Jean-François Goossens

Relation between intracellular acidification and camptothecin-induced apoptosis in leukemia cells.

Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3-0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed.

Synthesis, Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition Properties of Novel Indeno[2,1-c]quinolin-7-one and Indeno[1,2-c]isoquinolin-5,11-dione Derivatives

Indeno[2,1- c]quinolin-7-ones and 6 H-indeno[1,2- c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2- c]isoquinolin-5,11-dione bearing side chains at N-6 and C-8 positions ( 6a) was a potent human topoisomerase II inhibitor with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism of action. These indeno[1,2- c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II interfering anticancer molecules.

Apoptotic response of HL-60 human leukemia cells to the antitumor drug NB-506, a glycosylated indolocarbazole inhibitor of topoisomerase 1

The antitumor drug NB-506 is a glycosylated indolocarbazole derivative targeting topoisomerase I. This DNA-intercalating agent, which is currently undergoing phase I/II clinical trials, was shown to induce apoptosis in HL-60 human leukemia cells. We compared the cellular dysfunctions induced by NB-506 and the reference topoisomerase I poison camptothecin (CPT) at the nuclear, mitochondrial, and cytoplasmic levels. The two drugs NB-506 and CPT were almost equally toxic to HL-60 cells and produced similar cell cycle changes with a considerable increase in the fraction of cells with DNA content less than G1. The sub-G1 fraction, which can be considered as the apoptotic cell population, appeared more rapidly with CPT than with NB-506 but in both cases, the cell cycle perturbation was accompanied by a marked decrease in the mitochondrial transmembrane potential and the intracellular pH. In contrast, no change in the intracellular calcium concentration was detected. Treatment of HL-60 cells with NB-506 resulted in an increase in the activity of the intracellular protease caspase-3, as determined by a DEVD-based colorimetric assay and direct monitoring of poly(ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. The initiator caspase-8 was also stimulated by NB-506 but, as for caspase-3, the extent of the caspase activation was weaker with NB-506 compared to CPT. With both drugs, the protease activation resulted in DNA degradation, as independently confirmed via the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and characterization of internucleosomal DNA fragmentation. Collectively, these findings identify some of the molecular events leading to NB-506-induced apoptosis and as such, provide important mechanistic insights into the mode of action of topoisomerase I-targeted indolocarbazole antitumor drugs.

Targeting the gut microbiota with inulin-type fructans: preclinical demonstration of a novel approach in the management of endothelial dysfunction

Objective To investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction. Design We examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe−/−) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12 weeks with or without inulin-type fructans (ITFs) supplementation for the last 15 days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed. Results ITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe−/− mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation. Conclusions We demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.

Label‐free characterization of carbonic anhydrase—novel inhibitor interactions using surface plasmon resonance, isothermal titration calorimetry and fluorescence‐based thermal shift assays

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity‐based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka, kd, and KD), leads us to choose this methodology for the study of new potential inhibitors. Copyright

Chiral capillary electrophoretic determination of the enantiomeric purity of tetrahydronaphthalenic derivatives, melatoninergic ligands, using highly sulfated β‐cyclodextrins

Using cyclodextrin capillary zone electrophoresis (CD‐CZE), baseline separation of synthetic tetrahydronaphthalenic derivatives, potential melatoninergic compounds, was achieved. A method for the enantioresolution of these tetralins and determination of their enantiomeric purity was developped using anionic CDs (highly sulfated‐CD or highly S‐CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters such as the nature and concentration of the chiral selectors, buffer pH, organic modifiers, temperature and applied voltage were investigated. The use of charged CDs provides a driving force for our neutral compounds in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S‐β‐CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution of three tetralin compounds was obtained using 25 mM phosphate buffer at pH 2.5 containing 2.5% w/v of highly S‐β‐CD at 25°C with an applied field of 0.25 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of linearity, sensitivity, accuracy and recovery. The enantiomeric purity for the three molecules was determined and the detection limit of enantiomer impurities is about 0.3–0.6%.

Novel potent substance P and neurokinin A receptor antagonists. Conception, synthesis and biological evaluation of indolizine derivatives.

Exploration of SAR around dual NK(1)/NK(2) antagonist Cbz-Gly-Leu-Trp-OBzl(CF(3))(2) and its derivatives disclosed the essential requirements for more potent dual NK(1)/NK(2) binding. We report here the synthesis and the biological properties of a novel series of indolizine including pharmacophoric elements.

New Bis-catechols 5-lipoxygenase inhibitors

Three polyhydroxy-2-phenylnaphthalenes (1-3) and the oxy analogue of tetrahydroxypavinan (4) were prepared and evaluated for their antioxidant properties (inhibition of diphenylpycrylhydrazyl radical (DPPH), reduction of iron (III) ion) and inhibition of 5-lipoxygenase (5-LO) activity. Their three-dimensional structures were established on the basis of spectroscopic data and semiempirical calculations. Compounds 1 and 2 were found as potent 5-LO inhibitors as nordihydroguaiaretic acid (NDGA), whereas 4 is 2.5 times less potent than NDGA. The reliability of the 3-D structures with the 5-LO inhibition properties is discussed. Their antioxidant properties show that tested compounds are expected to act as redox inhibitors.

Evaluation of Decontamination Efficacy of Cleaning Solutions on Stainless Steel and Glass Surfaces Contaminated by 10 Antineoplastic Agents

OBJECTIVES The handling of antineoplastic agents results in chronic surface contamination that must be minimized and eliminated. This study was designed to assess the potential of several chemical solutions to decontaminate two types of work surfaces that were intentionally contaminated with antineoplastic drugs. METHODS A range of solutions with variable physicochemical properties such as their hydrophilic/hydrophobic balance, oxidizing power, desorption, and solubilization were tested: ultrapure water, isopropyl alcohol, acetone, sodium hypochlorite, and surfactants such as dishwashing liquid (DWL), sodium dodecyl sulfate (SDS), Tween 40, and Span 80. These solutions were tested on 10 antineoplastic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, irinotecan, cyclophosphamide, ifosfamide, doxorubicin, epirubicin, and vincristine. To simulate contaminated surfaces, these molecules (200ng) were deliberately spread onto two types of work surfaces: stainless steel and glass. Recovered by wiping with a specific aqueous solvent (acetonitrile/HCOOH; 20/0.1%) and an absorbent wipe (Whatman 903®), the residual contamination was quantified using high-performance liquid chromatography (HPLC) coupled to mass spectrometry. To compare all tested cleaning solutions, a performance value of effectiveness was determined from contamination residues of the 10 drugs. RESULTS Sodium hypochlorite showed the highest overall effectiveness with 98% contamination removed. Ultrapure water, isopropyl alcohol/water, and acetone were less effective with effectiveness values of 76.8, 80.7, and 40.4%, respectively. Ultrapure water was effective on most hydrophilic molecules (97.1% for cytarabine), while on the other hand, isopropyl alcohol/water (70/30, vol/vol) was effective on the least hydrophilic ones (85.2% for doxorubicin and 87.8% for epirubicin). Acetone had little effect, whatever the type of molecule. Among products containing surfactants, DWL was found effective (91.5%), but its formulation was unknown. Formulations with single surfactant non-ionics (tween 40 and span 80) or anionic (SDS) were also tested. Finally, solutions containing 10(-2) M anionic surfactants and 20% isopropyl alcohol had the highest global effectiveness at around 90%. More precisely, their efficacy was the highest (94.8%) for the most hydrophilic compounds such as cytarabine and around 80.0% for anthracyclines. Finally, the addition of isopropyl alcohol to surfactant solutions enhanced their decontamination efficiency on the least hydrophilic molecules. Measured values from the stainless steel surface were similar to those from the glass one. CONCLUSION This study demonstrates that all decontamination agents reduce antineoplastic contamination on work surfaces, but none removes it totally. Although very effective, sodium hypochlorite cannot be used routinely on stainless steel surfaces. Solutions containing anionic surfactant such as SDS, with a high efficiency/safety ratio, proved most promising in terms of surface decontamination.

A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing

Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico‐chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV‐detectable pI markers using a central composite design. The separation was performed in a fused‐silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol–water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.