Daniel Mansuy

JunD Reduces Tumor Angiogenesis by Protecting Cells from Oxidative Stress

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.

Ferrocenyl Quinone Methides as Strong Antiproliferative Agents: Formation by Metabolic and Chemical Oxidation of Ferrocenyl Phenols†

The modification of the biological effects of some biologically active molecules by ferrocene is an active field of study. The addition of a ferrocenyl moiety to selected polyaromatic phenols, amines, and amides can potentiate their antiproliferative effects against breast and prostate cancer cells. For example, hydroxytamoxifen, the active metabolite of the breast cancer drug tamoxifen, shows limited toxicity against hormone-refractory breast cancer cells (IC50 for MDA-MB231: 29 mm). However, when a phenyl group is replaced by a ferrocene moiety, the resulting “hydroxyferrocifen” displays a dramatic improvement in toxicity (IC50 = 0.5 mm). [6] In 2006, we proposed in this journal a novel mechanism of activation of ferrocene phenols: their ferrocene-mediated oxidation to quinone methide (QM) metabolites. This work was subsequently featured as part of a Highlight article. Quinone metabolites are potentially cytotoxic species, and, when ferrocene is present, QM formation takes place at comparatively mild (i.e. biologically relevant) oxidation potentials. Furthermore, the observation that some cancer cells are under heightened oxidative stress suggests that this class of redox-activated compounds may show an interesting selectivity profile. The formation of ferrocenyl QMs has been supported only by electrochemical experiments until now. We herein show that QMs do form upon metabolism of 1–4 by rat liver

Evidence for phosgene formation during liver microsomal oxidation of chloroform

Abstract When aerobically incubated with liver microsomes and NADPH, chloroform produces a stable adduct with cysteineas a nucleophilic trapping agent. The adduct was identified by thin layer chromatography, gas-liquid chromatography and combined gas chromatography-mass spectrometry as the reaction product of cysteine with phosgene.

Formation of nitrogen oxides and citrulline upon oxidation of Nω-hydroxy-L-arginine by hemeproteins

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: Involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).

Dual effects of macrolide antibiotics on rat liver cytochrome P-450: Induction and formation of metabolite-complexes: A structure-activity relationship

Previous studies have shown that the macrolide antibiotics, troleandomycin and erythromycin, are able to induce their own transformation into a metabolite forming an inactivated complex with rat liver cytochrome P-450. This paper reports the results of a study on the effects of several macrolide antibiotics including oleandomycin, erythromycin derivatives, josamycin, methymycin, tylosin, spiramycin and rifampicin, as well as antibiotics of other series, such as tetracycline and lincomycin, on rat liver cytochromes P-450 in vivo and in vitro. Only the antibiotics containing the desosamine and mycaminose amino sugars were able to give the dual effects already found with troleandomycin: induction of cytochrome p-450 and formation of an inhibitory cytochrome P-450--iron--nitrosoalkane metabolite complex in vivo or in vitro. From these studies, it appears that two structural factors are important for a macrolide antibiotic to lead to such effects: the presence of a non-hindered readily accessible N-dimethylamino group and the hydrophobic character of the molecule. These data are discussed in relation to the adverse effects observed during drug associations involving some of these macrolide antibiotics.

Alkane hydroxylation catalyzed by metalloporhyrins : evidence for different active oxygen species with alkylhydroperoxides and iodosobenzene as oxidants.

Abstract A comparative study of cyclohexane and n-heptane hydroxylations by cumylhydroperoxide and iodosobenzene, catalyzed by various metalloporphyrins, indicates that different active oxygen species, presumably the cumyloxy radical and a metal-oxo intermediate, are involved in these reactions.

Cytochrome P450 catalyzes the oxidation of Nω-hydroxy-L-arginine by NADPH and O2 to nitric oxide and citrulline

Rat liver microsomes catalyze the oxidative denitration of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides like NO and NO2-. Besides NO2- and citrulline, whose simultaneous formation is linear for at least 20 min, the formation of NO could be detected under the form of its P450 and P420-Fe(II) complexes by UV-visible and EPR spectroscopy. Classical inhibitors of NO-synthases, like N omega-methyl-and N omega-nitro-arginine, fail to inhibit the microsomal oxidation of NOHA to citrulline and NO2-. On the contrary classical inhibitors of hepatic cytochromes P450 like CO, miconazole, dihydroergotamine and troleandomycin, strongly inhibit this monooxygenase reaction. These results show that the oxygenation of NOHA by NADPH and O2 with formation of citrulline and NO can be efficiently catalyzed by cytochromes P450 (with rates up to 1.5 turnovers per min for the cytochromes of the 3A subfamily).

Particular ability of cytochromes P450 3A to form inhibitory P450-iron-metabolite complexes upon metabolic oxidation of aminodrugs.

The ability of 21 drugs containing an amine function to form inhibitory P450-iron-metabolite complexes absorbing around 455 nm was studied on liver microsomes from rats treated with various P450 inducers. These drugs belong to different chemical and therapeutic series and exhibit very different structures. In the case of eight compounds (diltiazem, lidocaine, imipramine, SKF 525A, fluoxetine, L-alpha-acetylmethadol, methadol and desmethyltamoxifen) whose oxidation by microsomes from rats treated with several inducers was studied, only dexamethasone (DEX)-treated rat microsomes and, to a lesser extent, phenobarbital (PB)-treated rat microsomes, were able to give significant amounts of 455 nm absorbing complexes. Ten of the 21 compounds studied gave such complexes with DEX-treated rat microsomes, while only three compounds gave complexes (in low amounts) with PB-treated rat microsomes only. For all compounds leading to complexes both with DEX- and PB-treated rat microsomes, much higher amounts of complexes were obtained with DEX-treated rat microsomes. DEX-treated rat microsomes also led to the most intense type I spectral interactions with most of the compounds studied, and very often exhibited the highest N-dealkylation activities towards the tertiary or secondary amine function of the drugs used. A few exceptions aside, there generally exists a qualitative relationship between the ability of P450 3As, induced by DEX, to bind and N-dealkylate amino compounds and their propensity to lead to 455 nm absorbing complexes. This was confirmed by in vivo experiments showing that rats treated with diltiazem, tamoxifen or imipramine accumulated large amounts of 455 nm absorbing complexes in their liver only after pretreatment with DEX and, to a lesser extent, with PB. This particular ability of P450 3As to oxidize amino drugs with formation of inhibitory P450-metabolite complexes could be of great importance for the appearance of drug interactions in man.

Preparation of functionalized polyhalogenated tetraaryl-porphyrins by selective substitution of the p-Fluorines of meso-tetra-(pentafluorophenyl)porphyrins

Abstract Reaction of various nucleophiles, such as primary and secondary amines in refluxing DMF, alkoxides in alcohol or THF and thioIs in the presence of NEt 3 in DMF, with meso-tetra-(pentafluorophenyl)porphyrin (or its Zn(H) and Fe(III) complexes) led to the selective replacement of the p-fluorine substituents of the starting porphyrin by NR 2 , OR or SR groups, in yields between 70 and 90%. The same regioselectivity was obtained by reaction of meso- tetra-(pentafluorophenyl) octabromoporphyrin with n-propylamine. Reaction with KCN gave a more complex reaction mixture.