RESUMO
4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.
Assuntos
Antineoplásicos/farmacologia , Caproatos/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Modelos Moleculares , Ratos , VorinostatRESUMO
⢠Myo-inositol hexakisphosphate (InsP(6)), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP(6) function in plants remain undefined. We now report the first evidence that InsP(6) can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. ⢠A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. ⢠Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-isomer (i.e. InsP(6)) on the basis of (1)H-, (31)P- and (13)C-NMR, (1)H-(1)H correlation spectroscopy (COSY), (1)H-(31)P heteronuclear single quantum correlation (HSQC) and (1)H-(13)C HSQC. Topical application of InsP(6) to early-stage somatic embryos indeed inhibits embryonic growth. ⢠Recently evidence has begun to emerge that InsP(6) may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.
Assuntos
Óvulo Vegetal/metabolismo , Ácido Fítico/isolamento & purificação , Pinus taeda/embriologia , Pinus taeda/crescimento & desenvolvimento , Sementes/embriologia , Bioensaio , Fracionamento Químico , Cromatografia Líquida , Genótipo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Noruega , Peptídeos/isolamento & purificação , Ácido Fítico/química , Pinus taeda/genéticaRESUMO
A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.
Assuntos
Germinação , Dissulfeto de Glutationa/farmacologia , Óvulo Vegetal/efeitos dos fármacos , Pinus/efeitos dos fármacos , Técnicas de Embriogênese Somática de Plantas/métodos , Sementes/efeitos dos fármacos , Tiossulfatos/farmacologia , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Germinação/efeitos dos fármacos , Glutationa/metabolismo , Glutationa/farmacologia , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/metabolismo , Oxirredução , Pinus/crescimento & desenvolvimento , Pinus/metabolismo , Pseudotsuga/efeitos dos fármacos , Pseudotsuga/crescimento & desenvolvimento , Pseudotsuga/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the undecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4-phenyl-3-butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP-stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA-Me and PBA both inhibit SP-stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA-Me and PBA dock at the ATP binding site of apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKs upstream of both JNK and p38 MAPK, with predicted interaction energies of -7.0 kcal/mol and -5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings suggest that AOPHA-Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition of JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule.
Assuntos
Anti-Inflamatórios/farmacologia , Caproatos/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Linhagem Celular , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Macrófagos/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Estaurosporina/farmacologia , Substância P/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pulsed infrared (nu approximately 2350 cm(-1)) laser excitation spectra of CO2 molecules embedded in helium droplets are reported. The spectra exhibit a sharp R(0) rovibrational line accompanied by a weak broader (deltanu approximately 10 cm(-1)) satellite band, which is shifted by 14 cm(-1) towards higher frequencies. We assign this satellite band to a simultaneous rovibrational excitation of a molecule and its helium solvation shell. The results are rationalized within a model, which includes coupling of the rotational states of a molecule and a ring of He atoms.