Antiproliferative and pro-apoptotic activity of novel phenolic derivatives of resveratrol.

Gloriosaols A-C, isolated from Yucca gloriosa (Agavaceae), are novel phenolic compounds structurally related to resveratrol. In the present study, we show that gloriosaols possess antiproliferative and pro-apoptotic activity on tumor cells of different histogenetic origin and that their cell growth inhibition potential is higher than that of resveratrol. Despite the close similarities in their structure, gloriosaols A-C exhibited different antiproliferative potency, as the EC(50) ascending order is: gloriosaol C, gloriosaol A, gloriosaol B. Further mechanisms of gloriosaol C cytotoxicity were elucidated in detail in U937 cells, the most sensitive of the cell lines tested. The effect of gloriosaol C on cell growth turned out to be strongly dependent upon the concentration. Gloriosaol C doses lower than the EC(50) value (8 mu-icroM) blocked the cell cycle in G(0)/G(1), with a concurrent decrease in the number of cells in the G(2)/M phases of the cell cycle. At higher doses, this arrest overlaps with the occurrence of apoptosis and necrosis. In the 10-25 microM range of doses, gloriosaol C caused cell death mainly by apoptosis, as measured by hypodiploidia induction, phosphatidyl serine externalization and disruption of mitochondrial transmembrane potential. A switch in the mode of death from apoptosis to necrosis occurred at doses of gloriosaol C higher than 30 microM. Gloriosaol C was found to induce production of reactive species dose-dependently, but also to counteract their elevation in stressed cells. Thus, the different fate of cells, that is cell cycle arrest or cell death, in response to different doses of gloriosaol C might be related to the extent of induced oxidative stress.

Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation.

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.

H(2)O(2) activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation.

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.

Echistatin induces decrease of pp125FAK phosphorylation, disassembly of actin cytoskeleton and focal adhesions, and detachment of fibronectin-adherent melanoma cells.

B16-BL6 mouse melanoma cells cultured on fibronectin-coated dishes were detached by treatment with echistatin, an RGD-containing disintegrin. Echistatin was active at micromolar concentrations and was not cytotoxic. Its effect was dose-dependent and reversible. Sequential morphological changes leading to rounding up of the cells were detected by phase-contrast microscopy and by immunofluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytoplasmic actin filaments were observed well before cell detachment occurred. Echistatin treatment down-regulated the phosphorylation of pp125FAK in fibronectin-adherent cells in a dose- and time-dependent fashion. The reduction of pp125FAK phosphorylation preceded cell detachment and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125FAK phosphorylation and that echistatin acts by inhibiting protein tyrosine kinase activity rather than activating protein tyrosine phosphatases.

Site-specific atherosclerotic plaques in the carotid arteries of middle-aged women from southern Italy: associations with traditional risk factors and oxidation markers.

BACKGROUND AND PURPOSE: Studies on cardiovascular disease have preferentially involved men because of the lower frequency of the disease in preelderly women. The aim of this analysis was to examine, with the use of a standardized ultrasound protocol, a cohort of women to differentiate early atherosclerotic lesions in different carotid segments in relation to traditional (lipoprotein abnormalities, high blood pressure, cigarette smoking) and nontraditional (oxidation markers) cardiovascular risk factors. METHODS: More than 5000 clinically healthy, middle-aged women (n=5062; age range, 30 to 69 years) living in the area of Naples in southern Italy participated in the Progetto Atena, a population-based study on the etiology of cardiovascular disease and cancer in the female population. A subsample of 310 participants underwent high-resolution B-mode ultrasound to assess intima-media thickness of common carotid artery and carotid bifurcation. RESULTS: Early atherosclerotic plaques (intima-media thickness >1.2 mm) were detected within the common carotid arteries in 37 women, in the carotid bifurcations in 77 women, and in both sites in 91 women. After age adjustment, common carotid plaques were found to be associated with higher systolic blood pressure (143 versus 138 mm Hg; P<0.05) and higher body mass index (29 versus 27 kg/m(2); P<0.01), while lesions at the carotid bifurcations were associated with higher LDL cholesterol (4.3 versus 3.8 mmol/L; P<0.01) and with smoking habit. Multivariate odds ratios for the presence of common carotid plaques were related to antibodies against oxidized LDL (odds ratio, 2.72; 95% CI, 1.46 to 5.07), and those for plaques at the bifurcation were related to lipid peroxides (odds ratio, 1.90; 95% CI, 1.04 to 3.47), and both relationships were independent of age, LDL cholesterol concentrations, body mass index, smoking habit, and systolic blood pressure. CONCLUSIONS: In a cohort of clinically healthy, middle-aged women, we found a site-specific association of traditional risk factors and oxidation markers with early atherosclerotic lesions in arterial segments differing in geometry, shear stress, extracellular matrix composition, and cell type populations.

Metal-ion catalyzed oxidation affects fibrinogen activity on platelet aggregation and adhesion.

Exposure of fibrinogen to the Fe3+/ascorbate oxidative system resulted in structural modifications and altered functionality of the glycoprotein. The overnight treatment of fibrinogen by oxidants caused a 20-fold increase of carbonyl content with respect to the native protein. Formation of dityrosines as well as loss of tryptophan following fibrinogen oxidation were observed. The occurrence of conformational changes of the fibrinogen molecule as a consequence of the oxidative treatment was also established. Oxidized fibrinogen showed a distinct capability from the native molecule to mediate platelet aggregation and adhesion. The percentage of ADP-induced platelet aggregation decreased as a function of fibrinogen oxidative damage. Further, both unstimulated platelets and ADP-activated platelets showed a reduced ability to adhere to oxidized fibrinogen than to the native protein. These results suggest that oxidative treatment alters fibrinogen domains involved in the recognition and the binding of this molecule by the platelet receptor GP IIb/IIIa.

Echistatin inhibits pp72syk and pp125FAK phosphorylation in fibrinogen-adherent platelets.

The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72syk and pp125FAK. The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72syk results strongly phosphorylated already after 15 min, whereas pp125FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72syk and pp125FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein IIb-IIIa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72syk and pp125FAK inactivation.

The mycotoxin fumonisin B1 inhibits integrin-mediated cell-matrix adhesion.

Fumonisin B1 (FB1), a mycotoxin produced by the corn fungus Fusarium moniliforme, causes a variety of animal diseases and is a suspected human carcinogen. The FB1 molecule bears remarkable structural resemblance to the long-chain sphingoid base backbones of sphingolipids. The toxicity and carcinogenicity of FB1 has been ascribed to its ability to inhibit ceramide synthase, a key enzyme in the metabolism of complex sphingolipids. In this study we have investigated whether the exposure of B16-BL6 mouse melanoma cells to FB1 affects cell growth and integrin-mediated cell matrix adhesion. Cell treatment with the highest tested dose (75 microM) of FB1 for 72 h induced an about 20% inhibition of cell growth. FB1 strongly affected B16-BL6 cell adhesion to immobilized fibronectin, by causing a dose-dependent inhibition of cell attachment to this substrate. FB1 also inhibited in a dose-dependent manner the adhesion of B16-BL6 cells to the immobilized anti-fibronectin receptor antibody, whereas it affected only to a low extent cell attachment to concanavalin A. Our results demonstrate that FB1 treatment alters integrin adhesive activity, thus affecting all cellular integrin-dependent functions.

Erythrocyte enzymes catalyze 1-nitropyrene and 3-nitrofluoranthene nitroreduction.

Nitroarenes are environmental contaminants produced during incomplete combustion processes. Nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study, we show that human red cells may also possess the metabolic competence to reduce 1-nitropyrene (NP) and 3-nitrofluoranthene (NF), the nitroarenes chosen as model compounds, to their corresponding amino derivatives, 1-aminopyrene (AP) and 3-aminofluoranthene (AF). The requirement of the cofactor couple NADH/FMN suggests that erythrocyte nitroreductase activity occurs via one electron transfer. The presence of oxygen strongly inhibited the haemolysate-catalyzed nitroarene reduction, whether measured as amine formation or nitroarene disappearance. Intermediate reactive species, that bind covalently to haemoglobin and/or other erythrocyte proteins, are formed during nitroreduction catalyzed by human haemolysate. In fact, the reduced metabolites AP and AF were released after mild acid hydrolysis of red cell proteins exposed to NP and NF, thus suggesting that sulphinamide adducts have been formed.

Arylation of sulfhydryl groups in vitro by the naturally occurring sesquiterpenoid benzoquinone avarone.

Avarone (AQ) is a naturally occurring sesquiterpenoid benzoquinone possessing antileukaemic activity. Its reactivity towards glutathione (GSH) and protein sulfhydryl (SH) groups was investigated. The stoichiometry of AQ reaction with GSH at [GSH]/[AQ] ratios lower than unity proved to be 1:2 (thiol:quinone), consistent with the formation of the corresponding hydroquinone (avarol) as well as a quinone-thioether in the reaction. Conversely, when the [GSH]/[AG] ratio was higher than unity, a hydroquinone-thioether was the only reaction product. AQ/protein interaction was also investigated by using bovine serum albumin (BSA) as model compound. As observed with GSH, arylation rather than oxidation of SH groups appeared to be the mechanism responsible for the AQ-induced depletion of protein SH groups. However, AQ proved to be less effective in depleting BSA sulfhydryls than that of GSH. AQ disappearance after BSA addition was greater than expected on the basis of the total SH groups depleted, if a stoichiometric ratio 1:2 (thiol:quinone) was assumed. It also occurred in the presence of BSA with blocked SH groups, thus suggesting that AQ may react with other nucleophilic protein residues, such as amino or imino groups. When HepG2 cells were exposed to AQ, depletion of both protein SH groups and GSH occurred. However, in contrast to the above, AQ proved to be more effective, probably because of its lipophilic nature, in depleting protein SH groups than GSH. Also, in intact cells AQ appeared to arylate both SH and other nucleophilic groups in proteins. This mechanism may play a major role in AQ-induced cytotoxicity.

Effect of avarol and avarone on in vitro-induced microsomal lipid peroxidation.

Lipid peroxidation was employed as an experimental model to study the antioxidant properties of avarol, a sesquiterpenoid hydroquinone and of its quinone, avarone. In the NADPH- or ascorbate-linked lipid peroxidation, avarol and avarone were shown to be more effective as inhibitors than in the t-BuOOH-dependent peroxidative process. However, in all three systems employed avarol was a more powerful inhibitor than avarone. The chemical structure of avarol, having an easily donatable hydrogen atom and its kinetics of inhibition suggested that the hydroquinone acted mainly as a radical scavenger. Conversely avarone appeared to interfere mainly with the initiation phase of lipid peroxidation. However, avarol and the semiquinone intermediate may contribute to the inhibitory action of the quinone. In fact avarone reduction to avarol has been shown to occur in the presence of reducing agents such as ascorbate or Fe(II) and to be catalyzed by NADPH-supplemented microsomes.

Characterization of the induction of rat hepatic microsomal drug-metabolizing enzymes by 1-nitropyrene metabolites, 1-aminopyrene and N-acetylaminopyrene.

The effect of 1-aminopyrene (1-AP) and N-acetylaminopyrene (1-NAAP) on rat hepatic microsomal monooxygenase system was investigated. Both drugs increased the total content of cytochrome P-450 (cyt. P-450). The substrate specificity and the electrophoretic pattern of 1-AP and 1-NAAP induced cytochrome(s) were compared with those of the major forms of cyt. P-450 induced by 3-methylcholanthrene (3-MC) and phenobarbital (PB). The results suggest that the form of cyt. P-450 induced by 1-AP and 1-NAAP resembles that one induced by 3-MC. Furthermore the abilities of liver microsomes from control or differently induced rats to ring hydroxylate and to activate 1-nitropyrene (1-NP) metabolites to species mutagenic for bacteria were compared. It was observed that: (1) 1-NAAP is a good substrate for microsome-mediated ring hydroxylation, whereas 1-AP is oxygenated only at a low extent; (2) 3-MC, 1-AP and 1-NAAP-stimulated microsomes are more active than control or PB-ones to ring hydroxylate 1-NAAP. As phenolic derivatives of 1-NAAP show high mutagenic activity, these results indicate that 1-AP and 1-NAAP induce toxification pathways of 1-NP in similar way, even if in less extent, as compared to 3-MC.

Inhibition of cyclophosphamide mutagenicity by beta-carotene.

Cyclophosphamide (CP) metabolites, rather than the parent compound, show mutagenic activity towards Salmonella typhimurium TA 1535 tester strain when S9 fraction from phenobarbital (PB)-induced rat liver is used as in vitro metabolizing system. On the other hand, inhibition of CP in vitro mutagenicity was observed by adding increasing amounts of beta-carotene (beta-C) to the system. A typical dose-dependent mutagenic response was observed by assaying 24 h urine samples of PB-induced rats injected i.p. with different amounts of CP. Addition of beta-C to urines of CP-treated rats failed to inhibit their mutagenicity. Conversely, a marked decrease in urine mutagenicity was observed when rats were simultaneously treated with the two drugs. These data show that beta-carotene partially inhibits, in vitro and in vivo, CP metabolism via hepatic mixed-function oxidase enzymes to mutagenic species.

Effect of enzyme inducers on metabolism of 1-nitropyrene in human hepatoma cell line HepG2.

We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.

Evaluation of concentration procedures of mutagenic metabolites from urine of diesel particulate-treated rats.

Several concentration procedures of mutagenic metabolites contained in the urine of diesel particulate-treated rats were compared. Mutagenicity was monitored by the Salmonella/microsome assay. The procedures tested were: lyophilization; filtration on XAD-2, XAD-7 or Sephadex LH-20 matrices; ultrafiltration; and extraction with organic solvents. Urine extraction with dichloromethane (DCM) gave almost quantitative recovery of activity while leaving salts and other polar compounds in the aqueous phase, and is the method recommended.

Role of rat liver inducible enzymes in in vitro metabolic transformation of 1-nitropyrene.

Liver enzymes (S9) of rats induced with various drugs differently affected the mutagenic response of 1-nitropyrene (1-NP) in vitro. The role of inducible enzymes in 1-NP conversion was evaluated by high-performance liquid chromatography (HPLC) analyses of 1-NP metabolites formed during incubation in vitro of the chemical with each S9 fraction. Methylcholanthrene (MC)-induced S9 preferentially results in 1-NP hydroxylation in position 6 or 8. Metabolic interconversions among 1-NP metabolites could be inferred from the HPLC patterns.

Effect of avarol, avarone and nine of their natural and synthetic derivatives on microsomal drug-metabolizing enzymes.

Avarol, a sesquiterpenoid hydroquinone, its quinone avarone, both main secondary metabolites from the marine sponge Dysidea avara and nine of their natural and synthetic derivatives were tested for ability to interact selectively with rat liver microsomal phenobarbital (PB)- or 3-methylcholanthrene (3-MC)-induced cytochrome (cyt.) P-450 isoenzymatic forms. Ethoxy- and pentoxyresorufin, aminopyrine and ethoxycoumarin were the specific substrates used for assaying cyt. P-450-dependent mono-oxygenase activities. All compounds were more effective in inhibiting the enzymatic activities measured in microsomes from PB-induced rat liver than those measured in microsomes from 3-MC-treated rats. Avarol and avarone, which were the most active inhibitors, act as mixed-type inhibitors of pentoxyresorufin-O-dealkylase activity. Mono- and diacetyl-derivatives of avarol, being deacetylated by rat liver microsomes, were almost as effective as the parent compound. Conversely, avarone derivatives, where one or two hydrogen atoms of the quinone ring have been substituted, were less effective inhibitors than the parent compound. The selective inhibition of PB-inducible cyt.P-450IIB by avarol and avarone was confirmed by their ability to inhibit the mutagenic activation of cyclophosphamide, as opposed to that of benzo[a]pyrene, which is activated mainly by the 3-MC-inducible cyt.P-450IA.

In vitro activation of isophosphamide and trophosphamide to metabolites mutagenic for bacteria.

The ability of S9 liver fractions from uninduced rats to activate isophosphamide (IP) and trophosphamide (TP) to metabolites mutagenic for bacteria was compared to that of S9 fractions prepared from rats pretreated in vivo with three inducers of hepatic monooxygenase. Pretreatment of rats with phenobarbital (PB) and Aroclor 1254 increased IP and TP mutagenic activation by S9 fractions as compared to control and 3-methylcholanthrene (3-MC)-induced rat liver S9. Furthermore, the effect of mixed-function oxidase inhibitors, such as alpha-naphthoflavone, metyrapone and SKF 525-A on S9-mediated mutagenic activation of IP and TP was investigated. The data obtained suggest the involvement of a PB-inducible form of cytochrome P-450 in the activation of IP and TP to mutagenic species.

Effect of liver enzyme inducers on metabolite excretion in rats treated with 1-nitropyrene.

Non-induced and phenobarbital (PB) or methylcholanthrene (MC) pretreated rats were injected with 1-nitropyrene (1-NP). Mutagenic activity of urine and feces samples were compared by the Salmonella/microsome assay. The highest, indirect-acting mutagenicity was associated with urines from MC-induced rats; HPLC analysis of organic extracts of urine samples showed that the differences in mutagenic response can be ascribed to different amounts of hydroxy derivatives of N-acetylaminopyrene excreted. Monohydroxy derivatives of 1-NP, being detected in the HPLC profiles of urine from PB-induced rats only, could be responsible of the higher direct-acting mutagenic activity of these samples as compared to urine from non-induced or MC-induced rats. The excretion rate of aminopyrene, the main metabolite of 1-NP identified in rat feces samples, was not affected by inducer pretreatment.

Biological availability of mutagenic compounds adsorbed onto diesel exhaust particulate.

Diesel particles were collected from the exhaust of a VW Golf diesel car by electrostatic precipitation. The particulate and its DCM extract were highly mutagenic in the Salmonella/microsome test in the presence and absence of metabolic activation; the highest response was observed with TA98 and TA1538 tester strains. The biological availability of particulate-associated mutagenic compounds was demonstrated by administering powder to rats and assaying, in vitro, the urine excreted within 24 h after treatment. The highest activity was obtained with TA98 in the presence of metabolic activation. Typical dose-effect responses were evident in urine of animals treated by all the administration routes tested (i.p., s.c. and per os), both in the presence and absence of a suspending vehicle. Concentration of mutagenic compounds present in urine of treated animals could be achieved by chromatography on Amberlite XAD-2 and XAD-7 resins. This study provides direct evidence for bioavailability to animal tissues of mutagens adsorbed onto diesel particulate, although part of the activity might be ascribed to nitroaromatic compounds formed during the collection of the powder. The present study is part of a more comprehensive work on diesel exhaust particulate, and results have to be considered in this light before any final conclusion can be drawn.