Alan Kambiz Fotoohi

Yehuda G Assaraf

Ali Moshfegh

Jamileh Hashemi

Gerrit Jansen

Godefridus J Peters

Catharina Larsson

Freidoun Albertioni
email: freidoun.albertioni@ki.se

Freidoun Albertioni<ce

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

Keywords: Methotrexate, 7-hydroxymethotrexate, resistance, leukemia, folylpolyglutamate synthetase, reduced folate carrier, thymidylate synthase, microarray, real-time quantitative RT-PCR MTX, methotrexate, 7-OHMTX, 7-hydroxymethotrexate, IC 50 , the drug concentration inhibiting cell folylpolyglutamate hydrolase, GARFT, glycinamide ribonucleotide formyltransferase, MRP, multidrug resistance-associated protein

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract

In vitro treatment of human T-cell leukemia cells with 7-hydroxymethotrexate , the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate.. To identify the genes associated with methotrexate and 7hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and antifolates, were down-regulated >2-fold in methotrexate -resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate -resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine β synthase, as well as the ATP-dependent folate exporters BCRP/ABCG2 and MRP1/ABCC1. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate -resistant subline, whereas in 7-hydroxymethotrexate -resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an antifolate resistance modality that are distinct from its parent drug methotrexate.

Introduction

During the past six decades, the rate of success in curing childhood acute lymphoblastic leukemia (ALL) has increased from less than 10% to more than 80%. This dramatic improvement is largely due to optimization of the use of existing chemotherapeutic agents.

Methotrexate (MTX) is the major anti-metabolite (Fig. 1) employed in combination chemotherapeutic regimens for childhood ALL. Treatment failure is mainly related to acquired drug resistance or selection of pre-existing molecular alterations facilitating antifolate resistance.

The mechanisms of action and resistance to MTX and its major metabolite 7hydroxymethotrexate (7-OHMTX) have been characterized in some detail [START_REF] Assaraf | Molecular basis of antifolate resistance[END_REF][START_REF] Fotoohi | Mechanisms of antifolate resistance and methotrexate efficacy in leukemia cells[END_REF]. Decreased cellular uptake via the reduced folate carrier (RFC/SLC19A1) [START_REF] Gorlick | Intrinsic and acquired resistance to methotrexate in acute leukemia[END_REF] or increased active efflux mediated by ABC transporters such as the multidrug resistance-associated proteins 1-5 (MRP 1-5), the breast cancer resistance protein (BCRP/ABCG2) [START_REF] Shafran | ABCG2 harboring the Gly482 mutation confers high-level resistance to various hydrophilic antifolates[END_REF][START_REF] Volk | Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter[END_REF] and P-glycoprotein (MDR or ABCB1) [START_REF] Gifford | P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene[END_REF] can lead to resistance to hydrophilic and lipophilic antifolates. The ability of cells to accumulate long chain MTX polyglutamates (MTX-PGs) is highly dependent on the activities of folylpolyglutamate synthetase (FPGS) and folylpolyglutamate hydrolase (FPGH). Furthermore, the susceptibility of cells to the cytotoxic effect elicited by MTX may also be influenced by mutations in the dihydrofolate reductase gene (DHFR) that result in a lower affinity for MTX, by increased activity of thymidylate synthase (TS), as well as by altered activities of other folate-and one-carbon metabolism enzymes.

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

The metabolite 7-OHMTX (Fig. 1) results from oxidation of MTX primarily by hepatic aldehyde oxidase. Intracellular conversion of MTX to 7-OHMTX has also been demonstrated in leukemia cells [START_REF] Newton | 7-Hydroxymethotrexate formation in a human lymphoblastic cell line[END_REF] . Although the affinity of DHFR for 7-OHMTX is markedly lower (>100-fold) than for MTX [START_REF] Farquhar | Synthesis and biologic evaluation of 7hydroxymethotrexate, 7-methylaminopterin, and 7-methylmethotrexate[END_REF] , the polyglutamated forms of this metabolite are more potent inhibitors of this enzyme [START_REF] Drake | Effects on dihydrofolate reductase of methotrexate metabolites and intracellular folates formed following methotrexate exposure of human breast cancer cells[END_REF][START_REF] Sholar | Inhibition of folate-dependent enzymes by 7-OH-methotrexate[END_REF] . Polyglutamated forms of MTX and polyglutamated conjugates of 7-OHMTX are also capable of inhibiting a number of other folate-dependent enzymes involved in de novo purine biosynthesis such as TS, glycinamide ribonucleotide formyltransferase (GARFT) and aminoimidazole carboxamide ribonucleotide formyltransferase (AICART).

We have recently reported that prolonged exposure of human leukemic T-cells to 7-OHMTX leads to antifolate resistance that attenuates the efficacy of MTX [START_REF] Fotoohi | Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy[END_REF] . While the primary mechanism of resistance to MTX was a marked reduction in RFC-mediated drug uptake, resistance to 7-OHMTX was due to a dramatic decrease (>98%) in FPGS activity, which enhanced resistance to short-term (4 hr) exposure to MTX more than 100-fold.

Materials and Methods

Materials

The HG-U133A GeneChip oligonucleotide microarray and all reagents required for microarray analysis were purchased from Affymetrix (Affymetrix Inc., Santa Clara, CA, USA [START_REF] Rask | Clinical and pharmacokinetic risk factors for high-dose methotrexate-induced toxicity in children with acute lymphoblastic leukemia--a logistic regression analysis[END_REF] . The conditions for sample collections and procedures and HPLC assay applied are according to previously published information [START_REF] Albertioni | Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using highperformance liquid chromatography[END_REF] . In short, plasma samples were obtained after intravenous infusion of MTX (5-8 g/m 2 ) during 24 hours. Blood samples were collected 20-23 h after beginning of MTX infusion (steady-state concentration), at 36 h and once every 6 hours afterward until plasma-MTX concentration fell below <0.2 M. Samples were generally drawn from a central venous catheter, while the steady-state samples were obtained from a peripheral vein.

2.3

Antifolate-resistant cells and tissue culturing MOLT-4 cells derived from a human T-cell ALL were purchased from American Type Culture Collection (Rockville, MD, USA). The generation and functional characterization of drug resistant derivatives have been previously reported [START_REF] Fotoohi | Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy[END_REF]. In short, this involved sequential exposure (12-18 steps each of 4 days) of parental MOLT-4 cells to gradually increasing concentrations of MTX (1-300 nM) or 7-OHMTX (50-30,000 nM). This resulted in sub-lines with greater than 50-fold increased resistance to MTX or 7-OHMTX relative to parental cells. Cells were cultured in RPMI-1640 medium (supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine) at 37 C under A c c e p t e d M a n u s c r i p t 6 a humidified air containing 5% CO 2 . For experimental studies cells were cultured in drugfree-medium for at least 3 passages, and harvested during the logarithmic phase growth at a density 0.8-1.5x10 6 /ml as determined by cell counting using a Coulter Multi-sizer (Coulter Electronics, Luton, United Kingdom).

. 4 I s o l a

t i o n a n d Q u a l i t y V e r i f i c a t i o n o f R N A
Triplicate inoculates of parental and resistant cells were thawed, washed and cultured in a standard medium (see above) for 4 passages, following which 10 7 cells of each cell type were collected by centrifugation. High quality RNA was extracted using the RNeasy Midi kit in accordance with the manufacturer's instructions (RNeasy Midi Handbook; Qiagen, KEBO Lab, Spånga, Sweden). A NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies Wilmington, DE) was used to determine RNA concentrations, and to verify its quality and purity, whereby RNA with an OD ratio of 1.99-2.0 at 260/280 was found acceptable.

Experimental Conditions and Analysis of Expression Arrays

The experimental procedures and analyses were in accordance with the instructions of the manufacturer (Technical manual of Affymetrix GeneChip products). In short, complementary DNA (cDNA) synthesized from 11 g of total RNA, was used for synthesis and isolation of biotin-labelled complementary RNA (cRNA), and fragmented to a mean size of ~ 50-100 nucleotides. Samples were subsequently analyzed in triplicate on Affymetrix U133A GeneChips containing 22,283 sets of probes for approximately 17,000 different species of mRNA. After hybridization for 16 h at 45 °C, washing and staining with streptavidin-R-phycoerythrin, the arrays were scanned in an Agilent Gene Array Scanner percentile of all measurements, and for each gene the raw data were divided by the median level of expression of the specific samples in question. Data from sets of probes designed to detect the expression of genes that failed to fulfil the criteria for detection (labelled "absent"

or "marginal" in all microarrays) and an intensity value less than 50 (to reduce background signals) were eliminated. For statistical calculations, analysis of variance (ANOVA) were used (p<0.05), and Benjamin and Hochberg false discovery rate were used for multiple testing correction. After the filtration steps, 863 probe sets with significantly altered expression levels between parental and/or MTX resistant and/or 7-OHMTX resistant cells remained for further analysis (Fig. 3). All microarray data were submitted to http://www.ncbi.nlm.nih.gov/geo/.

Quantitative Real-time PCR (qRT-PCR)

qRT-PCR was performed on cDNA samples of parental and resistant cells in three independent experiments to quantify the expression of FPGS, FPGH/GGH, RFC/SLC19A1, GART, AICART, MTR, MTHFR, MRP4, and MRP5. GAPDH was analysed as a positive control as well as used for normalization. The values obtained for resistant cells were subsequently divided with the values for parental cells to allow comparisons. Further details are available in Perkin-Elmer Instruction Manual of 1997.

Array-based Comparative Genomic Hybridization (array-CGH)

Genomic DNA was extracted from cultured cells using a commercial kit (Gen Elute, Sigma)

and used for analyses of gene copy number alterations by array-CGH. The experimental procedures and data analyses were according to previously published information [START_REF] Jonsson | High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization[END_REF] .

Results

As shown in Figure 2, as high as 30-40 M concentrations of 7-OHMTX, the primary catabolite of MTX, are readily attainable in the serum of childhood ALL patients treated with high-dose MTX [START_REF] Rask | Clinical and pharmacokinetic risk factors for high-dose methotrexate-induced toxicity in children with acute lymphoblastic leukemia--a logistic regression analysis[END_REF] . Furthermore, while the parental drug MTX is efficiently cleared from the serum, relatively high levels (several M) of the catabolite 7-OHMTX that exceed those of the parent drug by ~10-fold, persist for a long time in the serum.

Accordingly, we explored the pattern of gene expression by leukemic cells that had acquired resistance to MTX or 7-OHMTX. Wild type MOLT-4 cells and their sublines selected for resistance to MTX or 7-OHMTX were characterized regarding gene expression profiles (Fig. 3; Table 1) and DNA copy number alterations (Table 2). Gene expression levels were compared to gene copy numbers (Table 3) and verified for selected genes by qRT-PCR (Table 4).

Expression Profiles of 7-OHMTX and MTX-Resistant cells

Nine separate hybridizations were performed on the Human Genome U133A GeneChip representing triplicate inoculates of parental, 7-OHMTX-as well as MTX-resistant MOLT-4 cells. In total, 863 probe sets showed 2-fold or more difference in expression levels between the three lines analysed (Fig. 3). Analysis of the gene expression patterns revealed 176 genes in MTX-resistant cells and 365 genes in 7-OHMTX-resistant cells, the expression of which was more than 2-fold different from that in parental cells (p<0.05) (Fig. 3). Forty-eight of these genes were common to both drug-resistant cell lines. Functional classification revealed frequent dysregulation of genes encoding signalling proteins in both resistant derivatives studied (Table 1). In the MTX-resistant subline, the expression of genes encoding proteins profoundly, whereas in the 7-OHMTX-resistant cells, the most pronounced alterations were in transcripts coding for proteins involved in metabolism and cell proliferation (Table 1).

The expression levels of certain genes with functions related to folate metabolism, purine biosynthetic pathway and transport of related substances are detailed in Table 3. Both resistant cell lines exhibited 4-5-fold up-regulation of MTHFR. In addition, pronounced upregulation of more than 2-fold of MTR was detected in MTX-resistant cells, while 7-OHMTX cells overexpressed FPGH, SLC19A2, PFAS, and GCHFR. Prominent underexpression involved APRT in MTX-resistant cells, whereas in 7-OHMTX-resistant cells, ADA and CBS were expressed at very low or undetectable levels.

DNA Copy Number Alterations by Array-CGH

To characterize alterations in gene copy numbers in parental and antifolate-resistant cells, each of these cell lines were first compared to normal reference DNA using array-CGH.

Homozygous deletions common to all three cells were recorded at several chromosomal locations including 7q34, 9p21.2-p21.

Corroboration of Array Expression Levels by qRT-PCR

Analysis of the gene expression patterns of parental and resistant cells employing the

Affymetrix HG-U133A microarray revealed consistent differences with respect to 9 genes, some of which encode proteins involved in folate metabolism or purine biosynthesis. These differences were confirmed by qRT-PCR (Table 4). The levels were normalized against the endogenous housekeeping gene GAPDH, which showed comparable expression levels in parental and antifolate-resistant cells. These analyses revealed similar levels of expression determined by microarray as well as by qRT-PCR for all nine genes analysed. The results also confirmed the extensive up-regulation of MTR and MTHFR along with down-regulation

OHMTX-resistant cells (Table 4).

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

Discussion

Concentrations of 7-OHMTX, the primary catabolite of MTX, as high as 30-40 M, are readily attainable in the plasma of childhood ALL patients treated with high-dose MTX (Fig.

2) [START_REF] Rask | Clinical and pharmacokinetic risk factors for high-dose methotrexate-induced toxicity in children with acute lymphoblastic leukemia--a logistic regression analysis[END_REF] . Furthermore, although MTX itself is efficiently cleared from the plasma, relatively high levels of 7-OHMTX (i.e. several M), that exceed those of the parent compound MTX by approximately 10-fold, persist in the plasma for a long time. Accordingly, we herein explored the pattern of gene expression and gene dose alterations in human leukemia cells with acquired resistance to MTX or 7-OHMTX [START_REF] Fotoohi | Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy[END_REF] . Copy number alterations were detected in parental cells as well as in both antifolate-resistant cell lines (Table 2). The genomic regions of acquired gains and losses do not include the genes whose products are known to be significant in response to MTX (Table 3). These findings suggest that drug-induced changes in gene expression do not stem from copy number alterations, but may instead be due to transcriptional regulatory alterations. Furthermore, gene amplifications were not detected, thereby demonstrating that increases in copy numbers of drug-resistance mediating genes were neither induced by treatment with MTX nor with exposure to 7-OHMTX.

Expression of the RFC/SLC19A1 gene was down-regulated more than 2-fold in the MTXresistant cells, thereby confirming the role of this influx transporter as an important determinant of antifolate resistance [START_REF] Jansen | A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance[END_REF][START_REF] Rothem | Alterations in the expression of transcription factors and the reduced folate carrier as a novel mechanism of antifolate resistance in human leukemia cells[END_REF][START_REF] Rothem | Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines[END_REF][START_REF] Rothem | Reduced folate carrier gene silencing in multiple antifolate-resistant tumor cell lines is due to a simultaneous loss of function of multiple transcription factors but not promoter methylation[END_REF] . However, the major finding of the present investigation is that the dramatic loss of FPGS activity in the 7-OHMTX cells, presumably associated with decreased intracellular levels of folate, was accompanied by consistent alterations in the expression of genes capable of preserving intracellular pools of reduced folate and/or increase purine nucleotide biosynthesis. Thus, among the major differences observed between these cell lines were the following (Fig. whereas in MTX-resistant cells these levels were actually elevated 1.5-and 1.7-fold, respectively. These results are in accord with our recent findings that folate deprivation leads to a dramatic loss of BCRP/ABCG2 expression as well as retention of this folate influx transporter in the cytoplasm rather than sorting it to the plasma membrane [START_REF] Ifergan | Cytoplasmic confinement of breast cancer resistance protein (BCRP/ABCG2) as a novel mechanism of adaptation to shortterm folate deprivation[END_REF][START_REF] Ifergan | Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis[END_REF].

Furthermore, these findings are consistent with those reported recently by Evans and colleagues [START_REF] Kager | Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics[END_REF] who documented differential patterns of gene expression in various subtypes In agreement with the pronounced reduction observed in MTX uptake into MTX-resistant cells, diminished RFC mRNA expression was observed by microarray (2-fold) and qRT-PCT

(5-fold) in MTX-resistant leukaemia cells as compared to their parental counterpart (Table 4). However, the expression of RFC was not altered in ) that FPGS transcripts in antifolate-resistant ALL cell lines were highly aberrant as they underwent impaired splicing including exon skipping as well as intron retention. Hence, these novel alterations result in premature translation termination of FPGS, thereby leading to lack of FPGS activity and resistance to polyglutamatble antifolates. This mechanism was also corroborated in specimens from patients harbouring ALL, both at diagnosis and relapse. These findings provide a novel mechanistic basis for the loss of FPGS function in the absence of quantitative changes (i.e. decrease) in FPGS levels.

Members of the family of multidrug resistance proteins, notably MRP1-5/ABCC1-5, and BCRP/ABCG2 are known to export the folate antagonist MTX out of cells [START_REF] Assaraf | The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis[END_REF][START_REF] Chen | Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system[END_REF][27][28][29]. Levels of BCRP and MRP1-5 mRNAs were all slightly elevated (about 50%) in the MTX-resistant subline, but unchanged in 7-OHMTX cells (Table 3). and it is hence tempting to speculate here that elimination of vesicle-sequestered 7-OHMTX could be facilitated by VAMP8 overexpression. This finding warrants further studies to explore this interesting possibility.

Among the genes that encode proteins involved in the folate pathway, the expression of MTHFR was most profoundly altered in both of our antifolate-resistant cell lines (>4-fold upregulation) (Fig. 4). MTHFR plays a key role in folate metabolism by channelling singlecarbon units between nucleotide biosynthesis and methylation reactions. This enzyme converts 5,10-methylene-THF into 5-methyl-THF, the predominant circulating folate form that provides a methyl group for methylation of homocysteine to methionine. Moreover,

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

 7 Affymetrix). The arrays were first analyzed using GeneChip Operating Software (GCOS 1.4; Affymetrix), and data files were subsequently analyzed utilizing the GeneSpring 7.2 software. Normalization was performed with the same software employing default normalization parameters as follows: for each sample the raw data were divided by the 50th

  qRT-PCR reactions were carried out in 20 µl mixtures containing 9 µl cDNA template, 1 µl TaqMan® Gene Expression Assay and 10 µl TaqMan Fast Universal PCR Master Mix, No AmpErase UNG concentrated 2-fold. Amplifications involved 2 min at 50 °C (stage 1), and 10 min at 95 °C (stage 2), followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (stage 3), and were carried out in 96-well optical PCR plates (N 801-0560, Perkin Elmer) placed in an automated fluorometer (ABI PRISM 7700 Sequence Detection System, AppliedA c c e p t e d M a n u s c r i p t 8 Biosystems). The different mRNA species were normalized against GAPDH determined in the same samples.

A c c e p t e d M a n u s c r i p t 10 involved

 10 in DNA/RNA metabolism and membrane transport proteins were altered more

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

 11 within 5p, 6p, 7p, 7q, 8p, 12q, 17p, 17q, 18q and 22q as well as by loss in 10p.Cells resistant to 7-OHMTX differed from parental cells by gains involving 5p, 12q, 17p and loss of 1p, 10p, and 13q. To further delineate acquired alterations, profiles were compared visually between parental and drug-resistant cells as a complementary approach to the scoring of copy number alterations by CGH plotter plugin. This revealed that several of the distinguishing alterations in resistant cells were present as close to borderline alterations at the threshold in parental cells. In addition, array-CGH experiments were carried out whereby resistant cells were hybridized against parental cells. This approach identified gene copy number alterations outside the +/-0.25 threshold relative to parental cells including increase in 8pter-q24.3 in MTX-resistant cells, as well as increases in1q21.1-qter and 12q23.2-qter and decreases in 10p11.22-p11.23 and 13q22.1-qter in 7-OHMTX cells.

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

 12 RFC in MTX-resistant cells, as well as up-regulation of FPGH/GGH and MTHFR in 7-

  4): (a) Expression of ADA A c c e p t e d M a n u s c r i p t 14 encoding the major enzyme of purine catabolism was down-regulated 10-fold in 7-OHMTX cells but unchanged in MTX cells. (b) Consistently, the cystathionine β synthase (CBS) gene was not expressed at all in 7-OHMTX cells, while being expressed 2-fold lower than the normal levels present in MTX-resistant cells. (c) Expression of genes encoding for MTHFR and GART, enzymes involved in methyl-tetrahydrofolate and purine biosyntheses, respectively, were elevated 5-and 2-fold in 7-OHMTX-resistant cells and 4-and 1.5-fold in MTX-resistant cells. (d) Furthermore, the levels of mRNAs encoding for the ATP-driven folate efflux transporters MRP1/ABCC1 and BCRP/ABCG2 were decreased in gene expression by 30% and approximately 3-fold, respectively, in 7-OHMTX-resistant cells,

  of bone marrow cells from newly diagnosed patients with B-or T-ALL. These investigators found that 42-44 hr after MTX administration (0.8 g/m 2 ), T-ALL bone marrow cells formed long-chain (Glu4-7) MTX polyglutamates at a rate of only 355 pmol/10 9 cells, while the corresponding rate for hyperdiploid B-cell leukemia was as high as 3,170 pmol/10 9 cells. This difference was consistently associated with very low levels of FPGS mRNA in the T-ALL cells, as much as two orders of magnitude lower than the level present in B-lineage hyperdiploid cells, together with low level expression of the BCRP/ABCG2 gene in T-ALL cells. In contrast, the levels of expression of the AICART and GARFT genes, encoding key A c c e p t e d M a n u s c r i p t 15 enzymes involved in de novo purine biosynthesis were higher in the case of T-ALL than any other leukemia subtypes.

A c c e p t e d M a n u s c r i p t 16 Cells resistant to 7 -

 167 OHMTX displayed a dramatic up-regulation of VAMP8 (>700fold) ; VAMP8 is a vesicle-associated membrane protein involved in exocytosis,

  -THF is required for TS activity and thymidylate biosythesis and its depletion could lead to dUTP misincorporation into DNA and a consequent increase in the frequency of chromosome damage, thereby facilitating the genotoxic effects of MTX. It has been reported that a deactivating MTHFR allele (the MTHFR C677T variant allele) can enhance the risk for ALL relapse potentially by increasing cellular levels of 5,10 Methylene-THF and deoxythymidine monophosphate (dTMP), thereby enhancing DNA synthesis and counteracting MTX cytotoxicity. Thus, the significant increment in the expression of MTHFR mRNA in both of our antifolate-resistant cell lines may be related to cellular tolerance to MTX and 7-OHMTX. Although diminished transport and lack of polyglutamylation can readily explain the mechanisms underlying the significant increase in resistance to MTX and 7-OHMTX, respectively, some alterations in gene expression of resistant cells may have contributed to their tolerance.

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

 17 summary, the present characterization of the different patterns of gene expression profiles of MTX-and 7-OHMTX-resistant sublines of parental MOLT-4 leukemia cells selected by exposure to MTX and 7-OHMTX, provides a new perspective on differences in the pharmacodynamic consequences of HDMTX therapy. 7-OHMTX , the major metabolite of MTX exerts its cytotoxic effect by mechanisms that are distinct from those underlying the effects of the parent drug. Our current findings may facilitate the identification of the specific targets for MTX therapy and may provide additional insight into cellular responses to 7-OHMTX. The distinct patterns of gene expression profiles in cells that have acquired resistance to MTX or 7-OHMTX may indicate that part of the gene expression profile observed following exposure to high doses of MTX reported earlier [30] is related to exposure to 7-OHMTX, rather than to MTX itself. The differential expression of a relatively large number of genes by 7-OHMTX-and MTX-resistant cells indicates the occurrence of separate and independent effects. Thus, in relation with HDMTX therapy, where higher concentrations of 7-OHMTX can be detected in the plasma, the pattern of gene expression will be different from that when low concentrations of 7-OHMTX are present.

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

 20 Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. Cancer Res 1999;59:2532-5. [28] Wielinga P, Hooijberg JH, Gunnarsdottir S, Kathmann I, Reid G, Zelcer N, et al. The human multidrug resistance protein MRP5 transports folates and can mediate cellular resistance against antifolates. Cancer Res 2005;65:4425-30. [29] Zeng H, Bain LJ, Belinsky MG, Kruh GD. Expression of multidrug resistance protein-3 (multispecific organic anion transporter-D) in human embryonic kidney 293 cells confers resistance to anticancer agents. Cancer Res 1999;59:5964-7. [30] Cheok MH, Yang W, Pui CH, Downing JR, Cheng C, Naeve CW, et al. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nat Genet 2003;34:85-90.

Figure 1 .

 1 Figure 1. The chemical structures of methotrexate (MTX) and its major catabolite 7-

Figure 2 .

 2 Figure 2. Comparison of plasma levels for MTX and 7-OHMTX measured by HPLC in

Figure 3 .A c c e p t e d M a n u s c r i p t 21 Figure 4 .

 3214 Figure 3. A, Gene expression patterns and B, hierarchical clustering of parental cells

  Figure1

Figure4 A c c e p t e d M a n u s c r i p t

  Figure4

  0) Total 100% (n = 40) 100 (n = 136) 100 (n = 138) 100 (n = 227) A c c e p t e d M a n u s c r i p t

  levels are in relation to parental cells (MTX / parental or 7-OHMTX / parental) giving an arbitrary value of 1.0 for parental cells DNA copy number are in relation to normal reference DNA; n.a = no aberration detected Expression changes greater than 2-fold and copy number aberrations above thresholds are indicted in bold A c c e p t e d M a n u s c r i p t

Table 4 .

 4 Expression of selected genes by array and qRT-PCR in resistant as compared to parental cells. Expression values are in relation to parental cells (MTX / parental or 7-OHMTX / parental), giving an arbitrary value of 1.0 in parental cells as indicated

  ). TaqMan reagents and gene expression assays were obtained from Applied

	Biosystems (Stockholm, Sweden) including: folylpolyglutamate synthetase (FPGS, No:
	Hs00191956 m1), folylpolyglutamate hydrolase (FPGH/GGH, No: Hs00608257_m1),
	the reduced folate carrier (RFC/SLC19A1, No: Hs00161870_m1), glycinamide
	ribonucleotide transformylase (GART, No: Hs00531926_m1), 5-aminoimidazole-4-
	carboxamide ribonucleotide formyltransferase (AICART, No: Hs00269671_m1), 5-
	methyltetrahydrofolate-homocysteine methyltransferase (MTR, No: Hs00165188_m1),
	5,10-methylenetetrahydrofolate reductase (MTHFR, No: Hs00195560_m1), multidrug
	resistance-associated proteins 4 (MRP4, No: Hs00195260), and 5 (MRP5, No:
	Hs00194701_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, No:
	Hs99999905_m1).
	2.2	Comparison of plasma MTX and 7-OHMTX levels in MTX treated ALL patients
	Plasma levels of MTX and 7-OHMTX previously determined in 49 childhood ALL
	patients by HPLC

  3, 11p14.3-15.1 and 14q11.2, while gene amplifications were not found. The target interval in 14q11.2, spans the loci for TRAV20 (gene segment for T-cell receptor alpha variable 20) and TRA genes. The region 9p21.3 encompasses the CDKN2A gene locus encoding p16 and p14. In addition, regular gains or losses involved almost all chromosomes and were in most situations present in parental as well as drug-resistant cells (Table2). Alterations present in drug-resistant

cells are of interest since they may be related to the mechanism by which drug resistance has emerged. MTX-resistant cells were distinguished from parental cells by chromosomal

  7-OHMTX-resistant cells. Moreover, the level of FPGH mRNA was elevated more than 2-fold in 7-OHMTX-resistant cells. In

contrast, no difference in FPGS expression was detected in either of the resistant cell lines suggesting that other mechanisms, e. g., post-transcriptional modifications, may be responsible for the low activity of this enzyme in these cells. In a recent study it has shown (M. Stark, C. Wichman, I. Avivi and Y.G. Assaraf; "Aberrant splicing of folylypolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia", Blood 2009, in press

Table 1 .

 1 Genes up-or down-regulated more than 2-fold in resistant versus parental cells.

		Up-regulated genes	Down-regulated genes
	Functional category	MTX	7-OHMTX	MTX	7-OHMTX
	RNA / DNA metabolism	10% (4)	3% (4)	16% (22)	10% (23)
	Cell cycle	0% (0)	1% (1)	3% (4)	1% (2)
	Development	5% (2)	4% (5)	4% (6)	10% (23)
	Apoptosis	5% (2)	2% (3)	1% (1)	4% (9)
	Transporter	10% (4)	4% (5)	4% (6)	5% (11)
	Signaling	30% (12)	36% (49)	26% (35)	33% (75)
	Stress response	5% (2)	4% (5)	0% (0)	1% (2)
	Metabolism	10% (4)	22% (31)	17% (24)	14% (32)
	Growth	15% (6)	11% (15)	20% (27)	15% (34)
	Organization & Proliferation	0% (0)	4% (5)	9% (13)	7% (16)
	Other	10% (4)	9% (13)	0% (0)	0%

Table 3 . Expression levels of selected genes and copy number of cytoband involved.

 3

	Gene

The Children Cancer Foundation, The Cancer and Allergy Foundation, The Cancer Society in Stockholm, The King Gustaf V Jubilee Fund, Swedish Medical Society, Swedish Cancer Foundation, Swedish Research Council, Göran Gustafsson Foundation for Research in Natural Sciences and Medicine, and Karolinska Institutet Foundations


Gonzalo Sánchez-Duffhues

Marco A Calzado

Amaya García De Vinuesa

Giovanni Appendino

Bernd L Fiebich

Ulich Loock

Annette Lefarth-Risse

Karsten Krohn

Eduardo Muñoz

Denbinobin inhibits nuclear factor-κB and induced apoptosis via reactive oxygen species generation in human leukemic cells

Keywords: Denbinobin, 1, 4-Phenanthrenequinones, NF-κB, ROS, MAPKs, Apoptosis

) not yet fully understood. Because of the critical role of the transcription factor NF-κB and of ROS-induced activation of stress regulated kinases in tumourogenesis, we have investigated the effect of denbinobin on these pathways. We found that denbinobin is a potent inhibitor of TNFα and PMA-induced NF-κB activation, and that it can block the phosphorylation and degradation of IκBα by inhibiting TAK1 activity, an event lying upstream of IKK activation. Moreover, treatment with denbinobin not only elicited apoptotic signalling, including mitochondrial membrane dysfunction, activation of caspases and cleave of poly(ADP-ribose) polymerase, but also induced intracellular reactive oxygen species (ROS) generation and sustained activation of the mitogen activated kinases (MAPKs) ERK1+2, p38 and JNK1+2. The apoptotic effects of denbinobin could be prevented by pre-treatment with the intracellular ROS scavenger Nacetyl-L-cysteine, but not by pharmacological inhibition of MAPKs, suggesting that intracellular ROS generation underlies denbinobin-induced apoptosis, and that this effect takes place in a MAPKs-independent pathway. To define the structural elements critical for these activities, a series of phenanthrenequinones with different substituents in the phenanthrene-and/or in the quinone ring were prepared and assayed for NF-κB inhibition and ROS production. In this way, the major structure activity relationships and the structural elements critical for the activity of denbinobin could be established.

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

INTRODUCTION

The Phenanthrene system is relatively uncommon in natural products, and is usually generated by the oxidative coupling of stilbene precursors or by aromatization of diterpenoids. Some plant-derived phenanthrenequinones , and especially denbinobin, show interesting biological activities [START_REF] Kovacs | Natural phenanthrenes and their biological activity[END_REF]. Denbinobin (5-hydroxy-3,7-dimethoxy-1,4phenanthrenequinone) has been isolated from several orchidaceous plants, like Dendrobium nobile [START_REF] Lee | In vitro and in vivo antitumoral phenanthrenes from the aerial parts of Dendrobium nobile[END_REF], D. moniliforme [START_REF] Lin | Two phenanthraquinones from Dendrobium moniliforme[END_REF] and Ephemerantha lonchophylla [START_REF] Chen | Antioxidant principles from Ephemerantha lonchophylla[END_REF] and from a variety of Cannabis sativa [START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF]. Interestingly, the stems of D. moniliform and E. lonchophylla known as Shi-Hu in Chinese medicine has been used for a long time to treat respiratory infections, as a tonic to nourish the stomach, and to reduce fever. Also, the total synthesis of denbinobin has been reported [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF], and this compound has been shown to induce apoptosis in several cancer cell lines [START_REF] Yang | Molecular mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells[END_REF][START_REF] Kuo | Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction[END_REF][START_REF] Chen | Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells[END_REF]. Furthermore, we have recently shown that denbinobin inhibits HIV-1 reactivation through an NF-κB dependent pathway [START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF]. However, the molecular details involved in NF-κB inhibition and the induction of apoptosis are still unclear.

The imbalance between cell survival and death, a key feature of many neoplastic, degenerative, inflammatory and autoimmune diseases, may be caused by an aberrant turnover of reactive oxygen species (ROS), a process that, in turn, regulates the crosstalk between NF-κB and mitogen-activated protein kinases (MAPKs) [START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF][START_REF] Temkin | From death receptor to reactive oxygen species and c-Jun Nterminal protein kinase: the receptor-interacting protein 1 odyssey[END_REF]. NF-κB is a collective term used to describe members of the Rel family of transcription factor. The Rel family regulates transcription of large number of genes and is involved in the control of a variety of cellular processes, such as immune and inflammatory responses, development, cellular growth, apoptosis and HIV-1 gene expression [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF]. NF-κB is an inducible transcription factor made up of homo-and heterodimers of p50, p65 (RelA), p52, relB and crel subunits that interact with a family of inhibitory IκB proteins, of which IκBα is the best characterised. In most cell types, these proteins sequester NF-κB in the cytoplasm by A c c e p t e d M a n u s c r i p t 4 masking its nuclear localization sequence. Stimulation of cells with a variety of physiological or pathogenic stimuli leads to phosphorylation, ubiquitination, and the subsequent degradation of IκBα proteins [START_REF] Hacker | Regulation and function of IKK and IKK-related kinases[END_REF]. Phosphorylation of IκBα at serines 32 and 36 is a key step involved in the activation of NF-κB complexes. This event is mediated by IκB kinases (IKKs), which are formed by a high-molecular weight complex (IKC) containing at least two kinase subunits, (IKKα and IKKβ), and the associated modulatory protein NEMO/IKKγ [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF][START_REF] Hacker | Regulation and function of IKK and IKK-related kinases[END_REF]. Several upstream kinases, including TGFβ-activated kinase 1 (TAK1), have been described to regulate the activity of IKKs [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF]. TAK1 is a ubiquitin-dependent kinase that is activated by innate immune stimuli including bacterial components and pro-inflammatory cytokines such as interleukin-1 and TNFα and plays an essential role in innate immune signalling by activating both IKKs and MAPK-pathways [START_REF] Ninomiya-Tsuji | The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway[END_REF][START_REF] Sato | Essential function for the kinase TAK1 in innate and adaptive immune responses[END_REF][START_REF] Chen | Ubiquitin, TAK1 and IKK: is there a connection?[END_REF]. Therefore, it has been proposed that inhibition of TAK1 activity may be effective in preventing inflammation and tissue destruction promoted by pro-inflammatory cytokines [START_REF] Ninomiya-Tsuji | A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase[END_REF].

ROS metabolism has been proposed to play an important role in all types of programmed cell death; apoptosis, necrosis, and autophagy [START_REF] Temkin | From death receptor to reactive oxygen species and c-Jun Nterminal protein kinase: the receptor-interacting protein 1 odyssey[END_REF]. ROS are continuously produced during normal aerobic metabolism by the mitochondria electron chain and enhanced in response to many different physiological stimuli. In addition ROS are also produced by extramitochondrial cellular sources including endoplasmic reticulum, a lysosomal redox chain and plasma membrane Noxs [START_REF] Bartosz | Reactive oxygen species: Destroyers or messengers?[END_REF]. It has been demonstrated that lowand intermediated levels of ROS can protect cell from apoptosis by activating antioxidant mechanisms, including activation of the NF-κB pathway, which in turn up regulates antiapoptotic genes [START_REF] Gloire | NF-kappaB activation by reactive oxygen species: fifteen years later[END_REF]. Conversely, high levels of intracellular ROS results in a breakdown of the transmembrane mitochondrial potential (ΔΨ m ), a common feature of early apoptosis that precedes DNA fragmentation regardless the cell type and the apoptotic stimuli [START_REF] Zamzami | Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo[END_REF]. The disrupture of ΔΨ m is mediated by mitochondrial megachannel opening (permeability A c c e p t e d M a n u s c r i p t 5 transition, PT) [START_REF] Szabo | The high-conductance channel of porin-less yeast mitochondria[END_REF], which leads to the release of a host of pro-apoptotic proteins (cytochrome-c, Smac/Diablo) and the apoptotic inducing factor, which, in turn, mediates the activation of executor caspases [START_REF] Crompton | Mitochondrial intermembrane junctional complexes and their involvement in cell death[END_REF]. Furthermore, high levels of ROS may inhibit NF-κB activation and induce a sustained c-Jun N-terminal kinase (JNK) activation that promotes the apoptotic pathway [START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF][START_REF] Korn | Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase[END_REF][START_REF] Moodie | Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells[END_REF].

The MAPK signalling cascade was originally identified as an important pathway in the transduction of apoptotic signals initiated by stress or toxic stimuli [START_REF] Ichijo | From receptors to stress-activated MAP kinases[END_REF]. Major participants in this kinase cascade are two members of the MAPKs, JNK and p38 MAP kinase, as well as their upstream kinases, such as MAP kinase kinases (MKKs). In contrast, p42/44 MAPKs (ERKs) are most frequently associated with pro-survival activity. Recent studies have demonstrated the essential role of the kinase cascade as well as its downstream mitochondrial-dependent reactive oxygen species (ROS) production pathway during apoptosis induction by cellular stress [START_REF] Hatai | Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation[END_REF][START_REF] Tournier | Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway[END_REF][START_REF] Assefa | p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis[END_REF]. Some chemopreventive compounds as well as antineoplasic drugs may induce apoptosis through modulation of the MAPK pathways [START_REF] Hu | Activation of MAP kinases, apoptosis and nutrigenomics of gene expression elicited by dietary cancer-prevention compounds[END_REF][START_REF] Muller | Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review)[END_REF].

Leukemia, currently one of the most threatening hematological malignant cancers, has been found to be very sensitive to anticancer chemotherapeutic reagents which either interfere with the cell cycle or cause apoptosis. Thus, the manipulation of apoptosis has the potential to provide novel strategies for cancer chemotherapy by selectively activating this process in malignant cells. We report here that denbinobin is endowed with a potent prooxidant activity, related to the presence of a quinone ring. As a result, denbinobin inhibits TAK-1-dependent NF-κB activation and induces apoptosis on the human leukaemia cell line Jurkat. The compound induces the collapse of ΔΨm and the activation of caspases, suggesting that the main lethal apoptotic pathway induced by this phenanthrenequinone involves mitochondrial damage. described [START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF]. Denbinobin was identified according to its physical and spectroscopic properties [START_REF] Lin | Two phenanthraquinones from Dendrobium moniliforme[END_REF].

Synthesis of 1,4-phenanthrenequinones.

The method applied in the synthesis of 1,4phenanthrenequinones [START_REF] Kovacs | Natural phenanthrenes and their biological activity[END_REF][START_REF] Lee | In vitro and in vivo antitumoral phenanthrenes from the aerial parts of Dendrobium nobile[END_REF][START_REF] Lin | Two phenanthraquinones from Dendrobium moniliforme[END_REF][START_REF] Chen | Antioxidant principles from Ephemerantha lonchophylla[END_REF][START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF][START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF][START_REF] Yang | Molecular mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells[END_REF][START_REF] Kuo | Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction[END_REF][START_REF] Chen | Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells[END_REF][START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF][START_REF] Temkin | From death receptor to reactive oxygen species and c-Jun Nterminal protein kinase: the receptor-interacting protein 1 odyssey[END_REF][START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF][START_REF] Hacker | Regulation and function of IKK and IKK-related kinases[END_REF][START_REF] Ninomiya-Tsuji | The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway[END_REF][START_REF] Sato | Essential function for the kinase TAK1 in innate and adaptive immune responses[END_REF][START_REF] Chen | Ubiquitin, TAK1 and IKK: is there a connection?[END_REF][START_REF] Ninomiya-Tsuji | A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase[END_REF][START_REF] Bartosz | Reactive oxygen species: Destroyers or messengers?[END_REF][START_REF] Gloire | NF-kappaB activation by reactive oxygen species: fifteen years later[END_REF][START_REF] Zamzami | Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo[END_REF] and tri-and tetramethoxyphenanthrenes [START_REF] Szabo | The high-conductance channel of porin-less yeast mitochondria[END_REF][START_REF] Crompton | Mitochondrial intermembrane junctional complexes and their involvement in cell death[END_REF] used in this study has been previously described [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF] (Supplementary information). The synthesis of 1,4diacetoxy-7,8-dimethoxyphenanthrene [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF] is also described in supplementary material. (Harrow, UK). Anti-c-myc, anti-HA and anti-GFP epitopes were purchased from Roche (Manheim, Germany). The p38 kinase inhibitor SB203085 was purchased from Sigma-Aldrich, the JNK inhibitor SP600125 was from Calbiochem (Darmstadt, Germany) and the ERK inhibitor PD98059 was purchased from Alexis (Lausanne, Switzerland). [γ-32 P]ATP (3000 Ci/mmol) was from MP Biomedicals (Irvine, CA, USA). All other reagents were from Sigma.

Cell

Plasmids, transient transfections and luciferase assays:

The KBF-Luc contains three copies of the MHC enhancer κB site upstream of the conalbumin promoter, followed by the luciferase gene. The expression vectors for IKKα and IKKβ and the IKKα and β tagged GFP vectors have been described elsewhere [START_REF] Sebald | T cell receptor-induced lipid raft recruitment of the I kappa B kinase complex is necessary and sufficient for NF-kappa B activation occurring in the cytosol[END_REF]. The expression vector TRAF-2, TRAF-6, TAK-1-HA, TAB-1-myc were obtained from Dr. M.L. Schmitz (Giessen, Germany). To study the NF-κB transcriptional activities the Jurkat cells were transfected with the KBF-Luc plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. 24 h post-transfection, cells were pre-treated with denbinobin for 30 min, and treated or not with PMA for 6 hours. For the kinase overexpression experiments, 293T cells (5 x 10 5 cells/well) were transiently transfected with the expression vectors for TAK1/TAB1, TRAF-2, TRAF-6, IKKα and IKKβ, and the KBF-Luc plasmids. After 24 h, cells were treated with indicated concentrations of denbinobin for 6 h and luciferase activity measured. To determine NF-κB dependent transcription of the HIV-LTR-luc 5.1 cells were pre-incubated for 30 min with denbinobin and analogues as indicated, followed by stimulation with TNFα (2 ng/ml) for six h (supplementary Table I andII 2.5 Isolation of nuclear extracts and mobility shift assays. Jurkat cells (10 6 /ml) were pretreated with denbinobin at the indicated doses for 1 h and then were stimulated or not with PMA (50 ng/ml) for 2 hours. Cells were then washed twice with cold PBS and proteins from nuclear extracts isolated as previously described [START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF]. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA). For the electrophoretic mobility shift assay (EMSA), double stranded oligonucleotide containing the consensus site for NF-κB, 5´-AGT TGA GGG GAC TTT CCC AGG C-3´ or the consensus site for SP-1, 5´-ATT CGA TCG GGG CGG GGC GAG C-3´ (Promega) were end-labeled with [γ- 32 P]ATP. The binding reaction mixture contained 5 µg of nuclear extracts, 0.5 µg poly(dI-dC) (Amersham Biosciences Inc.), 20 mM Hepes pH 7, 70 mM NaCl, 2 mM DTT, 0.01% NP-40, 100 µg/ml BSA, 4% Ficoll, and 100,000 cpm of end-labelled DNA fragments in a total volume of 20 µl. After 30 min incubation at 4 °C, the mixture was electrophoresed through a native 6% polyacrylamide gel containing 89 mM Tris-base, 89 mM boric acid and 2 mM EDTA. Gels were pre-electrophoresed for 30 min at 225 V and then for 2 h after loading the samples. These gels were dried and exposed to X-Ray film at -80 °C.

2.6

Western Blot: Jurkat cells (10 6 cells/ml) were stimulated with the indicated compounds.

Cells were then washed with PBS and proteins extracted in 50 µl of lysis buffer (20 mM A c c e p t e d M a n u s c r i p t 9 Hepes pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na 3 VO 4 , 5 mM NaF, 1 mM DTT, leupeptin 1 µg/ml, pepstatin 0.5 µg/ml, aprotinin 0.5 µg/ml, and 1 mM PMSF) containing 0.5% NP-40. Protein concentration was determined by the Bradford assay (Bio-Rad) and thirty µg of proteins were boiled in Laemmli buffer and electrophoresed in 10% SDS/polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V; 4 °C) for 1 h. Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% non-fat dry milk overnight at 4 °C, and immunodetection of specific proteins was carried out with primary antibodies using an ECL system (GE Healthcare, Piscataway, NJ, USA).

2.8 IKK kinase assay. Cells were lysed in NP-40 lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, leupeptine (10 µg/ml), aprotinin (10 µg/ml), 1% (v/v) NP-40 and 10% (v/v) glycerol) for 15 min at 4 °C, and after centrifugation for 10 min at 13000 rpm, the supernatant was incubated with non-specific IgG and 25 µl protein A/G sepharose (preclearing) and incubated overnight on a spinning wheel.

After centrifugation, the supernatants were incubated with 2 µg of anti-IKK-γ antibody and 25 µl protein A/G sepharose and incubated for 2-4 h on a spinning wheel at 4 °C. The precipitate was washed three times in cold lysis buffer and three times in cold kinase buffer (20 mM Hepes/KOH pH 7.4, 25 mM ß-glycerophosphate, 2 mM DTT, 20 mM MgCl 2 ). The kinase assay was performed in a final volume of 20 µl kinase buffer containing 40 µM ATP and 2 µg of the purified substrate protein GST-IκBα . After incubation for 20 min at 30 °C, the reaction was stopped by the addition of 5x SDS loading buffer. After separation by SDS-PAGE the proteins were transferred to nitrocellulose membranes and IκBα phosphorylation was detected by western blot using the antibody anti-phospho-IκBα 5A5.

A c c e p t e d M a n u s c r i p t 10 2.9 Analysis of cell cycle analysis and determination of cytotoxicity. The percentage of cells undergoing chromatinolysis (subdiploid cells) was determined by ethanol fixation (70%, for 24 h at 4 °C). Then, the cells were washed twice with PBS containing 4% glucose and subjected to RNA digestion (RNAse-A, 50U/ml) and PI (20 µg/ml) staining in PBS for 1h at RT, and analyzed by cytofluorimetry. With this method, low molecular weight DNA leaks from the ethanol-fixed apoptotic cells and the subsequent staining allows the determination of the percentage of subdiploid cells (sub-G 0 /G 1 fraction). For cytotoxicity assays Jurkat cells were seeded in 96-well plates in complete medium and treated with increasing doses of denbinobin for the indicated times. Samples were then diluted with 300 µl of PBS and incubated 1 min at RT in the presence of PI (10 µg/ml). After incubation, cells were immediately analyzed by flow cytometry in an EPICS XL flow cytometer (Coulter, Hialeah, FL).

Cytofluorimetric analysis of mitochondrial transmembrane potential and ROS generation.

To evaluate the mitochondrial transmembrane potential (ΔΨ m ) and the superoxide anion generation (ROS), treated or untreated cells (5x10 5 /ml) were incubated in PBS with DiOC6(3) (green fluorescence) (20 nM) (Molecular Probes, Eugene, OR, USA) and dihydroethidine (HE) (red fluorescence after oxidation) (2 µM) (Sigma) for 20 min at 37 °C, followed by analysis on an Epics XL flow cytometer.

Differential effects of denbinobin on NF-κB activation and MAPKs signalling

pathways. We have recently found that denbinobin inhibits TNFα-induced HIV-1 reactivation by targeting activation of the NF-κB canonical pathway, but the molecular details of this activity remained elusive [START_REF] Sanchez-Duffhues | Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-kappaB-dependent pathway[END_REF]. To further confirm the anti-NF-κB activity of denbinobin, Jurkat cells were transiently transfected with a luciferase reporter construct under the control of a minimal promoter containing three binding sites for NF-κB. Activation by PMA increased the luciferase gene expression driven by this artificial promoter in Jurkat cells, and we found that denbinobin could effectively inhibit this promoter activity in a dosedependent manner (Fig. 1A). In addition, using electrophoretic shift assays, we confirmed the inhibitory effect of denbinobin on PMA-induced NF-κB binding to DNA (Fig. 1B). Kinetic experiments demonstrate that denbinobin also inhibited PMA-induced IκBα phosphorylation and degradation as well as p65 phosphorylation, suggesting that denbinobin mediates its anti-NF-κB activity by targeting a step upstream of IκBα phosphorylation in the canonical pathway of NF-κB activation. Conversely, denbinobin did not interfere with the activation of the MAPKs; ERK, p38 and JNK (Fig. 1C).

Since the degradation of IκB proteins was shown to occur after signal-induced phosphorylation of IκB proteins at specific serine residues catalysed by IKKs present in the IKC [START_REF] Karin | Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity[END_REF], we investigated whether the prevention of IκBα phosphorylation and degradation was due to an impaired kinase activity of the IKC. Endogenous IKC was isolated by immunoprecipitation with an anti-IKKγ Ab, and its activity was analysed by immune complex kinase assays using recombinant IκBα protein as substrate. Either PMA or TNFαstimulation in Jurkat cells clearly induced an increase in IKK activity compared with unstimulated cells, and this activity was dose dependently inhibited in the presence of In mammals, in vitro and over-expression studies suggest TAK1 is involved in TNFR1 and IL-1R/TLR-mediated signalling pathways upstream of IKKs and JNK [START_REF] Takaesu | TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway[END_REF]. Two mammalian TAK1 adaptor proteins, TAB1 and TAB2, were isolated as TAK1-interacting proteins by yeast two-hybrid screening and TAB1 interacts constitutively with TAK1 and induces TAK1 kinase activity when over-expressed [START_REF] Shibuya | TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction[END_REF]. To investigate the effects of denbinobin on TAK1 induced IKK activation in vivo we co-transfected 293T cells with plasmids encoding IKKα-GFP and IKKβ-GFP fusion proteins together with the expression vectors TAK1-HA and TAB1-myc. IKK-GFP fusion proteins were used to differentiate from endogenous IKKα/β proteins in which the phosphorylation is difficult to detect with the antibody used. Over-expression of IKKβ-GFP and IKKα-GFP alone also led to some autophosphorylation of these kinases that was not affected by denbinobin (data not shown). Taken together, these results strongly suggest that denbinobin inhibits the NF-κB activation pathway by targeting the TAK1 kinase. [START_REF] Ninomiya-Tsuji | A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase[END_REF]. However, denbinobin did not inhibit PMA-induced p38, JNK and ERK phosphorylations (Fig. 1C). Therefore, we investigated whether denbinobin alone was sufficient to activate these MAPKs. Jurkat cells we incubated with denbinobin (5 µM) for the indicated times, and the phosphorylation/activation status of JNK, p38 and ERK was investigated by western blots. As depicted in Fig. 3A, denbinobin induced a robust and sustained activation of the stress activated kinases, JNK and p38.

Denbinobin also activated ERK but to a lesser extent than JNK and p38. Since p38 and JNK are mainly activated by oxidative stress, we pre-incubated Jurkat cells with two different anti-oxidants, Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) and N-Acetyl cysteine (NAC), which reduce oxidant-mediated cell damage through different mechanisms [START_REF] Gillissen | Oxidant scavenger function of ambroxol in vitro: a comparison with Nacetylcysteine[END_REF][START_REF] Nowak | Antioxidant properties of Ambroxol[END_REF]. Interestingly, NAC, but not ambroxol, completely inhibited denbinobin-induced phosphorylation and activation of p38, JNK and ERK. Since Ambroxol is a weak inhibitor of superoxide-dependent auto-oxidation of substrates, it is likely that denbinobin induces MAPKs activation through generation of superoxides.

Denbinobin induced reactive oxygen species, loss of mitochondrial membrane potential and activation of caspases in Jurkat cells.

We have shown previously that quinone analogues induce apoptosis by generating high levels of intracellular ROS that, in turn, induce mitochondria depolarization [START_REF] Macho | Induction of apoptosis by vanilloid compounds does not require de novo gene transcription and activator protein 1 activity[END_REF][START_REF] Macho | Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells[END_REF]. Thus, to investigate the role of ROS and mitochondria function in denbinobin treated cells, we loaded Jurkat cells with a fluorochrome (dihydroethidium, DHE) that enters cells freely. Superoxide then oxidizes DHE into ethidium or a structurally similar product, which intercalates into DNA, producing 4A shows that cells treated with denbinobin had a very strong accumulation of intracellular ROS (78.9 % of the cells) compared to untreated cells (0.5 % of the cells). In parallel, the effects of denbinobin on the mitochondria inner transmembrane potential (ΔΨ m ) was investigated by using the fluorochrome DiOC 6 (3), a cationic probe that distributes passively between media, the cytosol and the mitochondria, where the final distribution of the fluorochrome depends mainly on the transmembrane potential. In Figure 4A it is shown that compared to control cells (6.22% of the cells), denbinobin-treated cells have a lower DiOC 6 (3) fluorescence (32,75% of the cells), which corresponds with a drop in ΔΨ m . Pretreatment with NAC completely abrogated denbinobin-induced ROS production and disruption of the ΔΨ m .

Interestingly , pre-incubation with CsA, a potent inhibitor of the mitochondrial permeability transition pore (PTP) [START_REF] Crompton | The involvement of cyclosporin A binding proteins in regulating and uncoupling mitochondrial energy transduction[END_REF], completely inhibited the disruption of the ΔΨ m induced by denbinobin and partially inhibited ROS generation (50% of the cells). Accordingly, CsA did not prevent prevent denbinobin-induced MAPKs activation. These results suggest that denbinobin can generate ROS from both mitochondrial and non-mitochondrial sources.

An immediate consequence of the ΔΨ m breakdown can be the release into the cytosol of pro-apoptotic factors (cytochrome c and AIF). Cytosolic cytochrome c next binds to and activates Apaf-1/caspase-9, triggering the activation of the effector caspases that cleaved poly(ADP-ribose) polymerase 1 (PARP-1) and initiating the apoptotic process [START_REF] Crompton | Mitochondrial intermembrane junctional complexes and their involvement in cell death[END_REF]. To study the activation of the caspases pathway, we thus incubated cells with denbinobin for six hours, a time at which the ΔΨm dissipation was evident (Fig. 4A), and then evaluated the activation of caspases-3, -7 and -8 and the processing of PARP by western blots. Figure 4B shows that the treatment of Jurkat cells with denbinobin led to the processing and activation of these Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death, but evidence has been mounting that this process is a molecularly regulated event that is induced by ROS and disruption of the ΔΨ m [START_REF] Nakagawa | Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death[END_REF]. To further discriminate whether denbinobin induced cell death by necrosis or apoptosis, we treated cells with increasing concentrations of denbinobin for 12 h, and the cultures were then split for PI uptake and cell cycle analysis studies. Loss of plasma membrane integrity allows the uptake of PI, while intact plasma membranes exclude it. Therefore, PI uptake is a live/dead assay and does not distinguish between apoptotic and necrotic cell death. Conversely, cell cycle analysis makes it possible to assess the percentage of subdiploid cells, an indirect marker of apoptosis. As depicted in Figure 5, denbinobin induced an increase in the percentage of death cells measured by PI uptake (Fig. 5, upper panel) that paralleled with the percentage of subdiploid cells (chromatinolysis) (Fig. 5, lower panel). These results clearly demonstrate that denbinobin induces apoptosis by a caspase-dependent pathway that is activated by an increase in the intracellular levels of ROS.

Denbinobin induced apoptosis through a MAPK-independent pathway.

Several lines of evidence suggest that prolonged JNK and p38 activation promotes apoptosis via the mitochondria-dependent pathway [START_REF] Nakano | Reactive oxygen species mediate crosstalk between NF-kappaB and JNK[END_REF][START_REF] Temkin | From death receptor to reactive oxygen species and c-Jun Nterminal protein kinase: the receptor-interacting protein 1 odyssey[END_REF]. Therefore, using specific MAPKs chemical inhibitors, we investigated the role of JNK, p38 and ERK on denbinobin-induced apoptotic events. To this aim, Jurkat cells were preincubated for 30 min with PD98059, SP600125 and SB203085, inhibitors of MEK, JNK and p38 respectively. These inhibitors were used at the concentrations that specifically inhibited denbinobin-induced ERK, JNK and p38 phosphorylation in Jurkat cells (data not shown). We found that none of the inhibitors tested and -8 as well as PARP cleavage (Fig. 6B) in Jurkat cells. Furthermore, the inhibition of ERK and JNK could enhance denbinobin-induced ΔΨ m disruption and processing of caspases 3 and 8, an observation that suggests a protective role for ERK and JNK on the apoptotic pathway activated by denbinobin.

Identification of the essential functional group in the phenanthrene ring of denbinobin.

In order to identify the pharmacophore responsible for biological activities of denbinobin a series of analogues with different substituents on the phenanthrene ring were analysed (Supplementary Table I). The IC50 anti-NF-κB activity of the compounds was analysed using the 5.1 cell line and ROS generation was measured in Jurkat cell by flow cytometry. Compound 1, a phenanthrenequinone having no substituents at the carbons analysed, retains the biological activities of denbinobin (2) although some loss of anti-NF-κB activity was observed. Removal of the OH group at C-3 (3) did not significantly affect the biological activities of denbinobin. In contrast, hydroxylation at C-8 in compound 3 [START_REF] Bartosz | Reactive oxygen species: Destroyers or messengers?[END_REF] results in a complete loss of activity. Single methoxylation at C-6 (5), C-7 (4) or C-8 [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF] showed similar activity to denbinobin (2) and enhanced the activity of compound 1, which was further increased when double methoxylation was introduced in these carbons (7, 8 and 9). In general, we observed that methoxylation at C-6, C-7 and C-8 enhanced the biological activities of 1,4-phenantrenequinones. Interestingly, methoxylation at C-3 is detrimental for the biological activities of this series since compound 10 that is compared to compound 4 lost the anti-NF-κB and pro-oxidant activities. While the detrimental activity of a single methoxylation at C-3 may be overcome by methoxylation at C-7 (2 and 3), single methoxylation at C-2 (19 and 20) and double methoxylation at C-3 and C-5 [START_REF] Temkin | From death receptor to reactive oxygen species and c-Jun Nterminal protein kinase: the receptor-interacting protein 1 odyssey[END_REF][START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF][START_REF] Hacker | Regulation and function of IKK and IKK-related kinases[END_REF][START_REF] Ninomiya-Tsuji | The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway[END_REF][START_REF] Sato | Essential function for the kinase TAK1 in innate and adaptive immune responses[END_REF][START_REF] Chen | Ubiquitin, TAK1 and IKK: is there a connection?[END_REF][START_REF] Ninomiya-Tsuji | A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase[END_REF] and at C- II). To further study if compound 24 is also endowed with the pro-apoptotic activities described above for denbinobin, we treated Jurkat cells with compounds 22-24 and found that only compound 24 was able to induce the activation of caspases 3 and 7 and the processing of PARP (Supplementary Fig. 1AS). Moreover, compound 24 also induced ROS production and disruption of the ΔΨ m (Supplementary Fig. 1BS).

DISCUSSION

The use of plant extracts to alleviate human pathologies is centuries old, and continues nowadays. Thus, natural product research has paved the way to develop single agent drugs for the treatment of many diseases. In this context, both anti-oxidant and pro-oxidant natural products are of special interest for the development of new anticancer agents. ROS are a class of radical or non-radical oxygen-containing molecules that display high reactivity with lipids, proteins and nucleic acids, and it has been demonstrated that cancer cells produce high levels of ROS and are constantly under oxidative stress [START_REF] Szatrowski | Production of large amounts of hydrogen peroxide by human tumor cells[END_REF]. Thus, elevated rates of H 2 O 2 generation have been detected in seven human cancer cell lines, and elevated levels of O 2 -in primary blood samples from patients with different types of leukaemia have been reported [START_REF] Devi | Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias[END_REF]. Furthermore, elevated oxidative modifications in DNA, proteins, and lipids have been detected in various primary cancer tissues, further suggesting that cancer cells are inherently under oxidative stress due to an elevate rate of proliferation [START_REF] Kong | A threshold concept for cancer therapy[END_REF].

Recently, it has been suggested that the increase in ROS during antineoplastic therapy can induce the expression of antioxidants like SOD, but that the overproduction of ROS can, in turn, exhaust the capacity of SOD and other adaptive antioxidant defences, eventually inducing cell death. A "threshold concept" for cancer therapy has been proposed to explain the dual effects of oxygen radicals [START_REF] Kong | A threshold concept for cancer therapy[END_REF]. In cancer cells, if ROS levels reach the "threshold level" that overwhelms the antioxidant capacity, irreversible damage occurs and apoptosis is initiated. In general, tumour cells are very sensitive to pro-oxidant compounds like phenolic flavonoids and other inhibitory quinone analogues [START_REF] Macho | Induction of apoptosis by vanilloid compounds does not require de novo gene transcription and activator protein 1 activity[END_REF][START_REF] Macho | Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells[END_REF][START_REF] Galati | Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties[END_REF], and we have demonstrated that denbinobin and related phenanthrenequinones induce severe oxidative damage in leukemic cells.

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

The mechanisms by which denbinobin induce ROS is unknown, but we show that its quinone moiety is critical for this activity, and the activity of the diacetylated quinol 23 could be due to an easy saponification and oxidation to the corresponding 1,4 phenanthrenenequinone (compound 7). In general, quinones can trigger a host of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Thus, quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical.

Another interesting possibility is that denbinobin may function as an inhibitory quinone analogue that interferes with CoQ-dependent cellular redox systems at the mitochondria and at the plasma membrane. We suggest that the interference of denbinobin with the CoQ binding site of these systems could lead to a redirection of the normal electron flow in the complex, generating an excess of ROS. Although our data suggest that denbinobin induces ROS from both mitochondrial and extra-mitochondrial sources, we believe that the main target of denbinobin is the mitochondria respiratory chain. Since denbinobin-induced apoptosis is independent of JNK and p38 activation, it is likely that the pro-oxidative environment induces a direct oxidation of thiol groups in mitochondrial PT pores allowing its opening and, as a consequence, the free distribution of solutes at both sides of the inner mitochondrial membrane, triggering the apoptotic pathway [START_REF] Costantini | Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites[END_REF]. This assumption is that denbinobin also affects the functionality of the mitochondria and induces apoptosis in lung and colon cancer cell lines [START_REF] Yang | Molecular mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells[END_REF][START_REF] Kuo | Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction[END_REF][START_REF] Chen | Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells[END_REF].

We have also demonstrated that denbinobin inhibits the activation of NF-κB, a key transcription factor that regulates the expression of anti-apoptotic and antioxidant genes such as SOD [START_REF] Papa | The NF-kappaB-mediated control of the JNK cascade in the antagonism of programmed cell death in health and disease[END_REF]. Therefore, the denbinobin-induced ROS increase is not balanced by NF-κBregulated antioxidant cellular defences. SAR analysis with the denbinobin analogues indicate that the NF-κB inhibitory activity of denbinobin is mediated by its quinone ring and the inhibition of NF-κB is probably the result of ROS accumulation. Although mild oxidative stress is required for NF-κB activation, it has also been demonstrated that, under certain circumstances, a strong pro-oxidative environment may inhibit NF-κB signalling [START_REF] Korn | Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase[END_REF][START_REF] Moodie | Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells[END_REF].

Kinase over-expression experiments suggest that TAK-1 may be the major target for denbinobin in the inhibition of the NF-κB pathway. Although TAK1 is critically involved in stress-activated cell signalling [START_REF] Chen | Stimulus-specific requirements for MAP3 kinases in activating the JNK pathway[END_REF][START_REF] Huangfu | Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappaB activation: TAO2 regulates TAK1 pathways[END_REF] a redox regulation for the activity of this kinase has not been described yet. However, Omori et al. have shown that genetic ablation of TAK1 induced ROS accumulation in keratinocytes and promoted skin inflammation [START_REF] Omori | TAK1 regulates reactive oxygen species and cell death in keratinocytes, which is essential for skin integrity[END_REF].

Interestingly, occasional exposure to field-grown orchids, as well as occupational contact with sawdust of phenanthrenequinones-containing tropical timbers, caused allergic contact dermatitis. In addition, animal models have demonstrated that phenanthrenequinones are potential allergens [START_REF] Hausen | Structure-activity relationships in allergic contact dermatitis. Part III. The sensitizing capacity of substituted phenanthrenequinones: a quantum-mechanical approach[END_REF]. It will be therefore interesting to investigate whether phenathrenequinones induces skin inflammation by inhibiting TAK-1 or by an immune mechanism that involves skin mast cells sensitization.

In conclusion, capitalizing on a series of mechanistic observation, we have elucidated the mechanism by which denbinobin, a constituent of the Chinese herb Shi Hu and of some varieties of Cannabis, induces ROS and inhibits NF-κB in leukemic cells, highlighting the 1. In electron rich molecules (many methoxy groups at the aromatic rings) the intermediate 9,10dihydroquinones 26 can also be isolated. Interestingly, in selected cases, the 9-position of the phenanthrene skeleton can be hydroxylated under these reaction conditions to the 9-hydroxy-1,4-phenanttrenquinones such as 15. The mechanism of this hydroxylation is not yet clear.

The details of this synthesis are described in a previous communication [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF]. In addition to the unsubstituted 1,4-benzoquinone (25a), the substituted 2-methoxy-1,4-benzoquines (25b), or 1,2-dimethoxy-1,4-benzyquinone (25c) can also be used as dienophiles in this Diels-Alder reaction. With 25b, a mixture of regioisomeric products is formed. In many cases, the isomers can be separated by silica gel chromatography [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF]. An alternative method for the introduction of oxygen substituents into ring A of the 1,4phenanthrenquinone is the Thiele-Winter reaction. In this reaction, the phenanthrenequinones are reacted with acetic anhydride in the presence of strong acids such as perchloric acid to afford the two regioisomeric acetates. In the case of the 8-benzyloxy-7-methoxy-1,4phenanthrenequinone [START_REF] Tournier | Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway[END_REF] [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF], the treatment with acetic anhydride and perchloric acid yields the tow regioisomeric tetraacetates 28 and 29 (Scheme 2). The benzyloxy group at C-8 is cleaved under these acidic conditions and the phenolic group thus generated is immediately acetylated. The tetraacetates are then saponified by treatment with sodium methoxide in methanol. In the presence of air, the resulting phenanthrene hydroquinones are oxidized to the corresponding 1,4-phenanthrenequinones. Treatment with diazomethane then selectively methylates the less hindered hydroxyl group at C-3. The resulting isomers are separated by chromatography to afford the pure monophenol 17 [START_REF] Krohn | Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones[END_REF].

O O R 6 R 5 R 4 R 3 R 1 R 2 O O R 1 R 2 + R 3 R 4 R 5 R 6 O O R 6 R 5 R 4 R 3 R 1 R 2 24 25 1-20 26 O O R 7 R 6 R 5 R 4 R 3 R 1
O O R 7 R 6 R 5 R 4 R 3 R 1 R 2 O O R 7 R 6 R 5 R 4 R 3

1

 1 Isolation of denbinobin. The isolation of denbinobin from a chemotype of Cannabis sativa (variety CARMA) cultivated at the I.S.I.C. (Rovigo, Italy) have been previously

  lines and reagents. Jurkat cells were grown at 37 °C and 5% CO 2 in supplemented RPMI 1640 medium containing 10% heat-inactivated FCS, 2 mM glutamine and antibiotics (Lonza, Basel, Switzerland). The 5.1 clone line is a Jurkat derived clone stably transfected with a plasmid containing the luciferase gene driven by the HIV-LTR promoter and was maintained in complete medium supplemented with G418 (200 µg/ml) (Sigma Co, St. Louis, MO, USA). The 293T cell line was obtained from the ATCC (Manassas, VA, USA) and cultured in complete DMEM (Lonza). The antibodies anti-IκBα (sc-371), anti-NFκB-p65 (sc-8008), anti-p-ERK1+2 (sc-7383, Tyr 204), the rabbit polyclonal anti-IKK-γ (sc-8330), and the goat polyclonal against p-IKK α/β (sc-21661, Ser 176) were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). The antibodies against anti-phospho-p65 (Ser 536), anti-phospho-IκBα (5A5, Ser 32/36), anti-JNK (9252), anti-p-JNK 1+2 (9255, Thr 183/Tyr 185) and anti-phospho p38 (9211, Thr 180/Tyr 182) were from Cell Signaling (Danvers, MA, USA). The mAb anti-tubulin and anti-ERK 1+2 (M5670) were purchased A c c e p t e d M a n u s c r i p t 7 from Sigma. The rabbit polyclonal α-caspase 3 was from Dako Diagnostic (Copenhaguen, DK), the antisera α-caspase 7, α-caspase 8 and the antibody anti-PARP were from Chemicon

8 mM

 8 ). For luciferase detection the cells were washed twice in PBS and lysed in 25 mM Tris-phosphate pH 7.8, 8 A c c e p t e d M a n u s c r i p t MgCl 2 , 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 m at RT in a horizontal shaker. Then the lysates were spun down and the supernatant was used to measure luciferase activity using an Autolumat LB 9510 (Berthold) following the instructions of the luciferase assay kit (Promega Co, Madison, WI, USA). Results are represented as RLU or fold induction over untreated control. Results represent mean ± SD of 4 different experiments.

  5 µM) (Fig.2A). Since denbinobin impaired TNFα-and PMA-induced activation of IKK activity, obvious direct candidates for the inhibitory activity of denbinobin would be either the IKKs itself or, alternatively, other kinases upstream IKKs. This possibility was assessed in 293T cells transiently transfected with the KBF-luc plasmid alone or in combination with expression vectors encoding IKKα, IKKβ, TRAF-2, TRAF-6 and TAK1/TAB1. Figure2Bshows that denbinobin could inhibit in a dose-dependent manner the NF-κB-dependent transcriptional activity induced by over-expression of TRAF-2, TRAF-6 and TAK1/TAB1, but not the activity induced by either IKKα or IKKβ.

  Fig. 2C shows that TAK1 can phosphorylate both exogenous IKKα-GFP and IKKβ-GFP proteins (IKKβ-GFP migrates slower than IKKα-GFP) and also phosphorylate the endogenous IκBα protein. Incubation of transfected cells with denbinobin resulted in the inhibition of IKKβ-GFP, IKKα-GFP and IκBα phosphorylation in a dose dependent manner.

13 3. 2

 132 A c c e p t e d M a n u s c r i p t Denbinobin induces a sustained activation of MAPKs that is inhibited by N-Acetyl cysteine. It has been shown that (5Z)-7-oxozeaenol, a selective TAK-1 inhibitor, prevents p38 and JNK activation in IL-1 stimulated cells

  A c c e p t e d M a n u s c r i p t 14 red fluorescence (HE staining). The cells were treated as indicated for six hours, and the percentage of cells expressing fluorescent Eth was determined by flow cytometry.

  Figure

A

  c c e p t e d M a n u s c r i p t caspases, and that the processing of PARP was completely inhibited by pre-incubation with NAC.

A

  c c e p t e d M a n u s c r i p t 16 could prevent denbinobin-induced ΔΨ m dissipation (Fig. 6A), activation of caspases -3, -7

  2 and C-3[START_REF] Szabo | The high-conductance channel of porin-less yeast mitochondria[END_REF] are not active even in the presence of methoxy groups at positions C-6, C-7 A c c e p t e d M a n u s c r i p t and C-8. Next, to analyse the role of the quinone structures, we made substitutions at C-1 and C-4 preserving functional methoxy groups at C-7 and C-8. Reductive methylation at C-1 and C4 converted compounds 4 and 7 into the inactive compounds 22 and 23. In contrast, inclusion of two acetoxy groups at C-1 and C-2 led to the active compound 24 that retains the same biological activities as compound 7 (Supplementary Table

  supported by the results obtained with the glutathione precursor NAC, which can prevent ROS generation, ΔΨ m dissipation and the activation of executor caspases. The role of PT in denbinobin-induced apoptosis was further confirmed by the observation that preincubation with CsA prevents denbinobin-induced ΔΨ m disruption. The mechanism by which A c c e p t e d M a n u s c r i p t 20 denbinobin induces ΔΨ m dissipation is not limited to leukemic cells, since it has been shown

A c c e p t e d M a n u s c r i p t 21 potentialFigure 1 .-

 211 Figure 1.-Denbinobin inhibits NF-κB activation pathway in Jurkat T cells. A, denbinobin

Figure 2 .A c c e p t e d M a n u s c r i p t 28 TRAF- 2 ,

 2282 Figure 2.-A, denbinobin suppresses the TNFα and PMA-induced activation of IKK in vivo.

Figure 3 .

 3 Figure 3.-A, Effects of denbinobin on MAPKs phosphorylation. Jurkat cells were incubated

Figure 4 .M a n u s c r i p t 29 cells; 23 .

 42923 Figure 4.-A, Effects of denbinobin in pre-apoptotic events. Jurkat cells were treated with

Figure 5 .-

 5 Figure 5.-Denbinobin induces cytotoxicity by apoptosis. Jurkat cells were treated with

Figure 6 .-

 6 Figure 6.-Denbinobin induces ΔΨm disruption and caspases activation by MAPK-

  Figure 1

Figure

  Figure

Figure

  Figure

Figure

  Figure

Figure

  Figure

2 Scheme 1 .

 21 Scheme 1. General Scheme of the Diels-Alder reaction of styroles[START_REF] Moodie | Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells[END_REF] with

Scheme 2 .

 2 Scheme 2. Thiele-Winter reaction and subsequent saponification, oxidation, and

A c c e p t e d M a n u s c r i p t 3 ForScheme 3 .Scheme 4 . 23 Synthesis of 1 , 4 -

 3342314 Scheme 3. Photocyclization of stilbenes 30 and 31 to the phenanthrenes 21 and 22.

R 1 R 2 A c c e p t e d M a n u s c r i p t 6 Supplementary Table 2 . 1 cells

 12621 Pro-oxidant and anti-NF-κB activities of 1,4-phenantrenes. 5.were pre-incubated for 30 min with increasing concentrations of compounds 21, 22 and 23 followed by stimulation with TNFα (2 ng/ml) for six h and the luciferase activity measured as indicated in Materials and Methods. The IC50 values were determined. For ROS measurements the cells were incubated with the 10 µM of each compounds and ROS determined by HE staining and flow cytometry detection. For screening purposes ROS was scored positive when the percentage of HE fluorescent cells was higher than 15% compared to untreated cells.

  Figure S1.-A, Effects of denbinobin analogues in pre-apoptotic events. A, Involvement of

CsA 5M 130 kDa 37 kDa 55 kDa Figure 4 caspase 7* PARP PARP* caspase 8 caspase 8* aPARP acaspase 8 Denbinobin 5mM 11.7% 9.35% 86.7% 5.23% DIOC6 Staining 49.2% 5.85% Sanchez-Duffhues et al.

		Control
		0.03%	0.47%	1.88%	4.43%	0.02%	5.8%
	HE Staining	6.19% 23.4%	93.3% 55.5%	4.10% 2.98%	89.6% 5.10%	0.85% 0.6%	81.9% 44.4%

Cell number 33.6% 1.9% 2.4% 7.14% 17.6% Log. Fluorescence Cell number Figure 5 Sanchez-Duffhues et al.

			Denbinobin
	Control	1 M	2.5 M	5 M	10 M
					34.4%
	2.03%	2.33%	4.53%	18.8%

Table 1 .

 1 Pro-oxidant and anti-NF-κB activities of 1,4-phenantrenequinones.5.1 cells were pre-incubated for 30 min with increasing concentrations of denbinobin (2) and analogues followed by stimulation with TNFα (2 ng/ml) for six h and the luciferase activity measured as indicated in Materials and Methods. The IC50 values were determined. For ROS measurements the cells were incubated with the 10 µM of each compounds and ROS determined by HE staining and flow cytometry detection. For screening purposes ROS was scored positive when the percentage of HE fluorescent cells was higher than 15% compared to untreated cells.

	1 H NMR (200 MHz): δ = 2.48 (s, 3H, CH 3 CO 2 ), 2.52 (s, 3H, CH 3 CO 2 ), 3.98 (s, 3H, OMe),
	4.03 (s, 3H, OMe), 7.31 (s, 2H, 2,3-H), 13 C NMR (500 MHz): δ = 21.0 (CH 3 CO 2 ), 21.7
	(CH 3 CO 2 ), 56.3 (OMe), 61.3 (OMe), 113.5 (CH-9), 118.5 (CH-10), 119.8 (CH-2), 121.5
	(CH-3), 121.9 (C-6a), 122.7 (C-10a), 123.6 (CH-5), 124.4 (CH-6), 126.2 (C-4a), 128.6 (C-
	1a), 143.8 (COAc), 144.8 (COAc), 145.5 (COMe), 150.0 (COMe), 169.2, (CH 3 CO 2 ), 169.3
	(CH 3 CO 2 ). IR (KBr): ν (cm -1 ) = 2960, 1754, 1600, 1465, 1374, 1203, 1083, 821, 738. MS
	(EI, 70eV): m/z (%) = 354.0 (30) [M + ], 270.1 (100), 49 (38), 312.1 (35). HRMS (EI, 70 eV):
	Calcd for C 21 H 15 NO 4 354.1103; found 354.1101.

5 Supplementary

Table 1 R 1

 11

						R 2	R 3	R 4	R 5	R 6	R 7	IC50 (µM)	ROS
		R 7			H	H	H	H	H	H	H	7.24	+
	R 6			O	H	OMe	OH	H	OMe	H	H	2.25	+
	R 5			R 1	H H	OMe H	H H	H H	OMe OMe	H H	H H	3.29 4.15	+ +
	R 4	R 3	O	R 2	H	H	H	OMe	H	H	H	4.36	+
					H	H	H	H	H	OMe	H	2.60	+
					H	H	H	H	OMe	OMe	H	3.07	+
					H	H	H	OMe	OMe	H	H	1.12	+
					H	H	H	OMe	H	OMe	H	2.82	+
					H	OMe	H	H	H	H	H	>25	-
					H	OMe	OMe	H	OMe	H	H	>25	-
					H	OMe	OMe	H	H	OMe	H	>25	-
					H	OMe	OMe	OMe	OMe	H	H	>25	-
					H	OMe	OMe	OMe	H	OMe	H	>25	-
					H	OMe	OMe	H	H	OMe	OH	>25	-
					OMe	H	H	H	OMe	OH	H	>25	-
					H	OMe	H	H	OMe	OH	H	>25	-
					OMe	H	H	H	OMe	OMe	H	>25	-
					OMe	H	H	OMe	H	OMe	H	>25	-

Table 1 R 1

 11

	R 1	R 2 R 2	R 3 R 3	R 4 R 4	R 5 R 5	R 6 R 6	R 7 R 7	IC50 (µM) IC50 (µM)	ROS ROS

Table 2 R 2

 22

	R 1		R 1	R 2	IC50 (µM)	ROS
		21	OMe	H	>25	-
	MeO	22	OMe	OMe	>25	-
	R 1	23	OAc	OMe	4.91	+

Table 2 R 2

 22

	R 2	R 1 R 1
	MeO MeO
	R 1 R 1