RESUMEN
Soluble epoxide hydrolase (sEH) serves as a potential target in inflammation-related diseases. Based on the bioactivity-guided separation, a new sesquiterpenoid inulajaponoid A (1) was isolated from Inula japonica with a sEH inhibitory effect, together with five known compounds, such as 1-O-acetyl-6-O-isobutyrylbritannilactone (2), 6ß-hydroxytomentosin (3), 1ß,8ß-dihydroxyeudesma-4(15),11(13)-dien-12,6α-olide (4), (4S,6S,7S,8R)-1-O-acetyl-6-O-(3-methylvaleryloxy)-britannilactone (5), and 1-acetoxy-6α-(2-methylbutyryl)eriolanolide (6). Among them, compounds 1 and 6 were assigned as mixed and uncompetitive inhibitors, respectively. The result of immunoprecipitation (IP)-MS demonstrated the specific binding of compound 6 to sEH in the complex system, which was further confirmed by the fluorescence-based binding assay showing its equilibrium dissociation constant (Kd = 2.43 µM). The detail molecular stimulation revealed the mechanism of action of compound 6 with sEH through the hydrogen bond of amino acid residue Gln384. Furthermore, this natural sEH inhibitor (6) could suppress the MAPK/NF-κB activation to regulate inflammatory mediators, such as NO, TNF-α, and IL-6, which confirmed the anti-inflammatory effect of inhibition of sEH by 6. These findings provided a useful insight to develop sEH inhibitors upon the sesquiterpenoids.
Asunto(s)
Epóxido Hidrolasas , Simulación de Dinámica Molecular , Epóxido Hidrolasas/química , Transducción de Señal , Regulación de la Expresión Génica , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin (7-demethylginkgetin, DGK), ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC 50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 µM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.
RESUMEN
Intestinal commensal fungi are vital to human health, and their metabolites play a key role in the reciprocal relationship. In the present work, eighteen alkaloids and seven monoterpenoids were isolated from the fermentation of the human intestinal fungus Penicillium oxalicum SL2, including seven undescribed alkaloids (penicilloxalines A-G), three undescribed monoterpenoids (penicilloxalines H-J), and fifteen reported compounds. The structures of the isolated compounds were identified by HRESIMS, 1D and 2D NMR, electronic circular dichroism spectra and quantum chemical calculations. Some metabolites displayed moderate agonistic effects against the pregnane X receptor (PXR), whereas (6R)3,7-dimethyl-6,7-dihydroxy-2(Z)-octenoic acid displayed a significant agonistic effect against the farnesoid X receptor (FXR) with an EC50 value of 0.43 µM, which was verified by investigating FXR downstream target genes and proteins, such as small heterodimer partner 1 (SHP1), fibroblast growth factor (FGF), and bile salt export pump (BSEP).
Asunto(s)
Penicillium , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares , Humanos , IntestinosRESUMEN
Intestinal commensal fungi are vital to human health, and their secondary metabolites play a key role in the reciprocal relationship. In the present study, the first example of 2,3-seco ergot alkaloids belonging to clavine-type were isolated from the fermentation of human intestinal fungus Aspergillus fumigatus CY018, including two pairs of diastereoisomers, secofumigaclavines A (3) and B (4) and secofumigaclavines C (5) and D (6), one analogue features a highly unsaturated skeleton, secofumigaclavine E (7), along with two known ones, fumigaclavines C (1) and D (2). Their structures were identified based on extensive spectroscopic data in a combination of quantum chemical calculations. Moreover, a single-step operation of semi-synthetic reaction based on riboflavin (RF)-dependent photocatalysis was performed to obtain the novel 2,3-seco ergot alkaloids 3 and 5 from their biosynthetic precursors 1 and 2. All the isolated compounds were evaluated for their anti-inflammatory activity. Among them, secofumigaclavine B (4) could bind to MD2 with a low micromole level of the equilibrium dissociation constant measured by surface plasmon resonance (SPR), and suppress TLR4-mediated NF-κB signaling pathway in RAW264.7 cells, resulting in its anti-inflammatory effect. Molecular dynamics revealed that amino acid residue Tyr131 played a key role in the interaction of secofumigaclavine B (4) with MD2. These findings suggested that secofumigaclavine B (4) could be considered as a potential candidate for the development of MD2 inhibitors.
Asunto(s)
Antiinflamatorios/uso terapéutico , Aspergillus fumigatus/efectos de los fármacos , Alcaloides de Claviceps/uso terapéutico , Antiinflamatorios/farmacología , Alcaloides de Claviceps/farmacología , HumanosRESUMEN
Inhibition of soluble epoxide hydrolase (sEH) is considered to be an effective treatment for inflammation-related diseases, and small molecules origin from natural products show promising activity against sEH. Two undescribed protostanes, 3ß-hydroxy-25-anhydro-alisol F (1) and 3ß-hydroxy-alisol G (2) were isolated from Alisma orientale and identified as new sEH inhibitors with IC50 values of 10.06 and 30.45 µM, respectively. Potential lead compound 1 was determined as an uncompetitive inhibitor against sEH, which had a Ki value of 5.13 µM. In-depth molecular docking and molecular dynamics simulations revealed that amino acid residue Ser374 plays an important role in the inhibition of 1, which also provides an idea for the development of sEH inhibitors based on protostane-type triterpenoids.
Asunto(s)
Alisma/química , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Triterpenos/farmacología , Inhibidores Enzimáticos/química , Epóxido Hidrolasas/química , Concentración 50 Inhibidora , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Farmacocinética , Extractos Vegetales/química , Extractos Vegetales/farmacología , Conformación Proteica , Triterpenos/químicaRESUMEN
As a part of our searching for natural human carboxylesterase 2 (human CES 2) inhibitors from traditional Chinese medicine, we found that the extract of Alisma orientale significantly inhibited human CES 2 in vitro. The investigation on A. orientale led to the isolation of a new protostane-type triterpenoid alismanin I (1). Its structure was determined according to HRESIMS, 1D and 2D NMR spectra. Alismanin I (1) displayed significantly inhibitory activity against human CES 2 with IC50 value of 1.31⯱â¯0.09⯵M assayed by human CES 2-mediated DDAB hydrolysis. According to its inhibition kinetic result, compound 1 was a noncompetitive type inhibitor, and its Ki was 3.65⯵M. Its inhibitory effect was confirmed in living cell level through a visual manner. The potential interaction mechanism of compound 1 with human CES 2 was also analyzed by circular dichroism (CD) spectrum and molecular docking.
Asunto(s)
Alisma/química , Carboxilesterasa/antagonistas & inhibidores , Carboxilesterasa/metabolismo , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Extractos Vegetales/farmacología , Carboxilesterasa/química , Dominio Catalítico , Dicroismo Circular , Inhibidores Enzimáticos/metabolismo , Humanos , Cinética , Extractos Vegetales/metabolismoRESUMEN
Two new protostane-type triterpenoids, 17-epi alisolide (1) and 24-epi alismanol D (2), were isolated from Alisma orientalis together with one known compound. Their structural elucidations were conducted by NMR, UV and HRESIMS spectroscopic analyses, and comparison with the literature data. All the isolated compounds were evaluated for inhibitory effects on HCE-2. Compound 2 displayed moderate inhibitory activity against HCE-2 with IC50 value of 23.1 µM.
Asunto(s)
Alisma/química , Triterpenos/química , Triterpenos/farmacología , Carboxilesterasa/antagonistas & inhibidores , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría UltravioletaRESUMEN
The bioactive substance investigation of Euphorbia ebracteolata obtained 17 compounds by various chromatographic techniques. Their structures were elucidated using widely spectroscopic data, including ESI-MS, HRESI-MS, CD, 1D- and 2D-NMR, which gave 5 new phenolic glucosides and 4 new monoterpenoids. The phenolic glucosides and monoterpenoids showed the inhibitory effect against the human carboxylesterase-2 (hCE-2) using a fluorescence bioassay in vitro, with the strongest inhibitor compound 4 (IC50 7.17µM). The antioxidant effects of these isolated compounds were evaluated using a DPPH scavenging assay. All of the phenolic acids displayed the DPPH scavenging effect, especially that eight compounds have better effect than vitamin C, with the IC50 values ranging from 4.52 to 7.52µM. Additionally, compounds 1-17 showed no cytotoxic effect against five human cancer cell lines by MTT assay.
Asunto(s)
Euphorbia/química , Glicósidos/química , Monoterpenos/química , Fenoles/química , Carboxilesterasa/antagonistas & inhibidores , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/aislamiento & purificación , Glicósidos/aislamiento & purificación , Humanos , Estructura Molecular , Monoterpenos/aislamiento & purificación , Fenoles/aislamiento & purificación , Extractos Vegetales/química , Raíces de Plantas/químicaRESUMEN
Phyllodium pulchellum (P. pulchellum) is a folk medicine with a significant number of bioactivities. The aim of this study was to investigate the effects displayed by alkaloids fractions, isolated from the roots of P. pulchellum, on neurotransmitters monoamine levels and on monoamine oxidase (MAO) activity. Six alkaloids, which had indolealkylamine or ß-carboline skeleton, were obtained by chromatographic technologies and identified by spectroscopic methods such as NMR and MS. After treatment with alkaloids of P. pulchellum, the reduction of DA levels (54.55%) and 5-HT levels (35.01%) in rat brain was observed by HPLC-FLD. The effect of alkaloids on the monoamines metabolism was mainly related to MAO inhibition, characterized by IC50 values of 37.35 ± 6.41 and 126.53 ± 5.39 µg/mL for MAO-A and MAO-B, respectively. The acute toxicity indicated that P. pulchellum extract was nontoxic.
RESUMEN
Objective: To study the anti-oxidative constituents of the aerial parts of Plumbago zeylanica. Methods: The ethanol extract of Plumbago zeylanica was separated and purified by various chromatographic techniques. On the basis of various spectroscopic data, the structures of isolated compounds were elucidated. ABTS+radical scavenging were carried out in antioxidant activity evaluation of the isolated compounds. Results: Eleven compounds were isolated and identified as cis-isoshinanolone-4-O-ß-D-glucopyranoside( 1),tachioside( 2),2,6-dimethoxy-p-hydroquinone-1-O-ß-D-glucopyranoside( 3),3-( ß-D-glucopyranosyloxy)-4-methoxybenzoic acid( 4),3'-O-ß-D-glucopyranosyloxy-plumbagic acid( 5),3'-O-ß-D-glucopyranosyloxy-plumbagic acid methyl ester( 6),plumbagic acid( 7),plumbagine A( 8),plumbagine C( 9),syringate-4-O-ß-D-glucopyranoside( 10) and 2-methyl-5-hydroxychromone( 11). Compounds 2,3,and5 displayed significant scavenging effect on ABTS+. Conclusion: Compounds 1 ~ 4,10,11 are obtained from this plant for the first time. Compounds 2,3,and 5 show significant anti-oxidative effects.
Asunto(s)
Medicamentos Herbarios Chinos , Plumbaginaceae , Glicósidos , Componentes Aéreos de las Plantas , TetrahidronaftalenosRESUMEN
Twelve new and 10 known protostane triterpenoids were isolated from the rhizome of Alisma orientale. Their structures were elucidated based on physical data analyses, including UV, HRESIMS, NMR experiments ((1)H, (13)C NMR, (1)H-(1)H COSY, HSQC, HMBC, and NOESY), and induced electronic circular dichroism. New compounds 1-12 were classified as protostanes (1-10), 29-norprotostane (11), and 24-norprotostane (12) by structure analyses. Furthermore, the inhibitory effects on human carboxylesterases (hCE-1, hCE-2) of compounds 1-22 were evaluated. Compounds 2, 6, 9, and 11 showed moderate inhibitory activities and were selective toward hCE-2 enzymes, with IC50 values of 8.68, 4.72, 4.58, and 2.02 µM, respectively. The inhibition kinetics of compound 11 toward hCE-2 were established, and the Ki value was determined as 1.76 µM using a mixed inhibition model. The interaction of bioactive compound 11 with hCE-2 was shown using molecular docking.
Asunto(s)
Alisma/química , Carboxilesterasa/antagonistas & inhibidores , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Triterpenos/aislamiento & purificación , Triterpenos/farmacología , Carboxilesterasa/metabolismo , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacocinética , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Extractos Vegetales/química , Rizoma/química , Triterpenos/química , Triterpenos/farmacocinéticaRESUMEN
Twelve new highly oxygenated lanostane triterpenoids and nine known ganoderic acids were isolated from the fruiting body of Ganoderma lucidum. The new compounds were lanostane nortriterpenoids with 27 carbons (1-5 and 8), lanostane nor-triterpenoids with 25 carbons (6 and 7), and lanostane triterpenoids (9-12) based on multiple spectroscopic data analysis, including HRESIMS, 1D-NMR, 2D-NMR, and CD. Compounds 1-5 were identified as rare nor-lanostanoids that contain a 17ß-pentatomic lactone ring. Compound 13, possessing a lactone ring, had been isolated previously. The P-glycoprotein (P-gp) inhibitory effects of compounds 1-21 were evaluated at a concentration of 20 µM using an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR). Compounds 1, 5, 18, and 20 and verapamil increased the accumulation of ADM in MCF-7/ADR cells approximately 3-fold when compared with the negative control. These data support the significant P-glycoprotein inhibitory activities of compounds 1, 5, 18, and 20. In silico docking analysis suggested these compounds had similar P-gp recognition mechanisms compared with those of verapamil (a classical inhibitor). Furthermore, in an in vitro bioassay, compounds 2, 4, 5, 6, and 18 showed moderate inhibitory effects against α-glucosidase compared with those of the positive control acarbose.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Inhibidores de Glicósido Hidrolasas/farmacología , Lanosterol/aislamiento & purificación , Lanosterol/farmacología , Reishi/química , alfa-Glucosidasas/efectos de los fármacos , Doxorrubicina/farmacología , Femenino , Cuerpos Fructíferos de los Hongos/química , Inhibidores de Glicósido Hidrolasas/química , Humanos , Lanosterol/química , Estructura Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Schisanlactone E (SE) is a major triterpene obtained from the plants of genus Kadsura. The aim of this research was to investigate the transformed metabolites of SE by fungi and evaluate the bioactivities of these products. After screening 10 strains of filamentous fungi, Cunninghamella blakesleana AS 3.970 was chosen as a potent organism to be used for the biotransformation of SE. 13 metabolites were obtained and determined to be new compounds through the use of spectroscopic data, including UV, 1D-, 2D-NMR, and HR-ESIMS. Furthermore, in an in vitro bioassay, metabolites 7 and 9 showed moderate inhibitory effects on the nitric oxide production in LPS-induced macrophages with IC50 values of 16.73, 5.91 µM, respectively; 9 could inhibit the proliferation of acetaldehyde-induced HSC-T6 cells, with the IC50 value of 21.4 µM. Preliminary findings on the structure-activity relationships for these metabolites were also discussed.
Asunto(s)
Cunninghamella/metabolismo , Kadsura/química , Macrófagos/efectos de los fármacos , Triterpenos/química , Animales , Biotransformación , Línea Celular , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Óxido Nítrico/biosíntesis , Relación Estructura-Actividad , Triterpenos/metabolismoRESUMEN
Three new phenolic constituents 1-3 were obtained from the 95% ethanol extract of the roots of Phyllodium pulchellum (Leguminosae). Their structures were elucidated on the basis of spectroscopic analyses, such as NMR, UV, IR, HR-ESI-MS, and CD. Furthermore, in an in vitro bioassay, all compounds were tested for inhibitory effects against the proliferation of acetaldehyde-stimulated HSC-T6 cells, and compound 3 exhibited potent inhibitory activity with the IC50 value of 7.6 µM.
Asunto(s)
Medicamentos Herbarios Chinos/aislamiento & purificación , Fabaceae/química , Fenoles/aislamiento & purificación , Acetaldehído/farmacología , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Fenoles/química , Fenoles/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/químicaRESUMEN
13F-1 is a 5-fluorouracil prodrug containing an Asn-Gly-Arg (NO2) COOCH3 tripeptide. 13F-1 might possess the activity against cancer growth by targeting Aminopeptidase N (APN/CD13). Our goal in this study was to evaluate the inhibitory effect of 13F-1 on the growth of human colonic carcinoma by both in vitro and in vivo studies. Experiments were performed in colonic carcinoma Colo205 cells, which highly express APN/CD13 on cell surface. The inhibition of 13F-1 on cancer cell growth was estimated by the colorimetric and clonogenic assays. The assays of Annexin V-FITC/PI and JC-1 fluorescence probe were employed to determine the apoptotic cells. Further experiment was performed in mice bearing Colo205 xenografts. 13F-1 was injected for three consecutive weeks. The specimens of Colo205 xenografts were removed for TUNEL staining and western blotting analysis. The expressions of APN/CD13 were analyzed by immunofluorescent flow cytometry and western blotting assays. 13F-1 significantly inhibited Colo205 cell proliferation. 13F-1 by injection delayed the expansion of Colo205 xenografts without significant toxicity to mice. The inhibitory effect of 13F-1 might arise from its role in apoptotic induction. Further analysis indicated that 13F-1 strongly inhibited APN/CD13 expression on cancer cell surface. In contrast, 5-FU did not affect APN/CD13 expression. These results indicated the mechanism of 13F-1 action that 13F-1׳s effect was associated with its role in suppression of APN/CD13 expression. Conclusion, 13F-1 could be developed as a promising agent for treatment of cancers with high expression of APN/CD13.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Antígenos CD13/metabolismo , Neoplasias del Colon/patología , Fluorouracilo/análogos & derivados , Fluorouracilo/metabolismo , Terapia Molecular Dirigida , Oligopéptidos/farmacología , Profármacos/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Femenino , Fluorouracilo/efectos adversos , Fluorouracilo/química , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Oligopéptidos/efectos adversos , Oligopéptidos/metabolismo , Profármacos/efectos adversos , Profármacos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rheumatoid arthritis (RA) is the rheumatism mainly manifested as disabling joint disease and mainly involves hands, wrists, feet and other small joints. Recurrent arthritis attacks, synovial cell hypertrophy and hyperplasia and bone and cartilage damages eventually lead to joint dysfunction and other complications, and there is no cure. Quercetin (QU) is a kind of natural flavonoids, with lipid-lowering, anti-inflammatory and other pharmacological activities, and minor toxic side effects. Thus, we assume that QU may be an adjuvant natural drug for treatment of RA. The possible mechanism is through regulation of NF-κB, to inhibit the transcription of joint synovitis factors, hinder the generation of inflammatory factors, and inhibit the inflammatory reaction; through inhibiting the activities of VEGF, bFGF, MMP-2 and other cytokines, to inhibit angiogenesis in multiple links and inhibit synovial pannus formation. QU may be an adjuvant natural drug for treatment of RA.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Citocinas/metabolismo , Inflamación/tratamiento farmacológico , FN-kappa B/metabolismo , Quercetina/uso terapéutico , Membrana Sinovial/efectos de los fármacos , Sinovitis/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Humanos , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Quercetina/farmacología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Sinovitis/metabolismoRESUMEN
In this study, two new compounds and six known compounds were isolated from the aerial parts of Pteris semipinnata. The chemical structures of these two new compounds were elucidated as 6ß,11α-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (1) and 7α,11α-dihydroxy-15-oxo-ent-kaur-16-en-19-oic acid (2) by the extensive spectral methods including 2D NMR and HR-MS techniques.
Asunto(s)
Diterpenos de Tipo Kaurano/aislamiento & purificación , Medicamentos Herbarios Chinos/aislamiento & purificación , Pteris/química , Diterpenos de Tipo Kaurano/química , Medicamentos Herbarios Chinos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
PURPOSE: Aminopeptidase N (APN/CD13) is highly expressed on the surface of cancer cells and is thought to be involved in cancer growth and metastasis. The research of APN/CD13 inhibitors is considered as a strategy of cancer treatment. We aimed to evaluate the efficacy of CIP-13F, a novel APN/CD13 inhibitor, using a Lewis lung carcinoma (LLC) implantation mouse model. METHODS: C57BL/6 mice were subcutaneously inoculated with LLC cells in anterior flank. Then, 0, 50 and 100 mg/kg of CIP-13F were injected via vena caudalis. Bestatin was used as the positive control. Administration of CIP-13F or bestatin was performed daily for 3 consecutive weeks. Mice were killed, and the tumors in anterior flank and metastasis nodules in lungs were examined. The assays of immunohistochemical staining, immunofluorescent flow cytometry and western blotting were performed to estimate the expression of APN/CD13 in LLC cells. We carried out the experiments of Annexin-V/PI staining, DNA fragmentation analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to determine apoptotic cells in LLC tissues. Using immunohistochemical staining with CD34, the antiangiogenesis of CIP-13F was evaluated in LLC tissue sections. RESULTS: CIP-13F treatment resulted in a significant delay of LLC growth in anterior flank. Examination of lungs showed that the number of metastatic nodules of LLC was also markedly decreased. The inhibitory effect of CIP-13F on LLC growth was further evidenced by the induction of LLC apoptosis, showing the increases in Annexin-V/PI staining cells, DNA fragmentation and TUNEL staining cells. Molecular analyses of LLC tissues in CIP-13F-treated mice suggested that the decrease in APN/CD13 expression by CIP-13F might account for its actions of mechanism. Further, the inhibition of angiogenesis in LLC tissues was determined, showing the decreases in microvessel density (MVD) and angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-α). CONCLUSION: Our results showed that CIP-13F effectively inhibited LLC growth and pulmonary metastasis in mice and suggested that CIP-13F is a potential drug for the treatment for cancers with positive APN/CD13 expression.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Piperidinas/farmacología , Animales , Antineoplásicos/farmacología , Antígenos CD13/metabolismo , Carcinoma Pulmonar de Lewis/enzimología , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/secundario , Procesos de Crecimiento Celular/efectos de los fármacos , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Inhibidores de Proteasas/farmacologíaRESUMEN
Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26-41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not ß-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Forbol 12,13-Dibutirato/farmacología , Animales , Línea Celular , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Pollos , Citoesqueleto/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Silenciador del Gen , Cobayas , Técnicas In Vitro , Cinética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/ultraestructura , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Miosinas/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Interferente Pequeño , RatasRESUMEN
Biotransformation of deoxyandrographolide (1) by Fusarium graminearum AS 3.4598 was investigated in this paper. And five transformed products of 1 by F. graminearum AS 3.4598 were obtained. Their chemical structures were characterized as 3-oxo-8α,17ß-epoxy-14-deoxyandrographolide (2), 3-oxo-14-deoxyandrographolide (3), 3-oxo-17,19-dihydroxyl-8,13-ent-labdadien-15,16-olide (4), 1ß-hydroxyl-14-deoxyandrographolide (5), and 7ß-hydroxyl-14-deoxyandrographolide (6) by spectral methods including 2D NMR. Among them, products 2, 4, and 5 are new.