OkPyo Zee

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.

Lead Compounds for Anti-inflammatory Drugs Isolated from the Plants of the Traditional Oriental Medicine in Korea

Effects of compounds isolated from medicinal plants in Korea on prostaglandin E2 (PGE2) production in rat peritoneal macrophages were examined, and mechanism of action of the active constituents was analyzed. The active constituents were as follows; tectorigenin and tectoridin isolated from the rhizomes of Belamcanda chinensis, platycodin D isolated from the roots of Platycodon grandiflorum, imperatorin isolated from the roots of Angelica dahurica, and hyperin isolated from the roots of Acanthopanax chiisanensis. These compounds inhibit the induction of cyclooxygenase-2 (COX-2), thus inhibiting PGE2 production. The chemically synthesized chalcone derivative, 2-hydroxy-4-methoxychalcone, also inhibits PGE2 production by suppressing COX-2 induction. In contrast, taiwanin C isolated from the roots of Acanthopanax chiisanensis inhibited PGE2 production by direct inhibition of COX-1 and COX-2.

Triterpenoid saponin fromviola hondoensis W. Becker et H Boss. and their effect on mmp-1 and type i procollagen expression

Bioassay-guided fractionation has led to the isolation of triterpenoid saponins such as Acutoside A (3-O-[O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranosyl] oleanolic acid) from the whole plants ofViola hondoensis. Among them, Saponin 1 exhibited potent inhibitory activity against matrix metalloproteinase (MMP)-1, and prevented the UV-induced changes in the MMP-1 expression. In addition, compound was isolated from this plant for the first time.

Apicularen A Induces Cell Death through Fas Ligand Up-Regulation and Microtubule Disruption by Tubulin Down-Regulation in HM7 Human Colon Cancer Cells

Purpose: Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A–induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A–induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo. Experimental Design: We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay. Results: Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G1-G0, DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, β-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A–treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by ∼72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 μg/kg/d). Conclusion: Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.

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.