[END_REF][START_REF] Charron | Analysis of deoxycytidine (dC) deaminase activity in herpes simplex virus-infected or HSV TK-transformed cells: association with mycoplasma contamination but not with virus infection[END_REF][START_REF] Neale | Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides[END_REF][START_REF] Tham | Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides[END_REF]. Already in 1985 it was observed that mycoplasma-encoded enzymes (e.g. TP), present in contaminated cell cultures, lead to decreased dTTP incorporation in lymphocytes [START_REF] Sinigaglia | Inhibition of [3H]thymidine incorporation by Mycoplasma arginini-infected cells due to enzymatic cleavage of the nucleoside[END_REF]. Recently, it has been demonstrated that A c c e p t e d M a n u s c r i p t 5 these enzymes, in particular the mycoplasma-encoded TP, can also interfere with the cytostatic activity of several chemotherapeutics, including 5-trifluorothymidine, in vitro [START_REF] Bronckaers | The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: Implications for cancer therapy[END_REF][START_REF] Jetté | Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection[END_REF][START_REF] Liekens | Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasmaencoded catabolic enzymes[END_REF]. Therefore we hypothesized that the elimination of mycoplasmas by antibiotics, suppression of mycoplasma-encoded enzymes in human tumour tissue or the development of mycoplasma-insensitive nucleoside analogue prodrugs may optimize treatment of cancer patients using purine and pyrimidine antimetabolites [START_REF] Liekens | Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasmaencoded catabolic enzymes[END_REF]. In the absence of such approaches, cancer patients may receive sub-optimal chemotherapeutic treatment.

This study was aimed at the development and assessment of TK-independent phosphoramidate prodrugs of 5-FdUrd that would also be insensitive to the TP-dependent inactivation of its free nucleoside analogue. From a wide variety of newly synthesized phosphoramidate prodrugs of 5-FdUrd, NUC-3073 was chosen for further in depth studies. This molecule contains a naphthyl and benzylalaninyl group to mask the charged 5'phosphate on 5-FdUMP.

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

Materials and Methods

Compound synthesis

The compound NUC-3073 [START_REF] Hatse | Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation[END_REF] The nucleoside 5-FdUrd (Carbosynth Ltd, Berkshire, UK) (7) was converted to the 5'-ProTide derivative by coupling with the phosphorochloridate derivative [START_REF] Beck | A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil[END_REF] in THF (Sigma-Aldrich) in the presence of N-methyl imidazole (NMI) (Sigma-Aldrich) to give the target compound NUC-3073 (1) (Scheme 3). The product was obtained as a mixture of two diastereoisomers as confirmed by the presence of two peaks in the 31 P and 19 F NMR spectra, and two closely spaced peaks detectable by HPLC.

Alternatively, the compound 1 was prepared using tBuMgCl (Sigma-Aldrich) (1M solution in THF). Due to the lack of selectivity towards the primary hydroxyl group, formation of 3'-Ophosphorylated derivative 8 was also observed (Scheme 4).

General

Anhydrous solvents were obtained from Sigma-Aldrich and used without further purification.

All reactions were carried out under an argon atmosphere. Reactions were monitored with analytical TLC on Silica Gel 60-F254 precoated aluminium plates (Merck Chemicals Ltd) and visualised under UV (254 nm) and/or with 31 Low and High resolution mass spectra were performed as a service by Birmingham University, using electrospray (ES). CHN microanalysis was performed as a service by MEDAC Ltd., Surrey.

Synthesis of

Synthesis of 5-Fluoro-2'-deoxyuridine-5'-O-[-naphthyl (benzyl-L-alaninyl)]

phosphate phosphate (1) (0.08 g, 0.130 mmol) in a solution of triethylamine (5 mL) and water (5 mL).

The reaction mixture was stirred at 35°C for 24 h and then the solvents were removed under reduced pressure. The residue was treated with water and extracted with dichloromethane (Fisher Scientific). The aqueous layer was concentrated and evaporated under reduced pressure, then the resultant crude material was purified by column chromatography on silica, Cells were grown at 37°C in a humidified incubator with a gas phase of 5% CO 2 .

eluting

Cell cultures

Cytostatic assays

Monolayer cells (U87 and U87.Hyor) were seeded in 48-well microtiter plates (Nunc TM , Roskilde, Denmark) at 10 000 cells/well. After 24 hours, an equal volume of fresh medium containing the test compounds was added. On day 5, cells were trypsinized and counted in a

Coulter counter (Analis, Suarlée, Belgium). Suspension cells (L1210/0, L1210/TK -, L1210.Hyor, CEM/0, CEM/TK -) were seeded in 96-well microtiter plates (Nunc TM ) at 60 000 cells/well in the presence of a given amount of the test compounds. The cells were allowed to proliferate for 48 h (L1210) or 72 h (CEM) and were then counted in a Coulter counter. The 50% inhibitory concentration (IC 50 ) was defined as the compound concentration required to reduce the number of viable cells by 50%.

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

Thymidylate synthase (TS) activity measurements

The activity of TS in intact L1210/0 and L1210/TK -cells was measured by evaluation of tritium release from [5-3 H]dUMP (formed in the cells from [5-3 H]dUrd or [5-3 H]dCyd) in the reaction catalysed by TS. This method has been described in detail by Balzarini & De Clercq [START_REF] Balzarini | Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells[END_REF]. Briefly, cell cultures (500 µL DMEM culture medium) were prepared containing ~ 3 x 10 6 L1210 cells and appropriate amounts of the test compounds (5-FdUrd and NUC-3073).

After 30 min, 2 h and 4 h pre-incubation of the cells with the compounds at 37°C, 1 µCi of [5-

3 H]dUrd or [5-3 H]dCyd was added to the cell cultures. After 30 min incubation, 100 µL of the cell suspensions were withdrawn and added to a cold suspension of 500 µL activated charcoal (VWR, Haasrode, Belgium) (100 mg/ml in TCA (Merck Chemicals Ltd) 5%). After 10 min, the suspension was centrifuged at 13 000 rpm for 10 min, after which the radioactivity in 400 µL supernatant was counted in a liquid scintillator using OptiPhase HiSafe (Perkin Elmer, Waldham, MA).

Human serum

The stability of the prodrug NUC-3073 in the presence of human serum was studied using in situ 31 P NMR (202 MHz). The ProTide NUC-3073 (1) (5.0 mg) was dissolved in a mixture of DMSO (Sigma-Aldrich)/D 2 O (Cambridge Isotope Laboratories) (0.05 mL/0.15 mL) and then subjected to 31 P NMR analysis at 37C. After the first spectrum (control) was recorded, the NMR sample was treated with human serum (Sigma-Aldrich) (0.3 mL) and immediately subjected to further 31 P-NMR experiments at 37C. 31 P-NMR data were recorded every 15 min over 12 h and 30 min. In order to improve the visualization of the results all the spectra were further processed using the Lorentz-Gauss deconvolution method. Data recorded were processed and analyzed with the Bruker (TopSpin 2.1 programme.

Buffer at pH 1

The stability of the prodrug NUC-3073 towards hydrolysis at pH = 1 was studied using in situ Data were processed and analyzed with the Bruker TopSpin 2.1 programme.

Buffer at pH 8

Cytostatic activity of 5-FdUrd and its prodrug NUC-3073 against TK-competent and TK-deficient tumour cell lines

The cytostatic activity of 5-FdUrd and NUC-3073 was determined in different TKexpressing and TK-deficient tumour cell lines. As shown in Table 1, 5-FdUrd is strongly dependent on the expression of TK for its cytostatic activity. Its IC 50 increased 4 000-fold for L1210/TK -cells (IC 50 : 3.1 µM) versus wild-type L1210/0 cells (IC 50 : 0.0008 µM) and by 50fold for CEM/TK -cells (IC 50 : 1.5 µM) versus CEM/0 cells (IC 50 : 0.028 µM). In contrast, the cytostatic activity of the 5-FdUrd prodrug NUC-3073 remained virtually unchanged in TKdeficient cells when compared with wild-type cells (IC 50 : 0.027 and 0.011 µM for L1210/TK - and L1210/0, and 0.32 and 0.089 µM for CEM/TK -and CEM/0 cells, respectively). Although the cytostatic activity of NUC-3073 was 3-to 10-fold inferior to 5-FdUrd against wild-type L1210/0 and CEM/0 cells, it proved 5-to 100-fold superior to 5-FdUrd in the TK-deficient tumour cell lines (Table 1).

Effect of mycoplasma infection of tumour cell cultures on the cytostatic activity of 5-

FdUrd and its prodrug NUC-3073

The L1210/0 cell cultures were infected with the mycoplasma species M. hyorhinis (cells designated: L1210.Hyor). 5-FdUrd lost its cytostatic activity against the mycoplasmainfected L1210.Hyor cells by 300-fold (IC 50 : 0.24 µM). Also, 5-FdUrd lost its cytostatic activity by 400-fold in U87.Hyor cell cultures when compared with uninfected U87 cells (Table 1). In sharp contrast, the 5-FdUrd prodrug NUC-3073 kept a similar cytostatic potential in both L1210/0 and L1210.Hyor cell cultures (IC 50 : 0.011 and 0.025 µM, respectively). A similar observation was made for this prodrug when evaluated for its cytostatic activity in U87 and U87.Hyor cell cultures (IC 50 : 0.035 and 0.039 µM,

A c c e p t e d

M a n u s c r i p t 18 respectively). Thus, whereas the free nucleoside 5-FdUrd markedly lost its cytostatic potential against Mycoplasma hyorhinis-infected tumour cell lines, the antiproliferative potential of its prodrug NUC-3073 was independent of the mycoplasma infection.

Phosphorolysis of 5-FdUrd and NUC-3073 by thymidine and uridine phosphorylases

5-FdUrd and its prodrug NUC-3073 were exposed to purified TP derived from E.coli or human erythrocytes, and to purified UP derived from human tumour tissue. Whereas E.coli and human TP rapidly converted dThd and 5-FdUrd to their free bases, NUC-3073 was completely stable in the presence of these enzymes (Fig. 2). Under similar experimental conditions, uridine was converted to uracil by human UP, but not by E.coli TP, or human TP.

When both compounds were exposed to UP, dThd and NUC-3073 were not affected by the enzyme, whereas 5-FdUrd was slightly hydrolysed (Fig. 2, panel C).

Inhibition of thymidylate synthase (TS) by 5-FdUrd and NUC-3073

The major target for the cytostatic activity of 5-FdUrd is thymidylate synthase (TS).

The activity of TS in intact tumour cells can be directly monitored by measuring the tritium release in intact L1210/0 cell cultures that were exposed to [5- in situ (Fig. 3). Indeed, whereas the inhibition of 3 H release was only 2-fold increased upon longer pre-incubation times of 5-FdUrd, the inhibitory potential of NUC-3073 increased 10fold (Fig. 3, panels A and B, and C and D). Interestingly, pre-incubation of the tumour cells with 5-FdUrd and NUC-3073 for at least 4 h results in TS inhibition in the intact tumour cells at drug concentrations that are very comparable with the 50% cytostatic activity concentrations of these drugs. Taken all data together, our observations indicate that the 5-FdUrd prodrug needs several metabolic conversion steps before reaching TS as the target enzyme for inhibition, and support the view that NUC-3073 acts as an efficient prodrug of 5-FdUrd to exert its eventual cytostatic activity.

The activity of TS in the presence of 5-FdUrd and NUC-3073 was also measured in intact L1210/TK -cells using [5-³H]dCyd as an externally supplied substrate (due to TK deficiency, [5-3 H]dUrd cannot be used). As demonstrated in Table 3 and Fig. 3 (panels E and F), the concentration of 5-FdUrd required to cause 50% inhibition of TS decreased by a factor 5 700 in TK-deficient L1210/TK -cells (IC 50 : 1.4 µM) when compared to wild-type L1210/0 cells (IC 50 : 0.0003 µM). In contrast, the inhibitory activity of NUC-3073 against TS remained virtually unchanged in L1210/TK -cells (IC 50 : 0.053 µM in L1210/TK -cells versus 0.013 µM in L1210/0 cells). 5.16 ppm), which lack the ester motif (Fig. 4). These intermediates did not persist as they were quickly metabolized to the aminoacyl phosphoramidate derivative (the final product in this assay) via the loss of the aryl group (steps a to c in Fig. 4). Due to the achirality at the phosphate the metabolite 11 appeared as a singlet (6.85 ppm). Thus, the enzymatic assay spectra showed a fast metabolism of the parent ~ 4.00 with complete conversion to the In order to support both, the putative mechanism proposed as well as the results obtained in the enzymatic assay, we decided to prepare the intermediate 11 via a synthetic route. Thus chemical hydrolysis of the compound 1 in the presence of triethylamine and water was performed. The product 11 obtained as a diammonium salt was next added to the assay's final sample (containing only the enzymatic metabolite 11 in TRIZMA), and 31 P-NMR analysis was investigated. The 31 P-NMR spectrum has shown exclusively one peak at 6.85 ppm; therefore our results have proved the proposed metabolic pathway and activation of ProTides.

Stability studies

In the current work also chemical stability studies on the prodrug NUC-3073 (1) have been performed by exposing the compound to human serum and to aqueous buffers (pH 1.0 and 8.0) using in situ 31 P NMR. Each experiment has been carried out dissolving the ProTide in the suitable deuterated solvent and analysing the samples at 37°C for about 14 h, acquiring scans at the regular time intervals. For a better resolution original spectra (lower graphs) and deconvoluted ones (upper graphs) are reported. The stability assay of the phosphoramidate NUC-3073 (1), after incubation in human serum, showed 73% of unchanged compound after 12 h and 30 min as shown in Fig. 6. When chemical hydrolysis was evaluated at extreme experimental conditions, i.e. at pH 1.0 and pH 8.0 at 37°C, a full stability of prodrug NUC-3073 (1) in both acidic and basic buffer conditions was observed. Spectra were recorded for 14 h acquiring scans every 12 min at regular intervals as shown in the Fig. 7 and8. The ProTide (1) examined at pH 1.0 showed constant doublet peaks of diastereoisomers at 4.35; 4.50 throughout the time of the assay (Fig. 7).

Also, at pH 8.0 the spectra displayed a persistent peak of the prodrug (1) at 4.48 and a single peak at 2.55 corresponding to a buffer peak (Fig. 8).

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

Discussion

Various mechanisms of tumour cell resistance towards fluoropyrimidines such as 5FU, 5-FdUrd and trifluorothymidine (TFT) have been described, including a decreased activity of crucial drug-activating enzymes (e.g. TK and orotate phosphoribosyltransferase), an increased activity of drug-inactivating enzymes (i.e. TP), an upregulation of the target enzyme (e.g. TS)

and/or defective facilitated diffusion of nucleosides [START_REF] Agarwal | Collateral resistance of a dideoxycytidine-resistant cell line to 5-fluoro-2'-deoxyuridine[END_REF][START_REF] Murakami | Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells[END_REF][START_REF] Kosaka | Effects of thymidine phosphorylase levels in cancer, background mucosa, and regional lymph nodes on survival of advanced gastric cancer patients receiving postoperative fluoropyrimidine therapy[END_REF][START_REF] Zhang | Mechanisms of resistance to fluoropyrimidines[END_REF][START_REF] Sobrero | Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2'-deoxyuridine in the HCT-8 human carcinoma line[END_REF]. Also, high TP levels found in several types of cancer tissue were reported to be predictive for a poorer prognosis upon treatment with fluoropyrimidines [41-43], although other studies have not confirmed these findings [43,44]. The study presented here was aimed at the development of a prodrug for 5-FdUrd, to circumvent possible resistance mechanisms and susceptibility to degradation by catabolic enzymes, present in the tumour micro-environment.

We describe herein NUC-3073, a phosphoramidate prodrug of 5-FdUrd that may fulfil these aims. After uptake into the tumour cells, NUC-3073 generates 5-FdUMP intracellularly upon enzymatic cleavage. Our stability studies and enzyme/serum studies by 31 P NMR technology revealed that the prodrug NUC-3073 is fully stable in acid and alkaline conditions, but subject to hydrolysis in the presence of carboxypeptidase Y, or to some extent by serum resulting in the formation of the nucleoside 5'-phosphoramidate derivative. Whereas TK is a key enzyme in the activation of 5-FdUrd, NUC-3073 was found to be much less dependent on TK to exert its cytostatic action in both murine (L1210) and human (CEM) cell cultures. Due to the lipophilic nature of ProTides, these molecules can deliver nucleoside-monophosphates directly into the intact tumour cell after conversion to their nucleoside phosphoramidate derivative by enzymes such as carboxyesterases or carboxypeptidases (i.e. carboxypeptidase Y), eliminating the need for an initial phosphorylation by specific nucleoside kinases such as cytosolic TK-1. In this regard, NUC-3073 may be an adequate tool for the treatment of tumour cells with a modified TK-1 activity (either acquired or inherent). Also, since TK-1

A c c e p t e d M a n u s c r i p t 24 expression is S-phase-dependent, it is expected that NUC-3073 can also efficiently deliver 5-FdUMP in tumour cells that are not in the S-phase of their replication cycle. Our TS activity studies revealed that NUC-3073 was able to inhibit TS in both wild-type and TK-deficient tumour cell lines, pointing again to an efficient intracellular delivery of the 5'-monophosphate of 5-FdUrd, and its virtual independence of cellular TK-1 for metabolic activation.

Normal tissue toxicity is always a concern in the development of anticancer drugs and in particular it might be a potential concern whether the 5-FdUrd prodrug may show an increased toxicity in non-proliferating cells or other cell types with low TK-1 activity. In such cell tissues, thymidine nucleotide pools are rather low, and thus, the released 5-FdUMP derivative and the di-and triphosphate drug metabolites may have a competitive advantage and exert a more pronounced cytotoxic activity. However, it should be kept in mind that in non-proliferating cells, TS activity is also markedly decreased due to lack of ongoing DNA synthesis. Thus, due to a lower degree of target enzyme (TS) activity in resting cells, it is expected that the released 5-FdUMP should not necessarily afford higher cell toxicity in such cells. It was indeed found that both 5-FdUrd and the 5-FdUrd prodrug NUC-3073 were markedly less toxic in confluent human cervix carcinoma HeLa/0 cell cultures compared with the toxicity values observed for proliferating L1210 and CEM cell cultures [minimal cytotoxic concentration (MCC) of NUC-3073 for HeLa: 2 µM]. Also, NUC-3073 was not found more toxic in confluent HeLa/TK -cells than in wild-type HeLa/0 cells (data not shown). Thus, evidence for increased toxicity of a prodrug such as NUC 3073 against nonproliferating tissue was not observed.

We also proved that NUC-3073 is unlikely to be inactivated by catabolic enzymes involved in nucleoside metabolism. Indeed, whereas 5-FdUrd is highly susceptible to enzymatic hydrolysis by TP resulting in the formation of 5-FU and 2'-deoxyribose-1phosphate, we found that its prodrug NUC-3073 is not a substrate for prokaryotic (i.e. E.coli) does not recognize NUC-3073 as a substrate, whereas 5-FdUrd is (poorly, but measurably) hydrolyzed by this enzyme. Several studies revealed that many tumour cells have elevated levels of TP, which also acts as an angiogenic factor [43,45]. Moreover, there are several reports on the preferential colonization of tumour tissue by mycoplasmas [START_REF] Huang | Mycoplasma infections and different human carcinomas[END_REF][START_REF] Pehlivan | Does Mycoplasma sp. play role in small cell lung cancer?[END_REF][START_REF] Pehlivan | Can mycoplasmamediated oncogenesis be responsible for formation of conventional renal cell carcinoma?[END_REF] which interfere with the cytostatic activity of several conventional chemotherapeutics in vitro through its encoded TP [START_REF] Bronckaers | The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: Implications for cancer therapy[END_REF][START_REF] Jetté | Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection[END_REF][START_REF] Liekens | Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasmaencoded catabolic enzymes[END_REF]. Our findings that 5-FdUrd, but not NUC-3073, markedly loses cytostatic activity when the tumour cells are infected by (TP-expressing) mycoplasmas, is in full agreement with these observations. Therefore, the administration of a TP-insensitive anti-cancer prodrug such as NUC-3073, demonstrated to be chemically stable at extreme pH conditions, may further improve cancer chemotherapy.

It should be mentioned that the isopropylalaninyl monoamidate phenyl mono ester prodrug of tenofovir (GS 7340) has been investigated for its pharmacokinetics in dogs [46]. This compound, being rather similar to NUC 3073 in terms of the prodrug moiety, had a halflife of 90 min in human plasma, and reached an oral bioavailability of  17% after a single oral dose of GS 7340 (10 mg/kg) to beagle dogs. There was an increased distribution of the parent drug to tissues of lymphatic origin compared to the commercially available prodrug tenofovir-DF (Viread) [46]. No increased side-effects for GS 7340 were reported when compared with the clinically used Viread. These observations revealed that there may be a potential clinical application for prodrugs such as NUC 3073.

In conclusion, we believe that ProTides, such as NUC-3073, may represent an interesting new approach towards the development of more resilient anti-cancer drugs. NUC-3073 may have at least several advantages over its parent drug 5-FdUrd: it exerts its cytostatic activity independently of TK and it is resistant to metabolic breakdown by TP, an enzyme that

  phosphorylation step has little therapeutic advantage. Hence, different

13 C

 13 P NMR spectra. Column chromatography was A c c e p t e d M a n u s c r i p t 7 performed on silica gel (35-70 M). Proton ( 1 H), carbon ( 13 C), phosphorus ( 31 P) and fluorine ( 19 F) NMR spectra were recorded on a Bruker Avance 500 spectrometer (Bruker UK, Coventry, UK) at 25°C. Spectra were auto-calibrated to the deuterated solvent peak and all NMR and 31 P NMR were proton-decoupled. Analytical HPLC was conducted by a Varian Prostar (LC Workstation-Varian prostar 335 LC detector) using Varian Polaris C18-A (Agilent Technologies, Edinburgh, UK) (10 M) as an analytic column.

1 )-M a n u s c r i p t 10 ( 3 1 ,

 11031 Alternative method. To a solution of 5-fluoro-2'-deoxyuridine (0.35 g, 1.42 mmol) in dry THF (15 mL), tBuMgCl (1M in THF, 0.40 mL, 1.56 mmol) was added dropwise. The reaction mixture was stirred for 30 min and then a solution of 1-naphthyl-(benzyl-L-alaninyl) phosphorochloridate (6) (1.14 g, 2.84 mmol) dissolved in anhydrous THF A c c e p t e d mL) was added dropwise. The reaction mixture was stirred at room temperature for 16 h and then evaporated under reduced pressure to give a crude residue. Purification by column chromatography on silica gel (Merck Chemicals) eluting with gradient of MeOH (0% to 5%) in CH 2 Cl 2 afforded the product 1 as a colorless solid (78 mg, 9%). (All the spectroscopic data of 1 are in agreement with data reported in section 2.1.4). Alongside with the desired product the 3'-ProTide analogue[START_REF] Longley | 5-fluorouracil: mechanisms of action and clinical strategies[END_REF] was isolated and characterized by NMR and HPLC.

A c c e p t e d M a n u s c r i p t 11 2. 1 . 6 .

 1116 Synthesis of 5-Fluoro-2'deoxyuridine-5'-O-[(L-alaninyl)] phosphate ammonium salt (11). 5-Fluoro-2'-deoxyuridine-5'-O-[(L-alaninyl)] phosphate ammonium salt was prepared by dissolving 5-fluoro-2'deoxyuridine-5'-O-[-naphthyl(benzyl-L-alaninyl)]

  A c c e p t e d M a n u s c r i p t 12 Murine leukaemia L1210/0 and human T-lymphocyte CEM/0 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). Human glioblastoma U87 cells were kindly provided by Dr. E. Menue (Institut Pasteur, Paris, France). Thymidine kinasedeficient CEM/TK -cells were a kind gift from Prof. S. Eriksson (currently at Uppsala University, Uppsala, Sweden) and Prof. A. Karlsson (Karolinska Institute, Stockholm, Sweden). Thymidine kinase-deficient L1210/TK -were derived from L1210/0 cells after selection for resistance against 5-bromo-2'-dUrd [33]. Infection of relevant cell lines with Mycoplasma hyorhinis (ATCC) resulted in chronically-infected cell lines further referred to as L1210.Hyor and U87.Hyor. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% foetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), 10 mM Hepes and 1 mM Sodium Pyruvate (Invitrogen).

13 2. 5 .

 135 Exposure of 5-FdUrd and NUC-3073 to E. coli-encoded TP and human-encoded TP and UPThe substrate specificity of TP towards natural thymidine (dThd) (Sigma-Aldrich), uridine (Urd) (Sigma-Aldrich), 5-FdUrd (Sigma-Aldrich) and NUC-3073 was investigated by high pressure liquid chromatography (HPLC) (Waters, Milford, MA

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

 14 by equilibration at 100% separation buffer). UV-based detection was performed at 258 nm.

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

 15 The enzymatic stability of the prodrug NUC-3073 towards carboxypeptidase Y (Sigma-Aldrich) was studied using in situ31 P NMR (202 MHz). The experiment was carried out by dissolving NUC-3073 (3.0 mg) in d 6 -acetone (Cambridge Isotope Laboratories, Andover, MA) (150 L) and adding TRIZMA (Sigma-Aldrich) buffer pH 7.6 (300 L). The resulting solution was placed in an NMR tube and a 31 P-NMR experiment at 25 C was recorded as the blank experiment. The enzyme Carboxypeptidase Y (0.2 mg) was dissolved in TRIZMA (150 and added to the solution of the phosphoramidate derivative in the NMR tube. Next, the tube was placed in the NMR machine, which was set to run a 31 P-NMR experiment (64 scans) every 4 min for 14 hours at 25 C. Data were processed and analyzed with the Bruker Topspin 2.1 program.

31 P

 31 NMR (202 MHz). The ProTide NUC-3073 (1) (2.6 mg) was dissolved in MeOD (Cambridge Isotope Laboratories) (0.1 mL) after which 0.5 mL buffer (pH = 1, prepared from equal parts of 0.2 M HCl and 0.2 M KCl (Sigma-Aldrich)) was added. Then, on this sample 31 P-NMR experiments were carried out at 37 C recording the data every 12 min for 14 h.

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

 16 stability of the prodrug NUC-3073 towards hydrolysis at pH = 8 was studied using in situ 31 P NMR (202 MHz). The ProTide NUC-3073 (1) (4.9 mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL buffer (pH = 8, prepared from a solution of 0.1 M Na 2 HPO 4 (Sigma-Aldrich), which was adjusted by 0.1 M HCl) was added. Next, on this sample 31 P-NMR experiments were carried out at 37C recording the data every 12 min for 14 h. Data were processed and analyzed with the Bruker TopSpin 2.1 programme.

  3 H]deoxyuridine ([5-3 H]dUrd) or [5-3 H]deoxycytidine ([5-3 H]dCyd). Indeed, after intracellular conversion of [5-3 H]dUrd or [5-3 H]dCyd to [5-3 H]dUMP, the C-5 tritium atom on the pyrimidine base is released during the TS-catalysed reductive methylation. The ability of 5-FdUrd and its prodrug NUC-3073 to inhibit tritium release from [5-3 H]dUrd and [5-3 H]dCyd was therefore evaluated in L1210/0 cell cultures at a variety of compound concentrations. 5-FdUrd proved to be a potent inhibitor of TS in situ. Its IC 50 for tritium release from [5-3 H]dCyd and [5-3 H]dUrd was around 0.0007 -0.0009 µM (Table 2). The inhibitory activity of NUC-3073 on tritium release was much less (~ 200-fold) pronounced than that of 5-FdUrd, especially after only 30 min preincubation of A c c e p t e d M a n u s c r i p t 19 the cells with the drugs (IC 50 : 0.16-0.19 µM). However, longer pre-incubation times of the cells (up to 4 h) with 5-FdUrd and NUC-3073 before measuring TS activity in the intact tumour cells revealed a much more pronounced inhibitory activity of the prodrug against TS

A c c e p t e d M a n u s c r i p t 20 3. 5 10 and 11 , 5 .

 20510115 Metabolism of 5-FdUrd phosphoramidatesAs shown in Fig.4, the putative mechanism of activation of ProTides inside the cell, after uptake, involves a first enzymatic activation step mediated by a carboxypeptidase-type enzyme that hydrolyzes the ester of the aminoacyl moiety (step a, compound 9). Next, a spontaneous cyclization followed by subsequent, spontaneous displacement of the aryl group (step b) and opening of the unstable ring mediated by water (step c) can give compounds respectively. The last step involves a hydrolysis of the P-N bond mediated by a phosphoramidase-type enzyme (step d) with release of the nucleoside monophosphate[START_REF] Congiatu | Novel potential anticancer naphthyl phosphoramidates of BVdU: separation of diastereoisomers and assignment of the absolute configuration of the phosphorus center[END_REF] in the intact cell[13;35].To prove the proposed metabolic scheme for NUC-3073 (1) and to determine whether the ester motif of the 5-FdUrd phosphoramidate derivative is cleaved-off or not, we carried out an enzyme incubation experiment designed to mimic the first stages of the putative activation in the intact tumour cells. The compound 1 was incubated with carboxypeptidase Y (also known as cathepsin A) in TRIZMA buffer and its conversion was monitored by31 P-NMR. Spectra were recorded for 14 h acquiring scans at the periodic intervals every 4 min as shown in Fig. For a better resolution original spectra (lower graphs) and deconvoluted ones (upper graphs) are shown. Two peaks at 4.07; 4.23 ppm in the 31 P-NMR spectrum correspond to two diastereoisomers of the parent compound 1. Upon the addition of cathepsin A, during the first 4 min of the experiment the compound 1 was hydrolyzed to the corresponding intermediate(s) 9 (4.95;

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

 21 intermediate within 26 min, which further stayed consistently present throughout the 14 h of the assay. The cleavage of the P-N bond releasing the nucleoside monophosphate was not detected in the enzyme experiment, as expected. This experiment indicates that the first activation step of ProTide NUC-3073 (1) may be sufficiently efficient, and therefore, may allow the eventual delivery of the nucleoside monophosphate metabolite in the intact tumour cells, following subsequent phosphoramidase-mediated cleavage of the amino acid to liberate FdUMP.

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

 22 spectra displayed a single peak inherent to the human serum at ~ 2.00 and the double peak of the parent at ~ 4.50 which after 4 h and 15 min was partly hydrolyzed to the aminoacyl phosphoramidate intermediate shown as a single peak at 7.20.

23

 23

A c c e p t e d M a n u s c r i p t 25 or

 25 mammalian (i.e. human erythrocyte) TP. Also, UP (derived from human tumour tissue)

A c c e p t e d M a n u s c r i p t 26 is

 26 often upregulated in tumours or may be externally expressed by mycoplasma infection of the tumour tissue. We wish to thank Ria Van Berwaer, Lizette van Berckelaer, Kristien Minner and Christiane Callebaut for their excellent technical assistance and Dr. Tarmo Roosild for generously providing human uridine phosphorylase hUPP1. Johan Vande Voorde acknowledges a PhD grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

A c c e p t e d M a n u s c r i p t 33 Table 2 .A c c e p t e d M a n u s c r i p t 34 Table 3 .Fig. 1 .Fig. 2 .Fig. 3 .

 332343123 Fig. 1. Structural formula of 5-FdUrd and its phosphoramidate prodrug NUC-3073.

Fig. 4 .

 4 Fig. 4. Proposed putative mechanism of activation of 5-FdUrd ProTides.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Carboxypeptidase-mediated cleavage of prodrug NUC-3073 (1) monitored by 31 P

Fig. 7 .

 7 Fig. 7. 31 P NMR spectrum of compound NUC-3073 (1) in buffer pH = 1.

Fig. 8 .Fig. 2

 82 Fig. 8. 31 P NMR spectrum of compound NUC-3073 (1) in buffer pH = 8.

2.1.3. Synthesis of 1-Naphthyl-(benzyl-L-alaninyl) phosphorochloridate (6). Anhydrous

	8
	to a stirred solution of 1-naphthyl dichlorophosphate (2) (2.50 g, 9.57 mmol) and L-alanine
	benzyl ester p-tosylate salt (NovaBiochem) (3.36 g, 9.57 mmol), in anhydrous DCM (Sigma-
	Aldrich) (50 mL). The reaction mixture was stirred at -78C for 30 min. and then allowed to
	slowly warm to room temperature and stirred for 1 h. A formation of the desired compound
	was monitored by 31 P-NMR. After the reaction was completed, the solvent was removed
	M under reduced pressure and the crude residue was purified by flash chromatography (hexane/EtOAc 7/3) to give the product (6) as an oil (1.82 g, 47%) [R f = 0.90 (hexane-EtOAc, 7:3)], 31 P-NMR (202 MHz, CDCl 3 , mixture of diastereoisomers):  P 7.92, 8.14 (Int.: 1.00:1.00); 1 H-NMR (500 MHz, CDCl 3 , mixture of diastereoisomers with a ratio of 1:1):  H 1.42-1.45 (m, 3H, CHCH 3 ), 4.20-4.23 (m, 1H, CHCH 3 ), 4.78-4.81 (m, 1H, NH), 5.09 (s, 2H, a n u s c r i p t OCH 2 Ph), 7.09-7.73 (m, 11H, ArH), 7.97-8.12 (m, 1H, ArH).
	1 h. Subsequently the reaction mixture was allowed to slowly warm up to room temperature for 3 h. A formation of the desired compound (2) was monitored by 31 P-NMR. After the reaction was completed, the resulting mixture was filtered and then evaporated in vacuo under nitrogen to afford the crude colorless oil as product, which was used without further purification in the next step (4.59 g, 84%) [R f = 0.93 (hexane-EtOAc (Fisher Scientific, Leicestershire, UK), 1:1)], 31 P-NMR (202 MHz, CDCl 3 ):  P 5.07; 1 H-NMR (500 MHz, A c c e p t e d
	CDCl 3 ):  H 7.52-7.71 (m, 4H, ArH), 7.86-7.89 (m, 1H, ArH), 7.95-7.98 (m, 1H, ArH), 8.16-
	8.19 (m, 1H, ArH).
	triethylamine (2.66 mL, 19.14 mmol) was added dropwise under argon atmosphere at -78 C

1-Naphthyl dichlorophosphate (2): Phosphorus oxychloride (1.94 mL, 20.81 mmol) was added to a solution of 1-naphthol (3.00 g, 20.81 mmol) in diethyl ether (Sigma-Aldrich) (70 mL) under argon atmosphere, then anhydrous triethylamine (2.9 mL, 20.81 mmol) was added dropwise at -78 o C and the resulting reaction mixture was stirred for

2.1.4. Synthesis of 5-Fluoro-2'-deoxyuridine-5'-O-[-naphthyl (benzyl-L-alaninyl)] phosphate (1). To

	9
	167.25; 1 H NMR (500 MHz, MeOD):  H 8.18-8.12 (m, 1H, ArH), 7.90-7.86 (m, 1H, ArH),
	7.72-7.67 (m, 2H, ArH, H-6), 7.55-7.47 (m, 3H, ArH), 7.45-7.27 (m, 6H, ArH), 6.16-6.06 (m,
	1H, H-1'), 5.13, 5.08 (2 x AB system, 2H, J = 12.0 Hz, OCH 2 Ph), 4.36-4.24 (m, 3H, 2 x H-5',
	H-3'), 4.15-4.03 (m, 2H, CHCH 3 , H-4'), 2.17-2.08 (m, 1H, H-2'), 1.79-1.67 (m, 1H, H-2'),
	1.38-1.34 (m, 3H, CHCH 3 ); 13 C NMR (125 MHz, MeOD):  C 174.9 (d, 3 J C-P = 4.3 Hz, C=O,
	M ester), 174.6 (d, 3 J C-P = 5.0 Hz, C=O, ester), 159.3 (d, 2 J C-F = 26.1 Hz, CH-base), 150.5 (d, 4 J C-F = 4.0 Hz, C=O, base), 147.9 (d, 2 J C-P = 7.4 Hz, C-Ar, Naph), 147.8 (d, 2 J C-P = 7.7 Hz, C-Ar, Naph), 141.7 (d, 1 J C-F = 233.9 Hz, CF), 141.6 (d, 1 J C-F = 233.8 Hz, CF), 137.2, 137.1, 136.2 (C-Ar), 129.7, 129.6, 129.5, 129.4, 129.0, 128.9, 128.1, 128.0 (CH-Ar), 127.9, 127.8 (C-Ar), 127.7, 127.6, 126.6, 126.5, 126.2, 125.7, 125.6, 125.4, 125.3, 122.6 (CH-Ar), 116.5 (d, 3 J C-P = 3.5 Hz, CH-Ar), 116.2 (d, 3 J C-P = 3.3 Hz, CH-Ar), 87.0, 86.9 (C-1'), 86.8, 86.7 (2x a n u s c r i p t d, 3 J C-P = 8.1 Hz, C-4'), 72.1, 72.0 (
	a solution of 5-fluoro-2'-deoxyuridine (Carbosynth) (0.25 g, 1.01 mmol) in dry THF (10 mL) at 0C under argon atmosphere, NMI (0.40 mL, 5.07 mmol) was added dropwise. The reaction mixture was allowed to stir for 30 min, and then a solution of 1-naphthyl-(benzyl-L-alaninyl) phosphorochloridate (6) (0.82 g, 3.04 mmol) dissolved in anhydrous THF (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 16 h and then evaporated in vacuo to give a residue that was re-dissolved in CH 2 Cl 2 (Fisher Scientific), washed twice with 0.5 M HCl (Sigma-Aldrich) (2 x 5 mL). The A c c e p t e d
	organic phase was purified by column chromatography on silica gel, eluting with CH 2 Cl 2 -
	MeOH (Fisher Scientific) as a gradient (0% to 5% MeOH) to afford the product 1 as colorless
	solid (47.0 mg, 8%) [R f = 0.19 (CH 2 Cl 2 -MeOH, 95:5)], (Found: MNa + , 636.1520.
	C 29 H 29 N 3 O 9 FNaP requires [MNa + ], 636.1523); 31 P NMR (202 MHz, MeOD, mixture of
	diastereoisomers 43% : 57%):  P 4.61, 4.25; 19 F NMR (470 MHz, MeOD):  F -167.45, -

C-3'), 68.1, 68.0 (CH 2 Ph), 67.8, 67.6 (2 x d, 2 J C-P = 5.2 Hz, C-5'), 51.9, 51.8 (CHCH 3 ), 40.9, 40.8 (C-2'), 20.5 (d, 3 J C-P = 6.5 Hz, CHCH 3 ), 20.3 (d, 3 J C-P = 7.6 Hz, CHCH 3 ); m/z (ES) 636 (MNa + , 100%), Reverse HPLC eluting with (H 2 O/MeOH from 100/0 to 0/100) in 45 min, showed two peaks of the diastereoisomers with t R 34.23 min and t R 34.59 min. Analytically calculated for C 29 H 29 FN 3 O 9 P: C, 56.77; H, 4.76; N, 6.85. Found: C, 56.57; H, 5.06; N, 6.72; UV (0.05 M phosphate buffer (Sigma-Aldrich), pH 7.4)  max = 271 nm ( max = 7050); Log P (octanol (Sigma-Aldrich): water) measured: 1.7, calculated 3.53 (Chemoffice 11.0).

  5-Fluoro-2'-deoxyuridine-3'-O-[-naphthyl (benzyl-L-alaninyl)] phosphate (8) was obtained as a colorless solid (15.0 mg, 2%) [Rf = 0.50 (CH 2 Cl 2 -MeOH, 95:5)], (Found: MNa + , 636.1525 C 29 H 29 N 3 O 9 NaPF requires [MNa

+ ], 636.1523);

31

P-NMR (202 MHz, MeOD):  P 3.79, 3.11; 19 F-NMR (470 MHz, MeOD):  F -168.62, -168.53; 1 H-NMR (500 MHz, MeOD):  H 8.24-8.15 (m, 2H, ArH), 7.94-7.88 (m, 1H, ArH), 7.73 (d, 1H, 3 J C-F = 7.9 Hz, H-6), 7.60-7.40 (m, 3H, ArH), 7.38-7.24 (m, 6H, ArH), 6.28-6.17 (m, 1H, H-1'), 5.14-5.11 (m, 2H, CH 2 Ph), 4.21-4.03 (m, 2H, CHCH 3 , H-3'), 3.93-3.63 (m, 3H, 2 x H-5', H-4'), 2.51-2.43 (m, 1H, H-2'), 2.32-2.23 (m, 1H, H-2'), 1.37 (d, 3H, J = 7.2 Hz, CHCH 3 , one diast.), 1.35 (d, 3H, J = 7.2 Hz, CHCH 3 , one diast.); 13 C NMR (125 MHz, MeOD): 174.6 (d, 3 J C-P = 4.8 Hz, C=O ester), 174.4 (d, 3 J C-P = 5.1 Hz, C=O ester), 159.1 (d, 2 J C-F = 26.0 Hz, C-base), 150.2 (d, 4 J C-F = 4.1 Hz, C=O, base), 148.0 (d, 2 J C-P = 7.2 Hz, C-Ar, Naph), 147.9 (d, 2 J C-P = 7.2 Hz, C-Ar, Naph), 141.4 (d, 1 J C-F = 233.0 Hz, CF), 141.2 (d, 1 J C-F = 233.0 Hz, CF), 137.5, 137.4, 136.3 (C-Ar), 130.8, 129.6, 129.5, 129.3, 129.2, 129.0, 128.9, 128.1, 128.0, 127.8, 127.6, 126.2, 126.1, 125.7, 122.6, 116.7, 116.3, 106.5 (CH-Ar), 87.5, 87.4 (C-1'), 86.6 (C-4'), 79.7 (d, 4 J C-P = 4.9 Hz, C-3'), 68.0 (C-5'), 62.6 (CH 2 Ph), 51.8 (CHCH 3 ), 39.9 (C-2'), 20.4, 20.3 (CHCH 3 ); m/z (ES) 613 (M + , 100%); Reverse-phase HPLC eluting with H 2 O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, l = 275 nm, showed two peaks of the diastereoisomers with t R 26.69 min. and t R 27.41 min. (23% : 72%).

Table 1 .

 1 This work is supported by a grant of NuCana BioMed, the Kamoshida S, Shiogama K, Shimomura R, Inada K, Sakurai Y, Ochiai M, et. al. Immunohistochemical demonstration of fluoropyrimidine-metabolizing enzymes in various types of cancer. Oncol Rep 2005;14:1223-30. 42. Ciaparrone M, Quirino M, Schinzari G, Zannoni G, Corsi DC, Vecchio FM, et. al. Gago F, Balzarini J, Liekens S. The dual role of thymidine phosphorylase in cancer development and chemotherapy. Med Res Rev 2009;29:903-53. 46. Lee WA, He GX, Eisenberg E, Cihlar T, Swaminathan S, Mulato A, Cundy KC. Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue. Antimicrob Agents Chemother 2005;49:1898-906. Cytostatic activity of 5-FdUrd and NUC-3073 as represented by the IC 50 value in different cell lines a 50% Inhibitory concentration or compound concentration required to inhibit tumour cell proliferation

	Concerted Actions of the K.U.Leuven (GOA 10/014) and the "Fonds voor Wetenschappelijk
	Onderzoek (FWO) Vlaanderen" (Credit n° G. 0486.08)


Ruth Hoffmann

Karin Von Schwarzenberg

Nancy López-Antón

Anita Rudy

Gerhard Wanner

Verena M Dirsch

Angelika M Vollmar
email: angelika.vollmar@cup.uni-muenchen.de

Helenalin bypasses Bcl-2-mediated cell death resistance by inhibiting NF-κB and promoting reactive oxygen species generation

Keywords: sesquiterpene lactone, apoptosis, chemoresistance, autophagy, ERstress

Evasion of cell death by overexpression of anti-apoptotic proteins, such as Bcl-2, is commonly observed in cancer cells leading to a lack of response to chemotherapy.

Hence, there is a need to find new chemotherapeutic agents that are able to overcome chemoresistance mediated by Bcl-2 and to understand their mechanisms of action. Helenalin, a sesquiterpene lactone (STL), induces cell death and abrogates clonal survival in a highly apoptosis-resistant Bcl-2 overexpressing Jurkat cell line as well as in two other Bcl-2 overexpressing solid tumor cell lines (mammary MCF-7; pancreatic L6.3pl). This effect is not achieved by directly affecting the mitochondriaprotective function of Bcl-2 in the intrinsic pathway of apoptosis since Bcl-2 overexpressing Jurkat cells do not show cytochrome c release and dissipation of mitochondrial membrane potential upon helenalin treatment. Moreover, helenalin induces an atypical form of cell death with necrotic features in Bcl-2 overexpressing cells, neither activating classical mediators of apoptosis (caspases, AIF, Omi/HtrA2, Apaf/apoptosome) nor ER-stress mediators (BiP/GRP78 and CHOP/GADD153), nor autophagy pathways (LC3 conversion). In contrast, helenalin was found to inhibit NF-κB activation that was considerably increased in Bcl-2 overexpressing Jurkat cells and promotes cell survival. Moreover, we identified reactive oxygen species (ROS) and free intracellular iron as mediators of helenalin-induced cell death whereas activation of JNK and abrogation of Akt activity did not contribute to helenalin-elicited cell death. Our results highlight the NF-κB inhibitor helenalin as a promising chemotherapeutic agent to overcome Bcl-2-induced cell death resistance.

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

Introduction

Chemotherapy remains a fundamental part in the treatment of cancer.

Chemoresistance of cancer cells, however, continues to be a major problem [START_REF] Fulda | Tumor resistance to apoptosis[END_REF].

Current anti-cancer treatments kill tumor cells by inducing apoptosis or other forms of programmed cell death [START_REF] Edinger | Death by design: apoptosis, necrosis and autophagy[END_REF]. Apoptosis can be triggered either at the cell surface (extrinsic pathway) or at the mitochondria (intrinsic pathway) [START_REF] Edinger | Death by design: apoptosis, necrosis and autophagy[END_REF]. Many standard chemotherapeutics act via mitochondria, inducing cytochrome c release into the cytosol , which results in the assembly of the apoptosome and the activation of caspase-9, initiating a caspase cascade leading to apoptotic cell death. In contrast, when AIF (apoptosis-inducing factor), Omi/HtrA2 (high-temperature-requirement protein A2) or endonuclease G are released into the cytosol, a caspase-independent cell death can occur [START_REF] Green | The pathophysiology of mitochondrial cell death[END_REF].

Members of the Bcl-2 (B-cell lymphoma) family are important regulators of the intrinsic pathway of apoptosis [START_REF] Reed | Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms, physiological roles, and therapeutic opportunities[END_REF][START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF]. They control mitochondrial outer membrane permeability promoting or inhibiting the release of mitochondrial proteins. Bcl-2, the first member of this family discovered, protects cells from apoptosis by inhibiting oligomerisation and pore formation induced by the pro-apoptotic Bcl-2 family members Bax and Bak or by sequestering pro-apoptotic BH3-only proteins. Since overexpression of Bcl-2 often confers chemoresistance, Bcl-2 is a promising therapeutic target [START_REF] Fulda | Tumor resistance to apoptosis[END_REF][START_REF] Yip | Bcl-2 family proteins and cancer[END_REF][START_REF] Vogler | Bcl-2 inhibitors: small molecules with a big impact on cancer therapy[END_REF]. Bcl-2 is also localized at membranes of the endoplasmic reticulum (ER) where it regulates Ca 2+ homeostasis by desensitizing cells to stimuli that cause the release of ER-sequestered Ca 2+ [START_REF] Pinton | Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum[END_REF][START_REF] Szegezdi | Bcl-2 family on guard at the ER[END_REF]. Autophagy is another strongly regulated cellular program, in which's regulation Bcl-2 is involved [START_REF] Levine | Bcl-2 family members: dual regulators of apoptosis and autophagy[END_REF]. Autophagy has been interpreted as a form of non-apoptotic death as well as a survival strategy during stress [START_REF] Hoyer-Hansen | Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium[END_REF]. Recently, a link between Bcl-2 and constitutive activation of nuclear factor B (NF-B) [START_REF] De Moissac | Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis[END_REF][START_REF] Regula | IKK beta is required for Bcl-2-mediated NF-kappa B activation in ventricular myocytes[END_REF][START_REF] Grimm | Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis[END_REF][START_REF] Heckman | NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells[END_REF], another crucial player for cell survival and apoptosis resistance in cancer [START_REF]Nuclear factor-kappaB in cancer development and progression[END_REF], has been described.

We could previously show that the sesquiterpene lactone helenalin, a secondary plant metabolite, induces apoptosis in Jurkat leukemia cells, which was caspase dependent and moreover cell death in Jurkat cells overexpressing Bcl-2 [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF].

Obviously, helenalin is able to overcome Bcl-2-mediated chemoresistance, however the mechanism is still unknown. Aim of this study, therefore, was to characterize the mechanism of cell death induced by helenalin in Bcl-2 overexpressing cancer cells.

Materials and Methods

Compounds

Helenalin, purchased from Axxora (Lörrach, Germany), was dissolved in DMSO (final concentration < 0.1%). Other chemicals were purchased from Merck (Darmstadt, Germany) (i.e. 3-MA, Etoposide, Necrostatin-1 Q-VD-OPh); Sigma-Aldrich (Taufkirchen, Germany) (BMS-345541, Deferoxamine mesylate, DMSO, N-acetyl-Lcysteine, Paclitaxel, Propidium iodide, ß-phenylethyl isothiocyanate); Axxora (Lörrach, Germany) (DCDHF diacetate, JC-1-iodide); Molecular Probes/Invitrogen (Karlsruhe, Germany) (goat anti-mouse IgGs), Bachem (Bubendorf, Germany) (Ac-DEVD-AFC, Ac-LETD-AFC), BIOTREND GmbH (Cologne, Germany) (Fura-2-AM, Bender MedSystems (Vienna, Austria) (Human Annexin V-FITC kit) or Repro Tech GmbH (Hamburg, Germany) (TNF-).

Cell culture

Human leukemia Jurkat T cells (J16), and Jurkat T cells transfected with vector control (Neo Jurkat) or Bcl-2 (Bcl-2 Jurkat) (both kindly provided by Drs. P.H.

Krammer and H. Walczak, Heidelberg, Germany) were cultured as previously described [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF]. MCF-7 cells were purchased from DSMZ (ACC 115). The highly metastatic human pancreatic cancer cell line L3.6pl was cultivated as originally described [START_REF] Rothmeier | The marine compound spongistatin 1 targets pancreatic tumor progression and metastasis[END_REF]. Bcl-2 overexpressing MCF-7 or L3.6pl cells (Bcl-2 MCF-7, Bcl-2 L3.6pl) and the corresponding empty-vector control cell lines (Neo MCF-7, Neo L3.6pl) were created by transfection with the pcDNA3 Bcl-2 vector (Addgene plasmid 8768) and empty vector pcDNA3 (Invitrogen/Molecular Probes, Karlsruhe, Germany), respectively, using the Amaxa ® Cell Line Nucleofector Kit ® V (LONZA Cologne AG, Cologne, Germany).

Quantification of cell death

The following protocols were used: A, Subdiploid-DNA content was quantified as previously described [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF][START_REF] Nicoletti | A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[END_REF]; B, Propidium iodide (PI, 5 µg/ml, 30 min at room temperature in the dark) staining was monitored by flow cytometry (FACSCalibur;

Becton Dickinson, Heidelberg, Germany). C, Annexin-V/PI double staining was performed using a human Annexin V-FITC Kit (Bender MedSystems, Vienna, Austria).

Clonogenic assay

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

The clonogenic assay was carried out as previously described [START_REF] Lopez-Anton | The marine product cephalostatin 1 activates an endoplasmic reticulum stress-specific and apoptosome-independent apoptotic signaling pathway[END_REF] and colonies were scored after 7 days of culture using the analysis system from S.CO LifeScience (Garching, Germany). The clonogenic assay with Neo L3.6pl and Bcl-2 L3.6pl cells was performed as previously described [START_REF] Rothmeier | The marine compound spongistatin 1 targets pancreatic tumor progression and metastasis[END_REF]. Cells were stained with crystal violet (0.5% crystal violet in 20% methanol) and quantified by absorption measurement (550 nm; SpectraFluor Plus TM (Tecan; Männedorf, Austria)).

Apaf-1 and AIF siRNA

Sense and antisense siRNA oligonucleotides corresponding to nucleotides 978-998 of Apaf-1 [START_REF] Lassus | Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization[END_REF] (AATTGGTGCACTTTTACGTGA), to the AIF nucleotides [START_REF] Park | Phytosphingosine in combination with ionizing radiation enhances apoptotic cell death in radiation-resistant cancer cells through ROS-dependent and -independent AIF release[END_REF] (GGAAATATGGGAAAGA-TCCdTdT) and oligonucleotides corresponding to a scramble sequence were purchased from Biomers.net GmbH (Ulm, Germany). Bcl-2

Jurkat cells were transfected with 3 µg of scramble, Apaf-1 or AIF siRNAs using the Amaxa ® Cell Line Nucleofector Kit ® V (LONZA Cologne AG, Cologne, Germany).

Cells were treated 48 h after transfection.

Measurement of cytochrome c release

Release of cytochrome c from mitochondria was analyzed as previously described [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF]. Purity of cytosolic fractions was assessed by incubating membranes with VDAC antibody (Cell signalling, Frankfurt, Germany)

Western blot analysis

Samples were treated as described before [START_REF] Lopez-Anton | The marine product cephalostatin 1 activates an endoplasmic reticulum stress-specific and apoptosome-independent apoptotic signaling pathway[END_REF] and membranes were incubated overnight at 4°C with the following specific antibodies: Apaf-1 (BD Transduction Laboratories, Heidelberg, Germany), Bcl-2, eIF2α (D-3), caspase- Nuclear extracts were prepared and EMSA was carried out using P 32 -labeled NF-B consensus sequence oligonucleotides (Promega, Heidelberg, Germany) as previously described [START_REF] Hoffmann | A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia-reperfusion[END_REF]. Gels were exposed to Cyclone Storage Phosphor Screens (Canberra-Packard, Schwadorf, Austria) followed by analysis with a phosphor imager station (Cyclone Storage Phosphor System, Canberra-Packard). The composition of p65/p50 of NF-B was confirmed by supershift analysis using antibodies against p65 and p50, respectively [START_REF] Kiemer | Induction of IkappaB: atrial natriuretic peptide as a regulator of the NF-kappaB pathway[END_REF].

Measurement of mitochondrial potential

The measurement was performed as previously described [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF]. Cells were stained with 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1) (Axxora, Lörrach, Germany) at a final concentration of 1.25 µM for 15 min at 37°C and analyzed by flow cytometry.

Caspase activity assay

Caspase activity was determined as described before [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF]. Caspase substrates: Ac-LETD-AFC for caspase-8 or Ac-DEVD-AFC (Bachem, Bubendorf, Germany) for caspase-3, respectively. Caspase activity was monitored by a plate-reading A c c e p t e d M a n u s c r i p t multifunction photometer (SpectraFluor PlusTM, Tecan, Männedorf, Austria) after 2 h at 37°C.

Transmission Electron Microscopy (TEM)

Jurkat and Bcl-2 Jurkat cells were left untreated (Co) or treated with helenalin for 8 hours and TEM was carried out as described before [START_REF] Lopez-Anton | Sesquiterpene lactones induce distinct forms of cell death that modulate human monocytederived macrophage responses[END_REF].

Statistical analysis

All experiments were performed at least three times.

Helenalin overcomes Bcl-2-mediated resistance

Overexpression of the antiapoptotic protein Bcl-2 in Jurkat T-cells confers death resistance against etoposide but not helenalin, confirming published data [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF].

Moreover, helenalin almost completely inhibited colony growth in Jurkat cells overexpressing Bcl-2. In contrast, the classical chemotherapeutic agent etoposide is not able to suppress the clonogenic survival of Bcl-2 overexpressing Jurkat cells (Fig. 1a). To verify that helenalin not only overcomes Bcl-2 resistance in leukaemia cells, we stably overexpressed Bcl-2 in the breast cancer cell line MCF-7 and the highly metastatic human pancreatic carcinoma cell line L3.6pl. Helenalin induced cell death in both cell lines (supplement fig. 1). Moreover, helenalin inhibited colony growth in Bcl-2 overexpressing L3.6pl cells that were clearly insensitive towards paclitaxel (Fig. 1b).

Helenalin does not abrogate mitochondrial function of Bcl-2 and acts independently of mitochondria and the apoptosome

Next, we focused on the mechanism leading to cell death in Bcl-2 overexpressing Jurkat cells. Helenalin did not alter Bcl-2 protein level, nor did it induce Bcl-2 phosphorylation as seen after paclitaxel treatment (Fig. 2a). Moreover, helenalin does not abrogate the function of Bcl-2 on mitochondria since the mitochondrial membrane potential was kept nearly intact and only minute amounts of cytochrome c

were released into the cytosol in Bcl-2 overexpressing compared to vector control (Neo Jurkat) cells treated with helenalin (Fig. 2b-c).

To underline these findings, transmission electron microscopy (TEM) was performed.

In wild-type Jurkat cells exposed to 20 µM helenalin mitochondria showed severe ultrastructural changes: all stages of desintegration were observed starting from swelling of the cristae and compaction of the matrix, up to mitochondrial "ghosts" with only residual inner membrane and cristae visible (Fig. 3, upper part). These structural findings concur with biochemical data: In wild-type Jurkat cells, loss of mitochondrial membrane potential, cytochrome c release and caspase activation was observed in response to helenalin [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF]. In contrast, in Bcl-2 overexpressing Jurkat cells exposed to 20 µM helenalin mitochondria remain structurally more or less uninfluenced (Fig. 3, lower part) which is in accordance with a lack of cytochrome c release and an intact Silencing of Apaf-1 via siRNA did not influence the induction of cell death by helenalin (Fig. 4a). This fact further suggests that helenalin-induced cell death occurs independently of the apoptosome and mitochondria. Accordingly, caspase activity measurements (Fig. 4b and supplement fig. 2 a,b) and employment of the pancaspase inhibitor Q-VD-OPh (Fig. 4c) negated a role for caspases in helenalininduced cell death in Bcl-2 overexpressing cells. The chemical β-phenylethyl isothiocyanate (PEITC) was used as positive control since it is known to induce a caspase-dependent cell death in Bcl-2 Jurkat cells [START_REF] Thomson | Phenethyl isothiocyanate triggers apoptosis in Jurkat cells made resistant by the overexpression of Bcl-2[END_REF].

Examining caspase-independent apoptotic factors such as the serine protease

Omi/HtrA2 and the endonuclease AIF by use of siRNA against AIF and the Omi inhibitor UCF showed that these factors are also not involved in helenalin-mediated cell death (supplement fig. 2c,d). Moreover, phosphatidylserine translocation to the outer cell membrane (analyzed by Annexin V staining), a classical sign of early apoptosis, was not visible in Bcl-2 overexpressing cells after helenalin treatment for 8 h, whereas the vector control cells (Neo Jurkat cells) were clearly Annexin V positive (Fig. 4d).

Potential mechanisms used by helenalin to bypass Bcl-2-mediated cytoprotection

We analyzed potential non-apoptotic events leading to helenalin-mediated cell death, such as autophagy and ER-stress. ER-stress induction has recently been linked to induction of autophagy [START_REF] Hoyer-Hansen | Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium[END_REF][START_REF] Li | The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells[END_REF]. Helenalin did not affect the expression of BiP/GRP78 and CHOP/GADD153, two critical UPR-upregulated proteins during ER-stress (Fig. 5a). Furthermore, increased Ca 2+ -levels, as seen after treatment with thapsigargin, an inhibitor of the endoplasmatic reticulum Ca 2+ ATPase (SERCA), was not observed after stimulation with helenalin (supplement fig. 3a,b). However, a strong transient activation of JNK, a rapid phosphorylation of eIF2α and a slightly reduced phosphorylation of mTOR in Bcl-2 overexpressing cells was detected upon helenalin treatment (Fig. 5b and supplement fig. 3b,c). All three events have been linked to induction of autophagy [START_REF] Hoyer-Hansen | Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium[END_REF][START_REF] Li | The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells[END_REF]. Nevetheless, no accumulation of LC3 II, commonly observed during autophagy, could be detected by Western blot analysis (Fig. 5c),

A c c e p t e d M a n u s c r i p t 10 and 3-MA, a well established inhibitor of autophagy [START_REF] Mizushima | Methods for monitoring autophagy[END_REF], partly diminished helenalininduced cell death after 16 h stimulation but not after 48 h (Fig. 5d). Furthermore TEM analysis of helenalin treated cells did not reveal significant occurrence of autophagolysosomes (Fig. 3).

We then examined mediators involved in signaling events of programmed necrosis.

Necrostatin-1 (Nec-1) inhibits RIP1 and acts as a specific inhibitor of necroptotic cell death [START_REF] Degterev | Expansion and evolution of cell death programmes[END_REF]. Exposure of Bcl-2 Jurkat cells to Nec-1 prior to helenalin treatment did not prevent cell death (Fig. 5e), arguing against necroptosis as a mode of cell death induced by helenalin. In contrast, Nec-1 abrogated necroptosis induced by TNF-α in the presence of cycloheximide and the pan-caspase inhibitor Q-VD-OPh in Jurkat cells (positive control) [START_REF] Degterev | Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury[END_REF] (Fig. 5e insert). An early JNK activation as observed in response to helenalin has been associated with programmed necrosis [START_REF] Degterev | Expansion and evolution of cell death programmes[END_REF]. Yet, JNK activation by helenalin does not contribute to cell death as indicated by the use of SP600125, a commonly used JNK inhibitor (activity was confirmed by analyzing the phosphorylation of the JNK downstream target c-Jun) (Fig. 5f and insert).

In addition, helenalin reduced Akt activation (supplement fig. 3d), which is an important survival factor in cancer cells [START_REF] Franke | PI3K/Akt: getting it right matters[END_REF]. However, in Bcl-2 overexpressing cells reduction of Akt activity does not seem to be involved in cell death as wortmannin, a potent inhibitor of Akt phosphorylation, does neither induce cell death itself nor adds to the toxicity of helenalin in Bcl-2 overexpressing Jurkat cells (supplement fig. 3e).

Role of NF-κB

Since a link of Bcl-2 and the NF-κB signaling pathway has been described for other cell lines [START_REF] De Moissac | Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis[END_REF][START_REF] Regula | IKK beta is required for Bcl-2-mediated NF-kappa B activation in ventricular myocytes[END_REF][START_REF] Grimm | Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis[END_REF][START_REF] Heckman | NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells[END_REF][START_REF] Ricca | bcl-2 overexpression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells[END_REF], we examined 1) whether Bcl-2 overexpressing Jurkat cells show increased NF-κB activity and if so, 2) whether helenalin is able to interfere with it. As shown in Fig. 6a, NF-κB-dependent gene transactivation as well as DNA binding activity were increased in Bcl-2 overexpressing Jurkat cells. NF-κB is known to be an important mediator of survival and death resistance in leukemia cells [START_REF] Kiessling | Inhibition of constitutively activated nuclear factor-kappaB induces reactive oxygen species-and irondependent cell death in cutaneous T-cell lymphoma[END_REF].

Helenalin significantly reduced NF-κB-dependent gene transactivation and DNA binding activity in Bcl-2 overexpressing cells (Fig. 6b). In addition, also the NF-κB inhibitor BMS-345541, acting on the IκB kinase complex, induced cell death in Bcl-2protected cells (supplement fig. 4a) pointing to NF-κB as an attractive target against Bcl-2-conferred cell death resistance. In search of a potential mechanism downstream of NF-κB we measured intracellular ROS level. Helenalin (20 µM)

A c c e p t e d M a n u s c r i p t increased intracellular ROS level comparable to H 2 O 2 (17, 6 µM), which was used as a positive control (Fig. 7a). An antioxidant such as NAC completely prevented and the radical scavenger Tiron attenuated cell death induced by helenalin in Bcl-2

overexpressing Jurkat cells indicating that the Bcl-2 resistance overcoming mechanism of helenalin requires ROS production (Fig. 7b). Moreover, helenalininduced cell death involves iron ions since the use of the iron chelator desferrioxamine (DFO) inhibits cell death induction by helenalin (Fig. 7c). Thus, ROS production by helenalin might partly depend on the Fenton reaction as reported by others [START_REF] Kiessling | Inhibition of constitutively activated nuclear factor-kappaB induces reactive oxygen species-and irondependent cell death in cutaneous T-cell lymphoma[END_REF]. Since BMS induced inhibition of NF-B only partly abrogated ROS response by helenalin (supplement fig. 4b) ROS induction by helenalin seems to occur both upstream as well as downstream of NF-B.

Discussion

The present paper highlights helenalin as a small molecule that is able to overcome Bcl-2-mediated chemoresistance in tumor cells. The major mechanisms that mediate this important feature are 1) the inhibition of Bcl-2-induced upregulation of NF-κB activity and 2) the generation of reactive oxygen species.

Sesquiterpene lactones (STL) such as helenalin comprise a large group of secondary plant metabolites with various biological activities that are linked to the high reactivity of their α,β-unsaturated carbonyl structures with thiol groups by a Michael-type reaction [START_REF] Schmidt | Helenanolide-type sesquiterpene lactones--III. Rates and stereochemistry in the reaction of helenalin and related helenanolides with sulfhydryl containing biomolecules[END_REF]. Interestingly, helenalin has recently gained considerable attention as a lead structure for the treatment of inflammation [START_REF] Lyss | The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65[END_REF][START_REF] Berges | Helenalin suppresses essential immune functions of activated CD4+ T cells by multiple mechanisms[END_REF], whereas the related STL parthenolide has been preferentially appreciated as experimental tumor drug [START_REF] Guzman | An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells[END_REF][START_REF] Kim | Susceptibility of cholangiocarcinoma cells to parthenolide-induced apoptosis[END_REF][START_REF] Steele | The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro[END_REF].

However, our work and several other groups attribute an in vitro as well as in vivo antitumor activity also to helenalin [START_REF] Dirsch | Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpression of Bcl-x(L) or Bcl-2[END_REF][START_REF] Lee | Sesquiterpene antitumor agents: inhibitors of cellular metabolism[END_REF][START_REF] Grippo | The cytotoxicity of helenalin, its mono and difunctional esters, and related sesquiterpene lactones in murine and human tumor cells[END_REF][START_REF] Dirsch | Cytotoxic sesquiterpene lactones mediate their deathinducing effect in leukemia T cells by triggering apoptosis[END_REF]. Both compounds have been shown to interfere with the signal transduction of NF-κB [START_REF] Lyss | The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65[END_REF][START_REF] Hehner | The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-kappa B by targeting the I kappa B kinase complex[END_REF] playing a pivotal role both in inflammation and cancer [START_REF] Ghosh | New regulators of NF-kappaB in inflammation[END_REF][START_REF] Baud | Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls[END_REF]. One of the most documented functions of NF-κB in cancer is its ability to promote cell survival by the induction of target gene products that inhibit the apoptotic machinery in both normal and malignant cells [START_REF] Chaturvedi | NF-kappaB addiction and its role in cancer: 'one size does not fit all[END_REF]. However, NF-κB can also prevent programmed necrosis by inducing genes that encode antioxidant proteins [START_REF] Luo | IKK/NF-kappaB signaling: balancing life and death--a new approach to cancer therapy[END_REF]. Thus, helenalin-induced ROS production in conjugation Interestingly, in most cases NF-κB is activated in malignant cells in response to inflammatory stimuli originating from the microenvironment [START_REF]Nuclear factor-kappaB in cancer development and progression[END_REF]. In addition, intrinsic mutations of the upstream regulators of NF-κB such as receptor activator of NF-κB (RANK), Notch1 or CD40 activity are discussed. Importantly, especially with respect to our work, a link between Bcl-2 and constitutive activation of NF-κB has been described in embryonic kidney cells as well as in myocytes [START_REF] De Moissac | Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis[END_REF][START_REF] Regula | IKK beta is required for Bcl-2-mediated NF-kappa B activation in ventricular myocytes[END_REF]. Moreover, downregulation of Bcl-2 was shown to attenuate NF-κB activation [START_REF] Regula | IKK beta is required for Bcl-2-mediated NF-kappa B activation in ventricular myocytes[END_REF]. However, others report that Bcl-2 inhibits NF-κB activity and subsequently apoptosis in serumdeprived embryonic kidney cells [START_REF] Grimm | Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis[END_REF].

In this study we show that Bcl-2 overexpressing Jurkat T cells have increased NF-κB activity compared to empty vector control cells. These data are supported by several other observations: 1) lymphoma cells with the t(14;18) translocation show high levels of NF-κB [START_REF] Heckman | NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells[END_REF] and 2) in breast cancer and leukemic (CLL) cells high NF-κB expression was associated with high Bcl-2 expression [START_REF] Viatour | NFkappa B2/p100 induces Bcl-2 expression[END_REF]. In fact, it has been shown

for MCF-7 breast cancer cells that Bcl-2 overexpression enhances NF-κB activity and induces MMP-9 transcription [START_REF] Ricca | bcl-2 overexpression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells[END_REF]. Thus Bcl-2 might not only prevent cell death by heterodimerizing with pro-apoptotic BAX or BAK proteins, subsequently abrogating the intrinsic mitochondrial pathway of apoptosis, but also by inducing a NF-κB-driven survival machinery. Using helenalin as tool we could confirm this notion. Helenalin is a known inhibitor of NF-κB [START_REF] Lyss | The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65[END_REF] and although the inhibitory action of STLs on NF-κB is undisputed, their molecular mechanism of action remains a matter of debate.

Surface plasmon resonance analysis supported earlier reports that helenalin exerts a direct inhibitory effect on the activated p65 moiety rather than targeting IκB kinase β (IKKβ) [START_REF] Lyss | The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-kappaB by directly targeting p65[END_REF][START_REF] Buchele | Surface plasmon resonance analysis of nuclear factor-kappaB protein interactions with the sesquiterpene lactone helenalin[END_REF]. Importantly, next to helenalin the NF-κB inhibitor BMS is able to block Bcl-2-mediated death resistance. This might open up a novel strategy to overcome Bcl-2-induced chemoresistance at least in leukemic cells.

Interestingly, helenalin does not act as specific Bcl-2 inhibitor since neither typical mitochondrial activation features such as cytochrome c release, disruption of mitochondrial membrane potential and caspase activation nor classical apoptosis (phosphatidylserine translocation) could be seen in response to helenalin but are reported for specific Bcl-2 inhibitors such as the compound ABT-737 [START_REF] Vogler | Bcl-2 inhibitors: small molecules with a big impact on cancer therapy[END_REF]. In contrast, helenalin is obviously able to bypass the survival mechanism of Bcl-2 at

A c c e p t e d M a n u s c r i p t 13 mitochondria. This is certainly a very important and promising feature with respect to chemoresistance that is frequently a consequence of deregulated apoptotic signalling.

Next to mitochondria, Bcl-2 may act also at the endoplasmic reticulum (ER)

influencing ER-stress-mediated cell death. The ER represents the most important storage site for Ca 2+ in the cell. Upon ER stress, high amounts of Ca 2+ can be released into the cytosol, mediating further downstream effects such as apoptosis [START_REF] Orrenius | Regulation of cell death: the calcium-apoptosis link[END_REF] or autophagy [START_REF] Hoyer-Hansen | Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2[END_REF]. We could not observe increased calcium level in response to helenalin treatment, although helenalin has been reported to decrease cellular glutathion levels [START_REF] Jodynis-Liebert | Effect of several sesquiterpene lactones on lipid peroxidation and glutathione level[END_REF], which is frequently associated with an increase of resting intracellular calcium, leading to cytotoxicity [START_REF] Powis | Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues[END_REF]. Furthermore, helenalin does not alter the expression of BiP/GRP78 and CHOP/GADD153, two markers for ER-stress [START_REF] Boyce | Cellular response to endoplasmic reticulum stress: a matter of life or death[END_REF][START_REF] Fulda | Cellular stress responses: cell survival and cell death[END_REF]. Also distinct hallmarks of autophagy such as conversion of LC3 I to membranebound LC3 II were missing [START_REF] Li | The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells[END_REF][START_REF] Mizushima | Methods for monitoring autophagy[END_REF]. Finally, the lack of phosphatidylserine translocation in helenalin-treated Bcl-2 overexpressing cells and the early onset of membrane permeability (PI positive staining) lead us to the assumption that helenalin induces cell death by necrosis. Necrosis often occurs when cell death programs such as apoptosis or autophagy are blocked [START_REF] Degenhardt | Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis[END_REF]. Thus, necrosis is a mechanism that overcomes resistance to apoptosis as has been observed in a variety of human tumors [START_REF] Edinger | Death by design: apoptosis, necrosis and autophagy[END_REF] and in apoptosis-defective breast carcinomas treated with an anthracylinebased therapy [START_REF] Olofsson | Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy[END_REF]. There is now evidence that necrosis, traditionally considered an accidental form of cell death, can be initiated or modulated by programmed control mechanisms. ROS, calcium-ions, poly-ADP-ribose polymerase (PARP), calcium activated non-lysosomal proteases (calpains) and cathepsins are some of the mediators of necrosis [START_REF] Hotchkiss | Cell death[END_REF].

Recently two forms of programmed necrosis have been described: necroptosis and PARP1-mediated necrotic death [START_REF] Degterev | Expansion and evolution of cell death programmes[END_REF]. Necroptosis is a form of programmed necrotic cell death under conditions where apoptosis is impeded [START_REF] Degterev | Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury[END_REF]. Although induction of autophagy has been observed in a number of cell lines by necroptotic signaling, autophagy seems to be a downstream consequence of necroptosis rather than a contributing factor. RIP1 translocates to mitochondria causing disruption of the bonding of ANT with cyclophilin D leading to rapid mitochondrial dysfunction that is associated with necroptosis [START_REF] Degterev | Expansion and evolution of cell death programmes[END_REF]. Necrostatins are well characterized inhibitors of RIP1 and therefore commonly used inhibitors of necroptosis. Since the RIP-1 *Graphical Abstract

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

 9 membrane potential (Fig.2b-c). Notably, the intermembrane space of the nuclear envelope drastically swells and numerous vesicles form in the cytoplasm in helenalin treated Bcl-2 overexpressing cells.

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

 12 an impaired antioxidative defense of helenalin-treated tumor might account for its success.

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

 14 necrostatin-1 did not influence helenalin-induced cell death and we did not observe induction of autophagy, helenalin induced cell death is not due to necroptosis. Another form of programmed necrosis, PARP1-mediated necrosis, is also unlikely to be induced by helenalin in Bcl-2 overexpressing Jurkat cells since the two major players of this pathway, AIF and JNK[START_REF] Degterev | Expansion and evolution of cell death programmes[END_REF], are not involved in helenalininduced cell death, shown by use of AIF siRNA and the JNK inhibitor SP600125.ROS represent important signalling molecules that help to transduce necrotic signals, e.g upon stimulation with TNFα[START_REF] Fulda | Cellular stress responses: cell survival and cell death[END_REF]. We could show that helenalin induces early ROS generation, which is responsible for cell death induction in Bcl-2 Jurkat cells since helenalin-mediated cell death was completely blocked by treatment with the antioxidant NAC and attenuated by the radical scavenger Tiron. Massive ROS generation can lead to both apoptosis and necrosis. Apoptosis induction by H 2 O 2 for instance is mediated by the release of cytochrome c and the activation of transcription factors like NF-κB, which may upregulate death proteins or produce inhibitors of survival proteins[START_REF] Fulda | Cellular stress responses: cell survival and cell death[END_REF]. As Bcl-2 overexpression protects cells from cytochrome c release and helenalin inhibits NF-κB, cells are potentially forced to switch to necrosis. Inhibition of constitutively active NF-κB causes the downregulation of ferritin heavy chain that leads to an increase of free intracellular iron, which can in turn induce massive production of ROS as reported in cutaneous T-cell lymphoma[START_REF] Kiessling | Inhibition of constitutively activated nuclear factor-kappaB induces reactive oxygen species-and irondependent cell death in cutaneous T-cell lymphoma[END_REF]. Our own data support this notion for Bcl-2 overexpressing leukemia cells as well: helenalin-induced cell death is significantly decreased following treatment with the iron-chelator DFO. Thus, the inhibition of NF-κB, free intracellular iron and ROS are mediators of helenalin-induced cell death in Bcl-2 overexpressing cells. However, helenalin might also stimulate ROS upstream of NF-B as a combination of BMS and helenalin partly abrogates ROS production. Neither inhibition of Akt nor JNK activation contributed to helenalin-mediated cell death in Bcl-2 Jurkat cells.In summary, helenalin abrogates Bcl-2-mediated chemoresistance not by directly targeting Bcl-2-induced mitochondrial resistance; helenalin rather inhibits augmented NF-κB activity in Bcl-2 overexpressing tumor cells and promotes production of ROS leading to necrosis.

Fig. 1 .

 1 Fig.1. Helenalin overcomes Bcl-2-mediated chemoresistance. A, Neo Jurkat or Bcl-2 Jurkat were either left untreated (Co) or treated with helenalin (Hel, 20 µM) or etoposide (Eto, 2 µM) for 24 h (cell death, left panel) or 2 h (colony growth, right panel). Cell death was quantified by measuring nuclei with a subdiploid DNA content and colony growth was quantified by a clonogenic assay; both assays are described under "Experimental Procedures". Results of colony growth are represented as the number of colonies referred to untreated cells (Co) (right panel). *, p < 0.001 (ANOVA, Bonferroni). B, Neo L3.6pl and Bcl-2 L3.6pl cells were either left untreated (Co) or stimulated with paclitaxel (Tax) or helenalin (Hel) for 2 h and a clonogenic assay was performed. *, p < 0.001 (ANOVA, Bonferroni). Data are expressed as mean  SEM (n=3). Bcl-2 protein level of Neo and Bcl-2 Jurkat cells, and Neo and Bcl-2 L3.6pl cells were analyzed by Western blot.

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .M a n u s c r i p t 18 (Fig. 6 .Fig. 7 .

 23451867 Fig.2. Helenalin does not abrogate mitochondrial function of Bcl-2. A, Bcl-2 Jurkat cells were treated with helenalin (Hel; 20 µM) for the indicated times. Paclitaxel (Tax; 1 µM, 16 h) was used as control. Bcl-2 expression pattern was analyzed by Western blot. B, Neo Jurkat and Bcl-2 Jurkat cells were treated with helenalin (Hel; 20 µM, 6 h) and mitochondrial membrane potential (MMP) was analyzed by staining with JC-1. Histograms of one representative experiment out of three are shown. C, For Western blot analysis of cytochrome c, Neo Jurkat and Bcl-2 Jurkat cells were treated with helenalin (Hel; 20 µM) for the indicated times. Cytosolic and membrane fractions were prepared. Purity of cytosolic fractions was verified by use of a VDAC antibody.

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  Figure

  Figure7

  Jurkat cells were stained with 2',7'-Dichlorodihydrofluorescein diacetate (DCDHF diacetate, 10 µM) for 30 minutes at 37°C in the dark and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany).

	2.9. Reporter gene assay
	M Bcl-2 and Neo Jurkat cells were co-transfected with a plasmid containing 5.7 kB of the human NF-κB promoter driving a firefly luciferase gene (pNF-κB-Luc, Stratagene, La Jolla, CA, USA; 4 µg) and a β-galactosidase plasmid (pβ-Gal, 6.83 kB, Promega, Mannheim, Germany; 506 ng) using the Nucleofector ® II device (LONZA Cologne AG, Cologne, Germany). Luciferase activity was measured with Orion II Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany) and normalized to β-galactosidase activity a n u s c r i p t 2.10. Electrophoretic mobility shift assay (EMSA)
	3 (Santa Cruz Biotechnology, Heidelberg, Germany), cytochrome c, JNK, pJNK (Thr183/Tyr185), VDAC, AKT, mTOR, Beclin-1, LC3B (G40), p-c-Jun (Ser63) II, p-eIF2α (Ser 51), A c c e p t e d
	caspase-9, pAKT (Ser473), p-mTOR (Ser2448) (Cell Signaling, Frankfurt, Germany),
	ANTI-GADD153/CHOP10 (Sigma, Saint Louis, Missouri, USA), BiP/GRP78 (BD
	Transduction Laboratories, Heidelberg, Germany). Equal protein loading was
	controlled by β-actin (Millipore, Billerica, MA, USA) staining of membranes.
	2.8. Measurement of ROS generation

6

Bcl-2

Acknowledgements

We would like to thank Romina Wiedmann (Center for Drug Research, Pharmaceutical Biology, Ludwig-Maximilians-University) for creating Bcl-2/Neo L3.6pl cells. The excellent technical assistance of Silvia Dobler and Cornelia Niemann is gratefully acknowledged.