Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug-DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed 'classical' pattern observed for intercalation, as proved by the DNA topoisomerase I-unwinding experiments, with relatively weak binding affinity (K(i)=1.2 x 10(5)M(-1)), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 10(5)M(-1) and binding site size of 2-3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug-DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.     

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.     

Induction of interleukin 8 release from the HMC-1 mast cell line: synergy between stem cell factor and activators of the adenosine A(2b) receptor

The HMC-1 mast cell line has both adenosine A(3) and A(2b) receptors on its surface, but only agonists of the A(2b) receptor are effective at releasing interleukin 8. Object of this study was to look for co-factors for adenosine A(2b) receptor activation. There was a powerful and statistically significant synergy for release of IL-8, both at the mRNA level (measured after 4 hr) and protein level (measured after 24 hr), between adenosine A(2b) receptor agonists and stem cell factor (SCF). Suitable concentrations for showing synergy were 100 ng/mL SCF and 3 microM 5'-N-ethylcarboxamidoadenosine (NECA). At these concentrations, the IL-8 released into the culture medium after SCF and NECA together was typically 3-5-fold greater in amount than the sum of the amounts of IL-8 released after exposure to the same concentrations of NECA and SCF separately. Since mast cells may be exposed to both adenosine and stem cell factor in the diseased lung, the synergy observed in this model system may have implications for asthma.     

Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design

Flavanols--a class of plant polyphenols abundant in tea leaves and grape seeds and skins--have been found to inhibit some matrix-proteases instrumental in inflammation and cancer invasion, such as leukocyte elastase (LE) and gelatinases. In order to establish the relationship between chemical structure and activity, 27 different flavonoids (antocyanidins, dihydrochalcones, dihydroflavonols, flavanolignans, flavanols, flavones, flavonols and isoflavones) and other compounds with anti-oxidant properties were evaluated for their potential in blocking LE and gelatinase activities. LE activity was measured using a chromogenic substrate: from comparison of the different levels of inhibition, it was deduced that a crucial role in inhibition might be played by a galloyl moiety or hydroxyl group at C3, three hydroxyl groups at B ring, one hydroxyl group at C4', and a 2,3-double bond. Gelatinase activity was measured using the gelatin-zymography assay, and its inhibition showed that three hydroxyl groups at the A or B ring, or, for non-planar molecules, a galloyl moiety at C3 could be determinant. This comparative study is proposed as a basis for designing new molecules with enhanced anti-proteolytic activities, and no or reduced side-effects, for use in hindering inflammation, cancer invasion and angiogenesis.     

Characterization of the mechanism underlying stonustoxin-mediated relaxant response in the rat aorta in vitro

Stonustoxin (SNTX) is a lethal factor isolated from the venom of the stonefish Synanceja horrida. Although SNTX exhibits a multitude of biological activities, the primary cause of death upon administration of the toxin is attributed to marked hypotension. We investigated the possible mechanisms underlying the vascular hyporeactivity of this novel toxin. Cumulative doses of SNTX (5-320 ng/mL) induced concentration-dependent relaxation in phenylephrine (PE)--precontracted rat aortic rings with intact endothelium. Endothelium removal abolished the relaxation induced by SNTX. Tetraethylammonium (TEA), an inhibitor of K(+) channels, partially inhibited SNTX-induced relaxation. Similarly, SNTX-induced relaxation was partially attenuated by the SP receptor antagonist (NATB), whereas the inducible iNOS inhibitor, AMT-HCl, completely abolished the relaxation caused by SNTX. From the results obtained, it can be postulated that a component of SNTX-mediated vasorelaxation is via binding of either SNTX or SP to the SP receptors that are located on the endothelial cells. Occupation of these SP receptors causes subsequent production of NO and activation of K(+) channels, thus leading to vasorelaxation of the rat aortic rings.     

Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1

The insulinotropic agent, exendin-4, is a long-acting analogue of glucagon-like peptide-1 (GLP-1) which improves glucose tolerance in humans and animals with diabetes, but the underlying mechanisms and the effects of exendin-4 on peripheral (muscle/fat) insulin action are unclear. Previous in vivo and clinical studies have been difficult to interpret because of complex, simultaneous changes in insulin and glucagon levels and possible effects on hepatic metabolism. Thus, the comparative effects of exendin-4 and GLP-1 on insulin-stimulated 2-[3H]deoxyglucose (2-DOG) uptake were measured in fully differentiated L6 myotubes and 3T3-adipocytes, including co-incubation with inhibitors of the PI-3-kinase (wortmannin) and mitogen-activated protein (MAP) kinase (PD098059) pathways. In L6 myotubes, there was a concentration-dependent and PI-3-kinase-dependent increase in insulin-stimulated 2-DOG uptake with exendin-4 and GLP-1, e.g. for exendin-4 the C(I-200) value (concentration of insulin required to increase 2-DOG uptake 2-fold) decreased from 1.3 +/- 1.4 x 10(-7)M (insulin alone, n=16) to 5.9 +/- 1.3 x 10(-8)M (insulin+exendin-4 0.1nM, n=18, P<0.03). A similar insulin-sensitizing effect was observed with exendin-4 in 3T3-adipocytes, but GLP-1 had no effect on adipocyte insulin sensitivity. In conclusion, this is the first direct evidence showing that exendin-4 increases insulin-stimulated glucose uptake in muscle and fat derived cells via a pathway that involves PI-3-kinase activation. Furthermore, the contrasting responses of exendin and GLP-1 in 3T3-adipocytes suggest that the peripheral insulin-sensitizing effect of exendin-4 (in contrast to the insulinotropic effect) does not involve the GLP-1 receptor pathway.     

L-arginine-induced relaxation of the rat isolated penile bulb.

The effects of L-arginine, the precursor in the synthesis of nitric oxide (NO), were investigated in the penile bulb isolated from saline (control) or lipopolysaccharide (20 mg/kg, i.p.)-treated rats. Four consecutive concentration-response curves for L-arginine were made at hourly intervals with the penile bulb. L-arginine (10(7)-10(-3) M) elicited a concentration- and time-dependent relaxation response in the control group. The NO synthase (NOS) inhibitors, N(G)-methyl-L-arginine (L-NMMA) and aminoguanidine, guanylate cyclase inhibitor, 1-H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) and protein synthesis inhibitor, cycloheximide, inhibited L-arginine-induced relaxation. In the lipopolysaccharide-group, L-arginine produced a pronounced non-time-dependent relaxation at the first concentration-response curve, which was not different from the fourth response of the control group. This response was also inhibited by aminoguanidine. These results show that L-arginine induced NO-mediated relaxation and suggest the presence of a biochemical pathway converting L-arginine to NO, which is probably an inducible type in the penile bulb.     

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.     

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells.

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, L-methamphetamine in comparison to rasagiline and its major metabolite, 1-R-aminoindan on oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen-glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen-glucose deprivation was reduced by 45-55%. L-methamphetamine, a major selegiline metabolite, but not 1-R-aminoindan, the major rasagiline metabolite, enhanced oxygen-glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen-glucose deprivation, to a combination of either selegiline and L-methamphetamine or rasagiline and 1-R-aminoindan, indicated that L-methamphetamine, but not 1-R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.     

Influence of nitric oxide on morphine-induced conditioned place preference in the rat central amygdala

Effects of intra-central amygdala injections of L-arginine, a nitric oxide (NO) precursor, and N(G)-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, on morphine-induced conditioned place preference in rats were investigated by using an unbiased 3-day schedule of place conditioning design. Animals receiving once daily injections of morphine (0.5-7.5 mg/kg, subcutaneously, s.c.) or saline (1.0 ml/kg, s.c.) showed a significant place preference in a dose-dependent manner. The maximum response was observed with 5.0 mg/kg of the opioid. Co-administration of morphine (5.0 mg/kg) with L-arginine (0.3, 1.0 and 3.0 microg/rat), but not with L-NAME (0.3, 1.0 and 3.0 microg/rat), during the acquisition of morphine-induced conditioned place preference increased morphine-induced conditioned place preference. The response to L-arginine was blocked by L-NAME preadministration. L-arginine and L-NAME by themselves did not induce conditioned place preference. When L-arginine or L-NAME at 0.3-3.0 microg/rat was administered 1 min before conditioned place preference testing, L-arginine but not L-NAME caused an increase in the expression of morphine-induced conditioned place preference, the effect that was blocked by L-NAME preadministration. A dose of L-arginine (0.3 microg/rat), but not L-NAME, during expression of morphine-induced conditioned place preference produced an increase in locomotion compared with that in the control group. It may be concluded that an increase in the NO levels in the central amygdala may have an effect on the acquisition and expression of morphine-induced conditioned place preference.