Inhibitory effects of 2-amino-3H-phenoxazin-3-one on the melanogenesis of murine B16 melanoma cell line.

Hyperpigmentations are a serious concern addressed by both the medical community and the cosmetic industry through the development of agents that block melanin biosynthesis. In this study, we found that 2-amino-3H-phenoxazin-3-one (APO), isolated from extracts of the edible mushroom Agaricus bisporus Imbach, exhibited potent inhibitory effects on melanogenesis in B16 cells, a murine melanoma cell line. APO inhibited melanin biosynthesis at 1,000 times lower concentrations (IC(50)=1.31+/-0.08 microM) than kojic acid (IC(50)=1.31+/-0.13 mM), without causing cellular toxicity. APO did not directly inhibit the enzyme activity of tyrosinase, the rate-limiting melanogenic enzyme. Further study showed that APO inhibited the protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF), a melanogenic transcription factor that regulates the expression of tyrosinase. These results suggest that APO is a promising depigmenting agent with both therapeutic and cosmetic value in preventing melanogenesis.

Creation of interferon-alpha8 mutants with amino acid substitutions against interferon-alpha receptor-2 binding sites using phage display system and evaluation of their biologic properties.

In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.

Anti-inflammatory and immunomodulatory properties of 2-amino-3H-phenoxazin-3-one.

Accumulating evidence suggests that nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are involved in the pathogenesis of various chronic inflammatory diseases and cancer. During the course of a screening program to identify natural anti-inflammatory substances, we isolated the compound 2-amino-3H-phenoxazin-3-one (APO) from an extract of the edible brown mushroom Agaricus bisporus IMBACH. APO inhibited NO production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharide (LPS) and interferon (IFN)-gamma (LPS/IFN-gamma) at low concentrations (IC(50)=1.5 microM) through reduced inducible NO synthase protein expression. PGE(2) production by LPS/IFN-gamma-stimulated macrophages was inhibited by APO at much lower concentrations (IC(50)=0.27 microM) than those required for the inhibition of NO production. Mechanistic analysis showed that APO inhibited both cyclooxygenase (COX)-1 and COX-2 enzyme activities with almost equal selectivity. Secretion of NO and the pro-inflammatory cytokine IL-6 by IFN-gamma-activated RAW264.7 cells, a murine macrophage-like cell line, was also dose-dependently reduced by APO. Furthermore, APO increased the secretion of the anti-inflammatory cytokine IL-4 by antigen-stimulated T cells and promoted the polarization of CD4(+) Th cells toward the anti-inflammatory Th2 phenotype at equimolar concentrations that inhibited NO production. Our results suggested that APO induced polarization toward the Th2 subset, at least in part through the down-regulation of IL-12 production. Thus, APO appears to have potent anti-inflammatory and immunoregulatory properties that may provide a promising therapeutic strategy for the treatment of T cell-mediated inflammatory autoimmune diseases as well as for bacteria-induced chronic-inflammatory diseases.

Cotreatment with interferon-alpha and -gamma reduces liver fibrosis in a rat model.

Background/Aims: Interferon-alpha is used widely to treat viral hepatitis. Interferon-gamma modulates a system attacking infected cells and also has an anti-fibrotic effect. A treatment with interferon-alpha and -gamma has undergone trials in eliminating hepatitis C virus. We investigated effects of cotreatment in a liver fibrosis model to explore anti-fibrotic effects. Methods: Rats were assigned to groups including normal controls (NC), CCl(4) controls, rat interferon-alpha treatment, rat interferon-gamma treatment, and cotreatment. All groups except normal controls received CCl(4) orally for 8 weeks. At the beginning of the third week of exposure, 6 weeks of treatment were initiated according to interferon group. Digitally analyzed immunohistochemistry, biochemical assays, and Northern analysis were performed. Results: Pixels (x10(5)) per field containing immunoreactive type III collagen (fibrotic density) in CCl(4) controls, interferon-alpha, interferon-gamma, and cotreatment groups respectively were [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text]. Liver hydroxyproline content correlated with fibrotic density, and was significantly low in the cotreatment group. Plasma hyaluronate and transaminase were significantly low in cotreatment and interferon-alpha groups. Northern blotting showed lowest mRNA expression for type I collagen, desmin, transforming growth factor (TGF)-beta1, and matrix metalloproteinase-2 mRNA in the cotreatment group; tissue inhibitor of metalloproteinase-1 and -2 mRNAs were significantly low in the interferon-gamma group. Conclusions: Cotreatment can suppress collagen and transforming growth factor-beta1 and has an overall anti-fibrotic effect without exacerbating inflammation.

Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice.

The equilibrium between the production and degradation of collagen is rigorously controlled by a number of growth factors and cytokines. Interferon alfa (IFN-alpha) is now widely used for the treatment of chronic hepatitis C, which can improve serum levels of fibrotic markers and the degree of hepatic fibrosis, not only in patients who responded to therapy but also in those in whom it is ineffective. These findings may suggest that IFN-alpha possesses direct antifibrotic effects in addition to its antiviral activity. However, in contrast to IFN-gamma, which has been shown to suppress collagen gene transcription, little is known about the mechanisms responsible for the antifibrotic effects of IFN-alpha. Here, we report that IFN-alpha, when administered into transgenic mice harboring the alpha2(I) collagen gene (COL1A2) promoter sequence, significantly repressed promoter activation and prevented the progression of hepatic fibrosis induced by carbon tetrachloride injection. Transient transfection assays indicated that IFN-alpha decreased the steady-state levels of COL1A2 messenger RNA (mRNA) and inhibited basal and TGF-beta/Smad3-stimulated COL1A2 transcription in activated hepatic stellate cells (HSC). These inhibitory effects of IFN-alpha on COL1A2 transcription were exerted through the interaction between phosphorylated Stat1 and p300. Blocking of the IFN-alpha signal by overexpressing the intracellular domain-deleted IFN receptor increased basal COL1A2 transcription and abolished the inhibitory effects of IFN-alpha. In conclusion, our results indicate that IFN-alpha antagonizes the TGF-beta/Smad3-stimulated COL1A2 transcription in vitro and suppresses COL1A2 promoter activation in vivo, providing a molecular basis for antifibrotic effects of IFN-alpha.

Antitumor activity of interleukin-18 against the murine T-cell leukemia/lymphoma EL-4 in syngeneic mice.

Interleukin (IL)-18 induces interferon (IFN)-gamma production by T cells and natural killer (NK) cells, and augments NK cell activity in mouse spleen cell cultures. It has recently been demonstrated that in vivo administration of IL-18 to mice results in considerable antitumor effects against syngeneic Meth A sarcoma. In this study, the antitumor effects of IL-18 against murine T-cell leukemia (EL-4) were evaluated. EL-4 proliferation was resistant in vitro to IL-18 and IFN-gamma. When 4 x 10(6) EL-4 cells were transplanted intravenously, the antitumor effects of IL-18 were not pronounced, and only a slight prolongation of the mean survival times was observed. The antitumor effects of IFN-gamma were even less apparent than those of IL-18. However, when mice were transplanted intravenously with 5 x 10(5) EL-4 cells, the extent of experimental visceral dissemination of EL-4 was markedly reduced in mice treated subcutaneously with IL-18, resulting in an increase in survival time with some mice even cured. Although IL-18 was highly effective at inhibiting the development of EL-4 lymphoma dissemination in C57BL/6 mice, it could not inhibit the development of dissemination in mutant C57BL/6 beige (bg/bg) mice lacking NK cell activity. The efficacy of IL-18 was also significantly reduced in nude mice lacking T cells. These results suggest that antitumor efficacy of IL-18 is mediated primarily by NK cells, but that T cells are also required for the complete antitumor efficacy of IL-18.