Usnea Acid as Multidrug Resistance (MDR) Reversing Agent against Human Chronic Myelogenous Leukemia K562/ADR Cells via an ROS Dependent Apoptosis.

PURPOSE: Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigated Usnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis. METHODS: CCK8 assay was used to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution, cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot. Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. RESULTS: In vitro study showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 µM) was decreased from 89.8% ± 4.7 to 32% ± 8.9 by combined with UA (4 µM). Adr-induced apoptosis and G1/G0 phase cell cycle arrest were remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. CONCLUSION: These data provide compelling evidence that UA is a promising agent against MDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.

Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV-796.

HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.

Lipid peroxidation induced by carbon tetrachloride and its inhibition by antioxidant as evaluated by an oxidative stress marker, HODE.

We have recently proposed total hydroxyoctadecadienoic acid (HODE) as a biomarker for oxidative stress in vivo. The biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert the oxidation products of linoleate to HODE. In the present study, this method was applied to measure the oxidative damage induced by the administration of carbon tetrachloride to mice and also to evaluate the capacity of antioxidant to inhibit the above damage. alpha-Tocopherol transfer protein knock out (alpha-TTP-/-) mice were used to evaluate antioxidant effect in the absence of alpha-tocopherol. The intraperitoneal administration of carbon tetrachloride to mice induced the increase in HODE in liver and plasma, which was followed by an increase in plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). F2-isoprostanes, another prevailing biomarker, were also increased similarly, but their concentration was approximately two to three orders of magnitude smaller than that of HODE. The lipophilic antioxidants such as gamma-tocopherol, gamma-tocotrienol and 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) were effective in suppressing the formation of HODE.

Potential serotonergic and noradrenergic involvement in the discriminative stimulus effects of the selective imidazoline I2-site ligand 2-BFI.

The functional significance of imidazoline I2 binding sites is unknown but microdialysis studies have indicated that the administration of I2-site ligands leads to an increase in extracellular levels of monoamines. The specific I2-site ligand 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) generates a cue in drug discrimination, thereby indicating functional consequences of I2-site ligand binding. In the present work, we explored the ability of selective noradrenergic and serotonergic ligands to substitute for 2-BFI. Hooded Lister rats were trained in two-lever operant chambers with condensed milk reward to distinguish 2-BFI (7 mg/kg) from saline vehicle, by pressing the correct lever to a predetermined success criterion. Training sessions were then interspersed with sessions in which animals were administered test substances and the proportion of lever presses on the 2-BFI-associated lever (substitution) recorded. Several agents exhibited significant partial substitution for 2-BFI: The monoamine-releasing agents D-amphetamine and fenfluramine dose-dependently substituted for 2-BFI, while norepinephrine (desipramine, reboxetine) and serotonin (clomipramine, citalopram) reuptake inhibitors substituted at one or more doses. Further investigation using specific receptor agonists and antagonists indicated a possible role for activation of alpha1-adrenoceptors but failed to support involvement of alpha2-adenoceptor, beta-adrenoceptor or 5-HT1A receptor activation. These results support the concept that the 2-BFI cue may contain both noradrenergic and serotonergic components.