Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation

Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression.     

Inhibition of mitosis by avarol, a natural product isolated from the sponge Dysidea avara

Avarol, a sesquiterpenoid hydroquinone, is a cytostatic agent, isolated from the sponge Dysidea avara. Autoradiographic studies show that in vivo (L5178y mouse lymphoma cells) avarol changes the labelling index in favour of the fraction of unlabelled cells (from 1.24 to 1.04). At concentrations below the 50% inhibition dose, the mitotic index increases from 6.5 +/- 0.5 to 10.4 +/- 0.8; at higher concentrations the formation of mitotic figures is almost completely suppressed. In vitro studies applying the methods of viscosimetry and electron microscopy demonstrate that avarol inhibits assembly of brain microtubule protein at an at least stoichiometric concentration ratio. Moreover, evidence is presented that the new antimitotic agent avarol inhibits protofilament elongation rather than lateral association of tubulin during protofilament formation. The results suggest that avarol interferes with polymerization of tubulin both in interphase and during mitosis.     

Avarol inhibits TNF-alpha generation and NF-kappaB activation in human cells and in animal models

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological properties including anti-inflammatory and antipsoriatic effects. In the present study we evaluated the pharmacological effect of avarol on some inflammatory parameters related to the pathogenesis of psoriasis. Avarol inhibited tumor necrosis factor-alpha (TNF-alpha) generation in stimulated human monocytes (IC(50) 1 microM) and TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB)-DNA binding in keratinocytes. In the mouse air pouch model, administration of avarol produced a dose-dependent reduction of TNF-alpha generation (ED(50) 9.2 nmol/pouch) as well as of interleukin (IL)-1beta, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) levels in pouch exudates. In the psoriasis-like model of 12-O-tetradecanoylphorbol-acetate-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 micromol/site) reduced edema, myeloperoxidase activity, IL-1beta, IL-2 and eicosanoid levels in skin. Histopathological study confirmed the inhibition of epidermal hyperplasia as well as leukocyte infiltration. The reduction of cutaneous TNF-alpha by avarol was also detected by immunohistochemical analysis. Avarol was also capable of suppressing in vivo NF-kappaB nuclear translocation, determined in mouse skin. Our results suggested that antipsoriatic properties of avarol previously described could be mediated in part by the downregulation of several inflammatory biomarkers, such as TNF-alpha and NF-kappaB in psoriatic skin.     

Sponge secondary metabolites: biochemical and ultrastructural localization of the antimitotic agent avarol in Dysidea avara

The secondary metabolite avarol, a potent cytostatic and antibacterial sesquiterpenoid hydroquinone, is present in large amounts only in the sponge Dysidea avara (2.7 g avarol/1 kg of fresh material). The present study was designed to determine the storage site of this compound within the organism. Light and transmission electron microscopic studies revealed that avarol is probably stored only in spherular cells. The compound is compartmented in intracellular cytoplasmic vesicles in a paracrystalline form, and therefore can have no inhibitory effect on the sponge cells. Quantitative analysis utilizing high-pressure liquid chromatography revealed that avarol is present at a concentration of 3.2 micrograms/10(6) spherular cells. It appears that avarol is released from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulating the bacteria with which the sponge is symbiotically associated.     

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

Antimutagenic activity of the novel antileukemic agents, avarone and avarol

The two antileukemic agents, avarone and avarol, were determined to be neither direct nor indirect mutagenic agents in the Ames microsomal test. Moreover, the two sesquiterpenoid compounds drastically reduced the mutagenic effect of benzo[a]pyrene in the same system. Subsequent enzymic studies demonstrated that avarone and avarol are powerful inhibitors of benzo[a]pyrene monooxygenase.     

Action of the antileukemic and anti-HTLV-III (anti-HIV) agent avarol on the levels of superoxide dismutases and glutathione peroxidase activities in L5178y mouse lymphoma cells

The antileukemic and anti-HTLV-III (anti-HIV) agent avarol, a sesquiterpenoid hydroquinone, was determined to be converted into its corresponding quinone derivative avarone via the semiquinone free radical. Its g-value was 2.0047; after hyperfine splitting the energy levels revealed 16 isotropic Hfs. The redox reaction products were identified at the pH values 4.0, 7.0 and 12.0 and the overall reaction pathways were formulated. In vivo experiments with L5178y mouse lymphoma cells in the ascites of mice revealed that the cytostatic potencies of avarol and avarone cannot be augmented by lowering the pH value. Incubation studies with L5178y cells in vitro showed that the intracellular levels of superoxide dismutases (SODases) and of glutathione (GSH) peroxidase activities significantly change after avarol administration. While both the Mn-SODase and the Cu/Zn-SODase activities dropped significantly, the GSH peroxidase activity increased inversely. From these experiments we assume that the anti-tumour and the antiviral effects of avarol/avarone may be due to an increase, induced by the drug, of the intracellular concentrations of superoxide radicals.     

Spectroscopic and biological properties of palladium(II) complexes of ethyl 2-quinolylmethylphosphonate

Spectroscopic (1H NMR, UV-visible) and biological (cytostatic, antiviral activity) studies of palladium(II) complexes of monoethyl 2-quinolymethylphosphonate (2-Hmqmp): dihalide adducts trans-Pd(2-Hmqmp)2X2, chelate Pd(2-mqmp)2.2H2O and ion-pair salt complexes [2-H2mqmp]+[Pd(2-Hmqmp)X3]- (X = Cl, Br), have been carried out in order to determine structural and biological properties of these biologically interesting complex compounds. The complexes were evaluated in vitro for their cytostatic activity against murine L1210 and human KB and T-lymphoblast Molt4/C8 and CEM/0 cell lines, and the results obtained were compared with those obtained for the complexes of diethyl 2-quinolylmethylphosphonate (2-dqmp). The L1210 cell was the most responsive line and complexes of diester 2-dqmp were more active than complexes of monoester 2-Hmqmp. A good relationship was observed between the cytostatic activity of the complexes and their lypophilicity or solubility. Some complexes exhibited significant cell growth inhibitory effects, but none of the them was more cytostatic than cisplatin. Both 2-dqmp and 2-Hmqmp complexes were also evaluated in vitro for their antiviral activity in different assay systems, comprising a broad spectrum of DNA and RNA viruses, but no specific antiviral effects were noted. In addition, the complexes did not show any specific anti-HIV activity.     

13C NMR spectroscopy of Methanobacterium thermoautotrophicum. Carbon fluxes and primary metabolic pathways

The flux of 13C-labeled carbons from the soluble metabolite 2,3-cyclopyrophosphoglycerate (CPP), a novel compound found in high concentrations exclusively in methanobacteria and methanobrevibacter, into carbohydrate-containing material has been deduced by solid-state 13C NMR spectroscopy which strongly argues for a role in gluconeogenesis for this unique metabolite. The turnover rates, but not the steady-state levels, of CPP labeled by 13CO2 or [13C]acetate depend dramatically on cell growth conditions. When the demand for carbohydrate synthesis is reduced (i.e. in stationary phase), the rates of CPP biosynthesis and degradation decrease 10-fold, and the disaccharide alpha, alpha-trehalose accumulates. Valinomycin, a metabolic inhibitor of Methanobacterium thermoautotrophicum growth, does not affect steady-state levels of CPP, but does decrease 13C uptake into the CPP pool. The effects of these different conditions on CPP labeling suggest stringent regulation of CPP linked to cellular metabolism. Labeling of CPP by [6-(13)C]glucose, which does not serve as an energy or carbon source for this organism, provides strong evidence that glucose is cleaved by the reverse of the gluconeogenesis pathway. This metabolic pathway linking glucose with triose phosphate type precursors and an analysis of the 13C NMR spectrum of CPP labeled by incubating cells with [U-13C]glucose have established that in vivo phosphoenolpyruvate synthetase must be reversible.     

GROWTH INHIBITION OF MURINE TUMOR CELLS, IN VITRO, BY PUROMYCIN, [6N]O2''-DIBUTYRYL 3'',5''-ADENOSINE MONOPHOSPHATE, OR ADENOSINE : Evidence of Commitment for Cell Division

The cytostatic effects of puromycm, [⁶N]O^2'-dibutyryl 3',5'-adenosine monophosphate, and adenosine on asynchronous and synchronous cultures of the murine mastocytoma, P815Y, have been studied. Cell growth was arrested after a minimum of one further division. A model is proposed for the inhibition of cell division in which the periods of inhibition and growth arrest are separated in time by one cell cycle.     

Structural characterization and cytostatic activity of chlorobischolylglycinatogold(III)

Based on the ability of bile acids for vectorializing the cytostatic activity of other agents, we have designed and synthesized a new bile acid cholylglycinato Au(III) complex, named Bamet-A1. It has been characterized by means of EA (elemental analysis), FT-IR, NMR, FAB-MS (fast atom bombardment-mass spectrometry) and Vis-UV techniques. This characterization allowed us to propose a structure of the type [Au CG(O) CG(N,O) Cl] for the neutral complex, which has the composition C522H84N2O12AuCl and is very soluble in water, methanol, ethanol and DMSO (dimethylsulfoxide). The study in aqueous solution suggested a redox process for its transformation, which is accompanied by the appearance of colloidal gold phase. The behavior in 4 mM NaCl water (in order to mimic the cytoplasmatic fluid) was similar to that observed in water, while in a 150 mM NaCl (similar to extracellular fluid and serum), the apparition of a dark blue precipitate was observed. This complex displays fluorescence, which does not change when incubated with DNA obtained from E. coli. Bamet-A1 was found to inhibit the growth of a variety of cell lines. The cytostatic effect was mild against human hepatoma HepG2, mouse hepatoma Hepa 1-6, rat hepatoma McA RH-7777 and human colon adenocarcinoma LS-174T, and stronger against mouse sarcoma S180-II and mouse leukemia L-1210 cells. The appearance of colloidal Au during the process of hydrolysis under physiological conditions may explains the low cytostatic activity.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

Free cytosolic Ca2+ measurements with fluorine labelled indicators using 19FNMR

Characterisation by 19F NMR of fluorine-labelled indicators of cytosolic free Ca2+ concentration (by 5FBAPTA) and pH (by Fquene) is described, together with the techniques used to load the cell suspensions with the indicators for NMR spectroscopy. Useful features of the 19F NMR indicators include direct identification of the intracellular cation bound to the indicators, internal calibration of [Ca]i and pHi from the spectra, and simultaneous measurements of two or more indicators in the same cell suspension. Perturbations of cellular functions by 5FBAPTA and quin 2 are very similar, but vary widely in different cell systems. The [Ca]i and pHi responses of normal and transformed cells to mitogens and growth factors in serum are compared with data from similar experiments using fluorescence indicators. The only major discrepancy in [Ca]i measurements using the two independent assays was observed in Ehrlich ascites tumour cells. These cells have a high intracellular Zn2+ content which substantially quenches the quin 2 fluorescence, but does not affect [Ca]i measurements by 5FBAPTA. The Zn2+ present in the cells is detected as a separate response in the 5FBAPTA spectrum. The time course of the Ca signal in 2H3 cells stimulated by antigen to release histamine by exocytosis has been defined using 5FBAPTA and quin 2. Extension of the 19F NMR technique to [Ca] i and pHi measurements in perfused organs is illustrated in rat heart and responses to pharmacological agents are demonstrated. Developments in prospect to improve sensitivity and to measure [Na]i with a new family of indicators are outlined.     

1H-cyclopenta[b]benzofuran lignans from Aglaia species inhibit cell proliferation and alter cell cycle distribution in human monocytic leukemia cell lines

Thirteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener all of them isolated from three Aglaia species (Aglaia duperreana, A. oligophylla and A. spectabilis) collected in Vietnam were studied for their antiproliferative effects using the human monocytic leukemia cell lines MONO-MAC-1 and MONO-MAC-6. Only rocaglamide type compounds showed significant inhibition of [3H-]thymidine incorporation and the most active compound didesmethylrocaglamide inhibited cell growth in a similar concentration range as the well-known anticancer drug vinblastine sulfate. Detailed structure-activity analysis indicated that the OH-group at C-8b which is a common structural feature of most naturally occurring rocaglamide compounds is essential for the described antiproliferative activity since replacement of this group by methylation led to a complete loss of the inhibitory activity for the resulting derivative. Rocaglamide derivatives rapidly inhibited DNA as well as protein biosynthesis of MONO-MAC-6 cells at concentrations well below those of actinomycin D or cycloheximide which were used as positive controls in the respective experiments. Didesmethylrocaglamide was furthermore able to induce growth arrest of MONO-MAC-1 cells in the G2/M and probably G0/G1-phase of the cell cycle with no morphological indication of cellular damage. Our data suggests that 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type act primarily by a cytostatic mechanism.     

Utilization of 1,3-Dioxolanes in the Synthesis of α-branched Alkyl and Aryl 9-[2-(Phosphonomethoxy)Ethyl]Purines and Study of the Influence of α-branched Substitution for Potential Biological Activity

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.     

Biosynthesis of domoic acid by the diatom Pseudo-nitzschia multiseries

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with alpha-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate-mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate.     

Platinum(II) and palladium(II) complexes with dithiocarbamates and amines: synthesis, characterization and cell assay

The [M(ESDT)Cl]n (M = Pd or Pt; ESDT = EtO2CCH2(CH3)NCS2, methylamino-acetic acid ethyl ester-dithiocarboxylate) species have been reacted with various amines (py, pyridine; PrNH2, n-propylamine; c-BuNH2, cyclobutylamine; en, ethylenediamine) in dichloromethane or chloroform with the aim to obtain mixed ligand complexes. The neutral complexes [M(ESDT)(L)Cl] (L = py, PrNH2 or c-BuNH2) and the ionic species ([M(ESDT)(L)2]Cl and [M(ESDT)(En)]Cl) have been isolated, and characterized by IR and proton NMR spectroscopies. The crystal structure of [Pd(ESDT)(PrNH2)Cl] has been determined by X-ray crystallography. The behaviour of the complexes in various solvents was described on the basis of the proton NMR spectra. The complexes and the dithiocarbamato intermediates have been tested for in vitro cytostatic activity against human leukemic HL-60 and HeLa cells.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.

Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.