JangJa Hong

Effects of hyperin, isoquercitrin and quercetin on lipopolysaccharide-induced nitrite production in rat peritoneal macrophages.

The extract of the root of Acanthopanax chiisanensis Nakai is used for the treatment of inflammation. To analyse the action mechanism of this extract, the effect of hyperin (quercetin‐3‐O‐β‐d‐galactose) isolated from the ethyl acetate fraction of the root of A. chiisanensis on nitrite production and induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS, 1 µg/mL)‐stimulated rat peritoneal macrophages were examined. The effect of the structurally related compounds, isoquercitrin (quercetin‐3‐O‐β‐d‐glucose) and quercetin (an aglycone of the two compounds) isolated from the extract of the leaves of Vaccinium koreanum Nakai was also examined to compare the effect. It was shown that hyperin inhibited the LPS‐induced iNOS expression and nitrite production. Of the three compounds, quercetin showed the most potent inhibitory activity. The phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK and c‐Jun N‐terminal kinase (JNK) were also inhibited by these compounds. These findings suggested that hyperin in the extract of the root of A. chiisanensis inhibits nitric oxide (NO) production through inhibition of the expression of iNOS by attenuation of p44/p42 MAPK, p38 MAPK and JNK, and thus participates in the antiinflammatory activity of the extract. Copyright

Nitric Oxide Production by the Vacuolar-Type (H+)-ATPase Inhibitors Bafilomycin A1 and Concanamycin A and Its Possible Role in Apoptosis in RAW 264.7 Cells

In the mouse leukemic monocyte cell line RAW 264.7, the vacuolar-type (H+)-ATPase (V-ATPase) inhibitors bafilomycin A1 and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric-oxide synthase mRNA and its protein and decreased cell growth and survival as determined by 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bafilomycin A1 and concanamycin A activated nuclear factor (NF)-κB and activator protein-1 and decreased the level of IκB-α and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-κB inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na+,K+-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor S-nitroso-N-acetyl-dl-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor NG-monomethyl-l-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitor-induced apoptosis in RAW 264.7 cells.

Apicidin, a histone deacetylase inhibitor, induces differentiation of HL-60 cells

The fungal metabolite apicidin (cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)) inhibited the growth of HL-60 cells in a concentration-dependent manner (100-1000 nM). At higher concentrations (>300 nM), cell death was induced. At 100 nM, it induced hyperacetylation of histone H4 time-dependently, while trichostatin A induced transient hyperacetylation. Apicidin (10-100 nM) increased the cells having nitroblue tetrazolium-reducing activity and expressing CD11b but not CD14 and CD15. The expression of CD11b by apicidin was long lasting, while that by trichostatin A was transient. In K562 cells, apicidin at 10-100 nM did not inhibit cell growth nor express CD11b, CD14 and CD15. Our findings indicate that apicidin inhibits proliferation and induces the early stage of differentiation of HL-60 cells.

Possible mechanism of action of the histone deacetylase inhibitors for the induction of differentiation of HL‐60 clone 15 cells into eosinophils

We have examined the effect of the histone deacetylase inhibitors apicidin, trichostatin A (TSA) and n‐butyrate on the histone acetylation and the differentiation of human eosinophilic leukemia HL‐60 clone 15 cells into eosinophils. Viability of the cells incubated with apicidin (100 nM), TSA (30 nM) or n‐butyrate (500 μM) did not change significantly, but higher concentrations of apicidin (300 nM) or TSA (100 nM) decreased the viability when examined at day 1. Apicidin (100 nM) as well as n‐butyrate (500 μM) induced continuous acetylations of histone H4 and lysine14 residue on histone H3, while TSA (30 nM) induced transient acetylations. After 6 days incubation, eosinophilic cells stained by Luxol‐fast‐blue were generated by apicidin (100 nM) and n‐butyrate (500 μM) but not by TSA (30 nM). Other markers for differentiation into eosinophils such as changes in intracellular structure, and expressions of integrin β7 and major basic protein, and the inhibition of cell proliferation were also induced by apicidin and n‐butyrate but not by TSA. Continuous acetylation of histone H4 achieved by repeated treatment with TSA (30 nM) at an interval of 12 h for more than three times induced such changes when examined on day 6. In addition, the induction was impaired by shortening the period of incubation with apicidin (100 nM) or n‐butyrate (500 μM). CCAAT/enhancer binding protein was continuously activated by apicidin (100 nM) and n‐butyrate (500 μM), but was transiently activated by TSA (30 nM). These findings suggest that the continuous acetylation of histones H3 and H4 is necessary for the differentiation of HL‐60 clone 15 cells into eosinophils.

Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells.

Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage‐like cell line, as determined by DNA fragmentation, the increase of annexin V‐stained cells, and the cleavage of poly(ADP‐ ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub‐G1 cells revealed that the DNA fragmentation occurred in a time‐ and concentration‐dependent manner at 0.021–2.1 μm of staurosporine. Staurosporine induced phosphorylation of p38 mitogen‐activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal‐regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3‐kinase (P13K) inhibitor LY294002 potentiated the staurosporine‐induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H‐89 potentiated the staurosporine‐induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro‐31–8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine‐induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and P13K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation.

Induction of apoptosis of RAW 264.7 cells by the cytostatic macrolide apicularen A

In RAW 264.7 cells, a mouse leukaemic monocyte cell line, apicularen A decreased cell growth and survival as assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay in a concentration‐dependent manner at 10–1000 nM. Apicularen B, an N‐acetyl‐glucosamine glycoside of apicularen A, was 10–100‐fold less effective than apicularen A. Apicularen A induced a DNA ladder, an increase in the percentage of sub‐G1 cells and annexin V‐binding cells, and promoted the activation of caspase as revealed by the cleavage of poly(ADP‐ribose) polymerase, indicating that apicularen A induced apoptosis in RAW 264.7 cells. In addition, apicularen A phosphorylated p44/42 mitogen‐activated protein kinase (MAPK) and p38 MAPK. The p44/42 MAPK inhibitor PD98059 rescued the cells from apicularen‐induced decrease in cell growth and survival as determined by the MTT assay, while the p38 MAPK inhibitor SB203580 augmented the effect of apicularen A. This suggested the activation of p44/42 MAPK to be pro‐apoptotic and the activation of p38 MAPK anti‐apoptotic in apicularen A‐treated RAW 264.7 cells.

Mechanism of the Eosinophilic Differentiation of HL-60 Clone 15 Cells Induced by n-Butyrate

n-Butyrate is one of the most powerful chemical inducers of the differentiation of human eosinophilic leukemia HL-60 clone 15 cells into mature eosinophils. We have recently reported that the mechanism by which HL-60 clone 15 cells differentiate into eosinophils by n-butyrate is that n-butyrate continuously inhibits histone deacetylase activity as a histone deacetylase inhibitor, resulting in continuous acetylation of histones. In this review, we discuss roles of histone acetyltransferase, histone deacetylase and histone deacetylase inhibitors in the differentiation of HL-60 clone 15 cells into eosinophils.

Inhibition of vacuolar-type (H+)-ATPase by the cytostatic macrolide apicularen A and its role in apicularen A-induced apoptosis in RAW 264.7 cells.

Apicularen A and the known vacuolar‐type (H+)‐ATPase (V‐ATPase) inhibitor bafilomycin A1 induced apoptosis of RAW 264.7 cells, while apicularen B, an N‐acetyl‐glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP‐dependent proton transport into inside‐out microsome vesicles, and inhibited the bafilomycin A1‐sensitive ATP hydrolysis. The IC50 values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A1. Furthermore, apicularen A inhibited the bafilomycin A1‐sensitive ATP hydrolysis more potently than apicularen B. F‐ATPase and P‐ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V‐ATPase, and thus induces apoptosis in RAW 264.7 cells.

Mechanism for the Differentiation of EoL-1 Cells into Eosinophils by Histone Deacetylase Inhibitors

Background: EoL-1 cells have a FIP1L1-PDGFRA fusion gene which causes the transformation of eosinophilic precursor cells into leukemia cells. Recently, we suggested that the induction of differentiation of EoL-1 cells into eosinophils by the HDAC inhibitors apicidin and n-butyrate is due to the continuous inhibition of HDACs. However, neither apicidin nor n-butyrate inhibited the expression of FIP1L1-PDGFRA mRNA, although both these inhibitors suppressed cell proliferation. Therefore, in this study, we analyzed whether the levels of FIP1L1-PDGFRα protein and phosphorylated-Stat5 involved in the signaling for the proliferation of EoL-1 cells are attenuated by HDAC inhibitors. Methods: EoL-1 cells were incubated in the presence of apicidin, TSA or n-butyrate. FIP1L1-PDGFRα and phosphorylated-Stat5 were detected by Western blotting. Results: Treatment of EoL-1 cells with apicidin at 100 nM or n-butyrate at 500 µM decreased the levels of FIP1L1-PDGFRα protein and phosphorylated-Stat5, while that with trichostatin A at 30 nM did not. Conclusions: The decrease in the level of FIP1L1-PDGFRα protein caused by apicidin and n-butyrate might be one of the mechanisms by which EoL-1 cells are induced to differentiate into eosinophils by these HDAC inhibitors.

Suppression of the antigen-stimulated RBL-2H3 mast cell activation by Artekeiskeanol A.

Effects of artekeiskeanol A, a newly isolated coumarin derivative from Artemisa keiskeana Miq. (Compositae), the extract of which is used for treatment of rheumatoid arthritis as a folk medicine, on the antigen-induced activation of RBL-2H3 cells were examined. RBL-2H3 cells were sensitized with dinitrophenol (DNP)-specific IgE, and then stimulated with the antigen DNP-conjugated human serum albumin (DNP-HSA). Artekeiskeanol A at 10 to 100 microM inhibited the antigen-induced degranulation in a concentration-dependent manner, the IC(50) value being 38.0 + or - 0.2 microM. Degranulation induced by thapsigargin or A23187 also was inhibited by artekeiskeanol A at 10 to 100 microM. The antigen-induced increase in the levels of mRNA for tumor necrosis factor (TNF)-alpha and interleukin (IL)-13 and phosphorylations of Akt, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p44/42 MAPK were also suppressed by artekeiskeanol A. Our findings suggested that the effectiveness of the extract of A. keiskeana might partly be due to the inhibition of mast cell activation by artekeiskeanol A.