Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response.

De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

Not flavone-8-acetic acid (FAA) but its murine metabolite 6-OH-FAA exhibits remarkable antivascular activities in vitro.

Flavone-8-acetic acid (FAA) has been proved to be a potent vascular-disrupting agent in mice. Unfortunately, FAA did not produce any anticancer activity in clinical trials. Previously, we had reported that FAA is metabolized by mouse microsomes into six metabolites, whereas it was poorly metabolized by human microsomes, with fewer metabolites formed in lesser amounts. Especially, 6-OH-FAA was not formed by human microsomes. In this work, two major available metabolites, 4'-OH-FAA and 6-OH-FAA, were tested and compared with the parent compound FAA for their potential antivascular activities in vitro. The ability of the products to induce morphological changes, disrupt preformed capillaries of EA.hy926 endothelial cells and inhibit tubulin polymerization in vitro was assessed. The action mechanism was determined using the RhoA and Rac1 inhibitors. At 25 µg/ml, 6-OH-FAA induced morphological changes and membrane blebbing, whereas 300 µg/ml of FAA and 4'-OH-FAA slightly changed the morphology without inducing membrane blebbing. At 300 µg/ml, 6-OH-FAA produced morphological changes that were 2.1-6.9-fold greater than that produced by FAA and 4'-OH-FAA, an effect that was consistent with its much greater inhibitory effect on tubulin polymerization compared with FAA and 4'-OH-FAA. 6-OH-FAA significantly disrupted the EA.hy926 cell capillaries. 6-OH-FAA activities were prevented in EA.hy926 cells pretreated with RhoA, but not Rac1, inhibitor. In this short communication we report for the first time that, in vitro, 6-OH-FAA, a mouse-specific FAA metabolite, exhibits significantly stronger antivascular activities compared with FAA and 4'-OH-FAA, which are mediated through the RhoA kinase pathway.

Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity.

Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

p70S6 kinase is a target of the novel proteasome inhibitor 3,3'-diamino-4'-methoxyflavone during apoptosis in human myeloid tumor cells.

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3'-diamino-4'-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser(136)), activation of caspases -8, -9, and -3 and cleavage of the caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr(389)-phosphorylation and (ii) protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent apoptosis and P70S6K degradation were simultaneously prevented by the caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the proteasome inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated proteasome inhibition and apoptosis of leukemia cells.

Aminopeptidase-N/CD13 is a potential proapoptotic target in human myeloid tumor cells.

The transmembrane metalloprotease aminopeptidase-N (APN)/CD13 is overexpressed in various solid and hematological malignancies in humans, including acute myeloid leukemia (AML) and is thought to influence tumor progression. Here, we investigated the contribution of APN/CD13 to the regulation of growth and survival processes in AML cells in vitro. Anti-CD13 monoclonal antibodies MY7 and SJ1D1 (which do not inhibit APN activity) and WM15 (an APN-blocking antibody) inhibited the growth of the AML cell line U937 and induced apoptosis, as evidenced by cell accumulation in the sub-G(1) phase, DNA fragmentation, and phosphatidylserine externalization. Isotype-matched IgG1 and the APN/CD13 enzymatic inhibitors bestatin and 2',3-dinitroflavone-8-acetic acid, were ineffective. Internalization of CD13-MY7 complex into cells was followed by mitochondrial membrane depolarization, Bcl-2 and Mcl-1 down-regulation, Bax up-regulation, caspase-9, caspase-8, and caspase-3 activation, and cleavage of the caspase substrate PARP-1. The broad-spectrum caspase inhibitor Z-VAD-fmk and the caspase-9- and caspase-8-specific inhibitors significantly attenuated apoptosis. CD13 ligation also induced apoptosis and PARP-1 cleavage in primary AML blasts, whereas normal blood cells were not affected. Overall, these data provide new evidence that CD13 can serve as a target for inducing caspase-dependent apoptosis in AML (independently of its APN activity). These findings may have implications for tumor biology and treatment.

Contributions of the D-Ring to the activity of etoposide against human topoisomerase IIα: potential interactions with DNA in the ternary enzyme--drug--DNA complex.

Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. Interactions between human topoisomerase IIα and etoposide in the binary enzyme--drug complex appear to be mediated by substituents on the A-, B-, and E-rings of etoposide. These protein--drug contacts in the binary complex have predictive value for the actions of etoposide within the ternary topoisomerase IIα--drug--DNA complex. Although the D-ring of etoposide does not appear to contact topoisomerase IIα in the binary complex, etoposide derivatives with modified D-rings display reduced cytotoxicity against murine leukemia cells [Meresse, P., et al. (2003) Bioorg. Med. Chem. Lett. 13, 4107]. This finding suggests that alterations in the D-ring may affect etoposide activity toward topoisomerase IIα in the ternary enzyme--drug--DNA complex. Therefore, to address the potential contributions of the D-ring to the activity of etoposide, we characterized drug derivatives in which the C13 carbonyl was moved to the C11 position (retroetoposide and retroDEPT) or the D-ring was opened (D-ring diol). All of the D-ring alterations decreased the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also weakened etoposide binding in the ternary enzyme--drug--DNA complex and altered sites of enzyme-mediated DNA cleavage. On the basis of these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex.

C5-DNA methyltransferase inhibitors: from screening to effects on zebrafish embryo development.

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.

Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells.

The implication of DNA methylation in cancer is today clearly established. Despite that nucleoside analogues are currently used for leukaemia treatment, their low stability in physiological conditions and their lack of selectivity arise the need for the identification of non-nucleoside DNA methyltransferase inhibitors. Here, we describe the synthesis and pharmacological characterisation of a novel class of DNA methyltransferase inhibitors: the 3-halo-3-nitroflavanones. We showed that 3-bromo-3-nitroflavanones 3b and 4a have a micromolar DNMT inhibition and an increased potency in a cell reporter model. Interestingly they are significantly more stable than the reference compounds and induce a low cytotoxicity, supporting them as new candidates for the development of non-cytotoxic cell-reprogramming epi-drugs for anticancer treatment.

Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning.

Electrospray tandem mass spectrometry of beta-nitroalkenyl meso-tetraphenylporphyrins.

Beta-nitroalkenyl meso-tetraphenylporphyrins [beta-TPPCHC(NO(2))R)], as free-bases and Zn(II) complexes, were studied by electrospray mass spectrometry (ESI-MS). Under this ionisation condition the [M + H](+) ions are formed. The fragmentation pattern of the resulting [M + H](+) ions were studied by electrospray tandem mass spectrometry (ESI-MS/MS). The ESI-MS/MS of beta- nitroalkenylporphyrins, either as free-bases or as Zn(II) complexes, show several interesting features, distinct from the typical behaviour of nitro compounds. For the studied compounds, common main fragmentation patterns are observed, namely characteristic losses of NO(2), HNO(2), 2OH, RNO(2), RCNO, RCNO(2), RCH(2)NO(2), C(6)H(5) plus NO(2) and the formation of the protonated macrocycle, [TPP + H](+) or [ZnTPP + H](+). However, depending on the presence or absence of the metal and the nature of the R substituent, important differences are observed on the relative abundances of the ions formed by the same fragmentation pathway. The presence of bromine in the alkenyl group leads to a peculiar behaviour, since the main fragmentation pattern corresponds to the combined elimination of the bromine atom with the typical nitro group fragments. When R = Br, the loss of the nitro group occurs in low relative abundance (11-16%). However, when R = CH(3), the relative abundance of the ion due to the loss of HNO(2) changes drastically from 100%, observed for the free-base porphyrin, to 29% in the case of the Zn(II) complex. These variations of the relative abundance of the fragment corresponding to the loss of the nitro moiety (typically considered as a diagnostic fragment) can induce to an erroneous interpretation of their MS/MS spectra. Some fragmentations are observed only for the free-base porphyrins, namely the loss of CH(NO(2)R and HNO(2) plus C(2)H(2), while the loss of OH, H(2)O, OH plus H(2)O and RCCH plus H(2)O is observed only for the complexes. Unusual and unexpected fragmentations are also observed, namely the losses of RCNO, RCNO(2) and HNO(2) plus C(2)H(2). This work demonstrates that valuable structural information about the beta-nitroalkenyl substituents linked to meso- tetraarylporphyrins can be achieved using MS/MS. These results can also be useful for the interpretation of the mass spectra of other nitroalkenyl substituted compounds.

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells.

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

Exploring the mechanism of inhibition of human telomerase by cysteine-reactive compounds.

Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR.

Correlation between electron-donating ability of a series of 3-nitroflavones and their efficacy to inhibit the onset and progression of aberrant crypt foci in the rat colon.

A series of five 3-nitroflavones were tested for their ability to inhibit the formation of colon aberrant crypt foci (ACF) induced by a s.c. injection of azoxymethane (C2H6N2O) in rats. Our aim was to relate the electron-donating effects of the 3-nitroflavones as characterized by their Hammett substitution constants with their efficacy in inhibiting ACF. In a first assay (initiation, protocol A) the 3-nitroflavone as well as the 4'-substituted nitro-, methoxy-, fluoro-, and hydroxy-3-nitroflavones were continuously present in the diet. In a second assay (postinitiation, protocol B) they were given for a period of 4 weeks after the last azoxymethane injection. The different substituents of the 3-nitroflavones at the 4'-position spanned a spectrum of Hammett constants (sigma(p+)), going from +0.79 for the electron-withdrawing group, NO2, to -0.92 for the electron-donating group, OH. For both protocols the percentages of inhibition plotted versus the Hammett substitution constants showed a linear correlation, the most efficacious ACF inhibition being produced by the molecules with the most electron-donating substituents. Moreover, the nitroflavones were not only chemoprotective during initiation of the ACF, but also therapeutic in the postinitiation progression assay. The above correlations may be of predictive value in the search for new chemoprotective agents. The overall molecular mechanism of the inhibition of ACF by the 3-nitroflavones under study appears to involve redox reactions.

Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors.

There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.

Metabolism of Flavone-8-acetic Acid in Mice.

Flavone-8-acetic acid (FAA) is a potent antivascular agent in mice but not in humans. Assuming that FAA was bioactivated in mice, we previously demonstrated that 6-OH-FAA was formed from FAA by mouse microsomes but not by human microsomes; its antivascular activity was 2.1- to 15.9-fold stronger than that of FAA, and its antivascular activity was mediated through the Ras homolog gene family (Rho) protein kinase A (RhoA) pathway. The present work aimed to study FAA metabolism in order to verify if 6-OH-FAA is formed in mice. Using synthesized standards and high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) analysis, we herein demonstrated, for the first time, that in vitro FAA and its monohydroxylated derivatives could directly undergo phase II metabolism forming glucuronides, and two FAA epoxides were mostly scavenged by NAC and GSH forming corresponding adducts. FAA was metabolized in mice. Several metabolites were formed, in particular 6-OHFAA. The antitumor activity of 6-OH-FAA in vivo is worthy of investigation.

Synthesis and biological study of a new series of 4'-demethylepipodophyllotoxin derivatives.

Etoposide (VP-16) is a potent human DNA topoisomerase II poison, derived from 4'-demethylepipodophyllotoxin, widely used in cancer chemotherapy. Continuous efforts have driven to synthesize new related compounds, presenting decreased toxic side effects, metabolic inactivation, drug resistance, and increased water solubility. Identified structure-activity relationships have pointed out the importance of the 4beta-substitution and of the configuration of the D ring. Here we report the synthesis of two novel series of derivatives of 4'-demethylepipodophyllotoxin. The first bears a carbamate chain in the 4 position (13a-f), whereas, in the second series, in addition to this chain, the lactone ring has been modified by shifting the carbonyl from position 13 to position 11 (27a-f). Moreover, an analogue of TOP-53 having this lactone modification has also been prepared (32). From this study, structure-activity relationships were established. Compounds 13a and 27a displayed potent cytotoxic activity against the L1210 cell line (10 to 20-fold higher than VP-16) and proved to be strong topoisomerase II poisons more potent than VP-16. From preliminary in vivo investigation of both compounds against P388 leukemia and orthotopically grafted human A549 lung carcinoma, it appeared that 13a and 27a constitute promising leads for a new class of antitumor agents.

Synthesis and biological evaluation of novel flavone-8-acetic acid derivatives as reversible inhibitors of aminopeptidase N/CD13.

The cell surface aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical role in angiogenesis. However, potent, selective, and, particularly, noncytotoxic inhibitors ot this protein are lacking, and the present work was undertaken with the aim of developing a new generation of noncytotoxic inhibitors that bind to APN/CD13. In this context, we have synthesized a series of novel flavone-8-acetic acid derivatives. Among the herein described and evaluated compounds, the 2',3-dinitroflavone-8-acetic acid (19b) proved to be the most efficient and exhibited an IC(50) of 25 microM which is 2.5 times higher than that of bestatin (1), the natural known inhibitor of APN/CD13. However, in contrast to bestatin (1), the dinitroflavone 19b did not induce any cytotoxicity to cultured human model cells. The presence of other substituents such as NO(2) or OCH(3) groups at the 3'- or 4'-position of the B phenyl group, or the existence of steric constraints (compounds 24 and 29), did not improve selectivity and potency. The flavone 19b affinity for APN/CD13 is not recovered with other proteases such as matrix metalloproteinase-9 (MMP-9), angiotensin converting enzyme (ACE/CD143), neutral endopeptidase (NEP/CD10), gamma-glutamyl transpeptidase (CD224), or the serine proteases dipeptidyl peptidase IV (DPPIV/CD26) or cathepsin G.

Synthesis and biological activity of sulfonamide derivatives of epipodophyllotoxin.

A series of novel 4beta-substituted sulfonamide derivatives of 4'-O-demethyl-4-desoxypodophyllotoxin has been synthesized. Their effects on human DNA topoisomerase II and, in some cases, on tubulin polymerization were evaluated. Compounds 8a, 8c, 8f, 8g, 8n, 8q, 8r, and 8s and the synthetic precursor 4 are potent topoisomerase II poisons that induce double-stranded breaks in DNA, with either improved or similar activity compared to etoposide. Only the amino precursor, compound 5, was slightly active in tubulin polymerization inhibition assays. We observed that the derivatives bearing an aromatic ring on the 4beta-sulfonamide substituent were either less cytotoxic or equivalent to the parent drug, while the sulfonamides containing an aliphatic side chain and the amino-sulfonamide derivatives, except 8d and 8g, exhibited increased cytoxicity compared to etoposide. In vivo, against the P388 leukemia and the A-549 orthotopic model of lung carcinoma, the most promising compounds were the morpholino- and the piperazino-containing sulfonamides derivatives 8r and 8s.

Flavones and polyphenols inhibit the NO pathway during apoptosis of leukemia B-cells.

We recently reported that resveratrol, a grape-derived polyphenol, in vitro induces the apoptosis of leukemic B-cells and simultaneously inhibits the production of endogenous nitric oxide (NO) through inducible NO synthase (iNOS) down-regulation. The same results were observed in the present study with not only acetate derivatives of polyphenols, particularly the pentaacetate of -viniferin (resveratrol dimer), but also with a synthetic flavone (a diaminomethoxyflavone) in both leukemia B-cell lines and B-cell chronic lymphocytic leukemia (B-CLL) patients' cells. Moreover, flavopiridol, another flavone already known for its pro-apoptotic properties in B-CLL cells, was also found to down-regulate both iNOS expression and NO production. Thus, inhibition of the NO pathway during apoptosis of leukemia B-cells appears a common mechanism for several compounds belonging to two distinct families of phytoalexins, the flavones and grape-derived polyphenols.

The inducible NO synthase is downregulated during apoptosis of malignant cells from B-cell chronic lymphocytic leukemia induced by flavopiridol and polyphenols.

Downregulation of iNOS and NO is a pathway common for flavones and polyphenols, two distinct families of phytoalexins. Our data suggest that inhibition of the NO pathway could be one of the mechanisms involved in the proapoptotic properties of these phytoalexins in leukemia B-cells.