RESUMO
Hirsuteine is one of the major bioactive tetracyclic indole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks, possessing a wide range of pharmacological activities including neuroprotective, anticonvulsant, antihypertensive, sedative and hypnotic, and so forth. The present study was undertaken to assess the metabolism and plasma pharmacokinetics of hirsuteine in rats. After oral administration of hirsuteine at the dose of 30 mg/kg, 13, 21, and 8 metabolites were detected in rat plasma, urine, and bile by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, respectively. Furthermore, plasma concentrations of hirsuteine and its four metabolites, 4-hirsuteine N-oxide, 3,4-dehydrohirsuteine, 11-hydroxyhirsuteine, and 11-hydroxyhirsuteine-11-O-glucuronide were simultaneously quantified in rat plasma, using carbamazepine as the internal standard. The linear calibration curve of hirsuteine was in the concentration range of 0.005-5.0 µg/ml. The lower limit of quantitation in the rat plasma was 5 ng/ml for hirsuteine. This study is the first to comprehensively investigate the metabolism process of hirsuteine and the pharmacokinetic profiles of hirsuteine and its major metabolite, and will provide a scientific basis to further elucidate the pharmacodynamic material basis and therapeutic mechanism of Uncaria prescriptions.
Assuntos
Cromatografia Líquida de Alta Pressão , Ratos , Animais , Espectrometria de MassasRESUMO
OBJECTIVE: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. METHODS AND RESULTS: Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. CONCLUSIONS: The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.
Assuntos
Metilação de DNA , Células Endoteliais/metabolismo , Epigênese Genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Superfície Celular/genética , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic ß-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Medicina Regenerativa/métodos , Animais , Células da Medula Óssea/citologia , HumanosRESUMO
This study investigated the metabolic fate of kakkalide (irisolidone 7-xylosylglucoside), a major isoflavone found in extracts of Pueraria lobata flowers, and in rat urine, bile, and feces. Using HPLC/UV or LC/MS/MS methods, seven metabolites, tectorigenin-7-O-glucuronide, tectorigenin-7-O-sulfate, tectorigenin-4'-O-sulfate, 6-OH biochanin A-glucuronide, irisolidone-7-O-glucuronide, tectorigenin, and irisolidone were identified in rat urine after oral administration of kakkalide. Furthermore, irisolidone-7-O-glucuronide was found in bile, and irisolidone and kakkalide were found in feces. An HPLC/UV method for simultaneous quantification of all the metabolites and kakkalide in urine, bile, and feces was developed using daidzein or apigenin as the internal standard. Over a 72-h period, 13.2 ± 2.8â% of the kakkalide was excreted as seven metabolites in urine. Over the same time period, irisolidone-7-O-glucuronide excretion in bile accounted for 3.8 ± 1.1â% of the dose, while kakkalide and irisolidone excretion in feces accounted for 2.1 ± 0.7â% and 0.7 ± 0.1â% of the dose, respectively. The results indicate that urine is the primary route of kakkalide elimination in vivo and that extensive metabolism may be one of the reasons for the low bioavailability of kakkalide.
Assuntos
Bile/química , Fezes/química , Glicosídeos/farmacocinética , Isoflavonas/farmacocinética , Administração Oral , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Flavonoides/isolamento & purificação , Flavonoides/metabolismo , Flavonoides/farmacocinética , Flavonoides/urina , Glicosídeos/isolamento & purificação , Glicosídeos/metabolismo , Glicosídeos/urina , Isoflavonas/isolamento & purificação , Isoflavonas/metabolismo , Isoflavonas/urina , Masculino , Pueraria/química , Ratos , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
Two new oleanane-type triterpenoid saponins, kakkasaponin II (1) and kakkasaponin III (2), were isolated from the methanol extract of the flowers of Pueraria thomsonii (Leguminosae), together with seven known oleanane-type triterpenoid saponins, phaseoside IV (3), sophoradiol monoglucuronide (4), kakkasaponin I (5), kaikasaponin III (6), soyasaponin I (7), soyasaponin III (8), and soyasaponin IV (9). The structures of 1 and 2 were elucidated by spectroscopic methods including IR, ESI-TOF-MS, and 1D and 2D NMR experiments.
Assuntos
Medicamentos de Ervas Chinesas/isolamento & purificação , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/isolamento & purificação , Pueraria/química , Saponinas/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Flores/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Ácido Oleanólico/química , Saponinas/químicaRESUMO
It is considered that cancer stem cells have the same characteristics as normal stem cells, such as drug-resistance, self-renewal, differentiation, and tissue-formation. Normal stem cells depend on their surroundings, a niche. Cancer stem cells may also depend on their own niche. Because cancer stem cells are resistant to present remedies, it is important to find a remedy targeting cancer stem cells. The remedy must not only target cancer stem cells themselves but also target the niche surrounding the cancer stem cells.
Assuntos
Células-Tronco Neoplásicas/fisiologia , HumanosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria Flos (PF), a traditional herbal medicine, is botanically from the dried flowers of Pueraria lobate (Willd.) Ohwi. (Chinese: ) or Pueraria thomsonii Benth. (Chinese: ). It has a long history of thousands of years in China for awakening the spleen, clearing the lungs, relieving alcohol. AIM OF THE REVIEW: This review aims to report the up-to-date research progress in ethnopharmacology, phytochemistry, pharmacology and toxicology, metabolism and therapeutic application of PF, so as to provide a strong basis for future clinical treatment and scientific research. MATERIALS AND METHODS: Relevant information on PF was collected from scientific literature databases including PubMed, CNKI and other literature sources (Ph.D. and M.Sc. dissertations and Chinese herbal classic books) by using the keyword "Puerariae". RESULTS: Briefly, phytochemical research report has isolated 39 flavonoids, 19 saponins and 25 volatile oils from PF. Flavonoids and saponins are the most important bioactive compounds, and most of the quality control studies focus on these two types of compounds. Modern pharmacological studies have revealed their significant biological activities in relieving alcoholism, hepatoprotective, anti-tumor, anti-inflammatory, and anti-oxidation, which provides theoretical support for the traditional use. CONCLUSIONS: Comprehensive analysis showed that pharmacological activity of most purified compounds from PF had not been reported. Kakkalide, tectoridin and their deglycosylated metabolites (irisolidone and tectorigenin) has been focused on excessively due to their higher content and better activities. This leads to low development and resources waste. Interestingly, PF made a breakthrough in the field of food. Many kinds of fat-lowering foods such as PILLBOX Onaka have been popular in Japan market, which received extensive attention. Therefore, we suggest that future research can be paid attention on the development of the plant's function in the field of food and medicine, as well as the transformation from experimental to clinical.
Assuntos
Medicamentos de Ervas Chinesas , Pueraria , Saponinas , Pueraria/química , Etnofarmacologia , Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/análise , Flores/química , Saponinas/análise , Compostos Fitoquímicos/farmacologia , Medicina Tradicional ChinesaRESUMO
Cell reprogramming reverts cells to multipotent, preprogrammed states by re-establishing epigenetic markers. It can also induce considerable malignant phenotype modification. Because key events in cancer relapse and metastasis, including epithelial-mesenchymal transition phenotypes, are regulated primarily by reversible and transient epigenetic modifications rather than the accumulation of irreversible and stable genetic abnormalities, studying dynamic mechanisms regulating these biological processes is important. Transcription factors for induced pluripotent stem cells and non-coding microRNAs allow pluripotent phenotype induction. We present the current knowledge of the possible applications of cell reprogramming in reducing aggressive phenotype expression, which can induce tumor cell hibernation and maintain appropriate phenotypes, thereby minimizing relapse and metastasis after surgical resection of gastrointestinal cancer.
Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Epigênese Genética/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Gastrointestinais/genética , Animais , Transformação Celular Neoplásica/patologia , Neoplasias Gastrointestinais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
The purpose of the study is to compare alkaloid profile of Uncaria rhynchophylla hooks and leaves. Ten oxindole alkaloids and four glycosidic indole alkaloids were identified using HPLC-diode array detection (DAD) or LC-atmospheric pressure chemical ionization (APCI)-MS method, and a HPLC-UV method for simultaneous quantification of major alkaloids was validated. The hooks are characterized by high levels of four oxindole alkaloids rhynchophylline (R), isorhynchophylline (IR), corynoxeine (C) and isocorynoxeine (IC), while the leaves contained high level of two glycosidic indole alkaloids vincoside lactam (VL) and strictosidine (S). The presented methods have proven its usefulness in chemical characterization of U. rhynchophylla hooks and leaves.
Assuntos
Alcaloides/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por Electrospray , Uncaria/química , Indóis/química , Óxidos/química , Oxindóis , Folhas de Planta/químicaRESUMO
Two novel flavonoid glycosides, 6"'-dihydrophaseoylspinosin (1) and 6â³,6"'-diferuloylspinosin (2), were isolated from the MeOH extract of Semen Ziziphi Spinosae, together with six known flavonoids, isovitexin-2â³-O-ß-(6-O-E-feruloyl)glucopyranoside (3), spinosin (4), isospinosin (5), 6"'-feruloylspinosin (6), swertisin (7), and isovitexin-2â³-O-ß-d-glucopyranoside (8). The structures of 1 and 2 were elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D NMR, and 2D NMR experiments.
Assuntos
Flavonoides/isolamento & purificação , Glicosídeos/isolamento & purificação , Apigenina/química , Apigenina/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Flavonoides/química , Glicosídeos/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
A novel spinosin derivative, 6â´-(4â´'-O-ß-D-glucopyranosyl)-vanilloyl spinosin (1) was isolated from the methanol extract of Semen Ziziphi Spinosae, together with five known flavonoids, swertish, spinosin, 6â´-feruloylspinosin, isospinosin and kaempferol 3-O-α-L-rhamnopyranosyl-(1 â 2)-O-[O-α-L-rhamnopyranosyl-(1 â 6)]-ß-D-glucopyranoside, and two alkanoids, zizyphusine and 6-(2',3'-dihydroxyl-4'-hydroxymethyl-tetrahydro-furan-1'-yl)-cyclopentene[c]pyrrole-1,3-diol. The structure of compound 1 was elucidated by spectroscopic methods including UV, IR, ESI-TOF-MS, 1D, and 2D NMR techniques.
Assuntos
Medicamentos de Ervas Chinesas/química , Flavonoides/isolamento & purificação , Flavonoides/química , Glicosídeos/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Kakkalide is a major isoflavonoid from the flowers of Pueraria lobata (Willd.) Ohwi, possessing the protective effect against ethanol-induced intoxication and hepatic injury. The metabolism of kakkalide was investigated in rats. Thirteen metabolites were isolated by using solvent extraction and repeated chromatographic methods and identified by using spectroscopic methods including UV, IR, mass spectrometry, NMR, and circular dichroism experiments. Four new compounds were identified as irisolidone-7-O-glucuronide (M-1), tectorigenin-7-O-sulfate (M-2), tectorigenin-4'-O-sulfate (M-3), and biochanin A-6-O-sulfate (M-4) together with nine known compounds identified as irisolidone (M-5), tectorigenin (M-6), tectoridin (M-7), 5,7-dihydroxy-8,4'-dimethoxyisoflavone (M-8), isotectorigenin (M-9), biochanin A (M-10), genistein (M-11), daidzein (M-12), and equol (M-13). The metabolic pathway of kakkalide was proposed, which is important to understand its metabolic fate and disposition in humans.
Assuntos
Flavonoides/química , Flavonoides/isolamento & purificação , Glicosídeos/metabolismo , Isoflavonas/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Flavonoides/metabolismo , Flavonoides/urina , Flores/química , Glucuronídeos/química , Glucuronídeos/isolamento & purificação , Glucuronídeos/urina , Glicosídeos/isolamento & purificação , Glicosídeos/farmacocinética , Glicosídeos/urina , Inativação Metabólica , Isoflavonas/isolamento & purificação , Isoflavonas/farmacocinética , Isoflavonas/urina , Espectroscopia de Ressonância Magnética , Masculino , Redes e Vias Metabólicas , Estrutura Molecular , Pueraria/química , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/isolamento & purificação , Ésteres do Ácido Sulfúrico/urina , Espectrometria de Massas em TandemRESUMO
PURPOSE: To evaluate oleanolic acid derivatives on liver disease related bioactivities, 29 oleanolic acid derivatives of several series were tested for their inhibitory activity on hepatitis C viral protease and for their cytotoxic effects on Hep G2 cells. RESULTS: The amino derivatives showed potent cytotoxicity, among which, the beta-amino isomer exhibited more distinct cytotoxicity than the alpha-isomer. The cytotoxicity of hemiesters and hemiamides varied as the chain lengths varied. The oxalic and malonic hemiesters showed weaker cytototoxicity than oleanolic acid, while those with longer chain lengths showed higher cytotoxicity. Contrary to the cytotoxic activity, the free amino derivatives showed little inhibitory activity on HCV protease. All the hemiesters and hemiamides showed high activity against HCV protease. CONCLUSION: The findings that addition of amino-group enhanced the cytotoxicity and that introduction of acidic group increased the inhibition on HCV protease may be useful for further design and synthesis of triterpene derivatives as drug candidates for liver diseases.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Hepacivirus/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/tratamento farmacológico , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
Recently we developed a mathematical model for microbial growth in food. The model successfully predicted microbial growth at various patterns of temperature. In this study, we developed a program to fit data to the model with a spread sheet program, Microsoft Excel. Users can instantly get curves fitted to the model by inputting growth data and choosing the slope portion of a curve. The program also could estimate growth parameters including the rate constant of growth and the lag period. This program would be a useful tool for analyzing growth data and further predicting microbial growth.
Assuntos
Bactérias/crescimento & desenvolvimento , Microbiologia de Alimentos , Modelos Logísticos , Modelos Biológicos , Software , Animais , Interpretação Estatística de Dados , Leite/microbiologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Circulating aldosterone concentrations occasionally increase after initial suppression with angiotensin II (Ang II) converting enzyme inhibitors or Ang II type 1 receptor blockers (ARBs), a phenomenon referred to as aldosterone breakthrough. However, the underlying mechanism causing the aldosterone breakthrough remains unknown. Here we investigated whether aldosterone breakthrough occurs in human adrenocortical H295R cells in vitro. We recently reported that bone morphogenetic protein (BMP)-6, which is expressed in adrenocortical cells, enhances Ang II- but not potassium-induced aldosterone production in human adrenocortical cells. Accordingly, we examined the roles of BMP-6 in aldosterone breakthrough induced by long-term treatment with ARB. Ang II stimulated aldosterone production by adrenocortical cells. This Ang II stimulation was blocked by an ARB, candesartan. Interestingly, the candesartan effects on Ang II-induced aldosterone synthesis and CYP11B2 expression were attenuated in a course of candesartan treatment for 15 d. The impairment of candesartan effects on Ang II-induced aldosterone production was also observed in Ang II- or candesartan-pretreated cells. Levels of Ang II type 1 receptor mRNA were not changed by chronic candesartan treatment. However, BMP-6 enhancement of Ang II-induced ERK1/2 signaling was resistant to candesartan. The BMP-6-induced Smad1, -5, and -8 phosphorylation, and BRE-Luc activity was augmented in the presence of Ang II and candesartan in the chronic phase. Chronic Ang II exposure decreased cellular expression levels of BMP-6 and its receptors activin receptor-like kinase-2 and activin type II receptor mRNAs. Cotreatment with candesartan reversed the inhibitory effects of Ang II on the expression levels of these mRNAs. The breakthrough phenomenon was attenuated by neutralization of endogenous BMP-6 and activin receptor-like kinase-2. Collectively, these data suggest that changes in BMP-6 availability and response may be involved in the occurrence of cellular escape from aldosterone suppression under chronic treatment with ARB.
Assuntos
Córtex Suprarrenal/fisiologia , Aldosterona/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Compostos de Bifenilo , Proteína Morfogenética Óssea 6 , Linhagem Celular , Genes Reporter , Humanos , Imuno-Histoquímica , Cinética , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetrazóis/farmacologia , TransfecçãoRESUMO
Two new isomeric alkaloids, 18,19-dehydrocorynoxinic acid B (1) and 18,19-dehydrocorynoxinic acid (2), were isolated from the CHCl3 extract of the leaves of Uncaria rhynchophylla, together with four known rhynchophylline-type alkaloids, corynoxeine (3), isocorynoxeine (4), rhynchophylline (5), and isorhynchophylline (6), and an indole alkaloid glucoside, vincoside lactam (7). The structures of compounds 1 and 2 were elucidated by spectroscopic methods including UV, IR, HREIMS, 1D and 2D NMR, and CD experiments. The activity assay showed that compounds 3-6, with a C-16 carboxylic ester group, and 7 exhibited inhibitory activity on lipopolysaccharide (LPS)-induced NO release in primary cultured rat cortical microglia (IC 50: 13.7-19.0 microM). However, only weak inhibitory activity was observed for compounds 1 and 2, with a C-16 carboxylic acid group (IC 50: >100 microM).
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Lipopolissacarídeos/farmacologia , Óxido Nítrico/biossíntese , Plantas Medicinais/química , Uncaria/química , Alcaloides/química , Animais , Medicamentos de Ervas Chinesas/química , Microglia/efeitos dos fármacos , Estrutura Molecular , Folhas de Planta/química , RatosAssuntos
Neoplasias Gastrointestinais/terapia , Células-Tronco Neoplásicas , Animais , Antígenos CD13 , Ciclo Celular , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/ultraestrutura , Células HCT116 , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos , Microscopia , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/ultraestrutura , Proteínas Nucleares , Complexo de Endopeptidases do Proteassoma , Proteínas Repressoras , Células Tumorais CultivadasRESUMO
We investigated the relationship between methylomic [5-methylation on deoxycytosine to form 5-methylcytosine (5mC)] and transcriptomic information in response to chemotherapeutic 5-fluorouracil (5-FU) exposure and cisplatin (CDDP) administration using the ornithine decarboxylase (ODC) degron-positive cancer stem cell model of gastrointestinal tumour. The quantification of 5mC methylation revealed various alterations in the size distribution and intensity of genomic loci for each patient. To summarise these alterations, we transformed all large volume data into a smooth function and treated the area as a representative value of 5mC methylation. The present computational approach made the methylomic data more accessible to each transcriptional unit and allowed to identify candidate genes, including the tumour necrosis factor receptor-associated factor 4 (TRAF4), as novel therapeutic targets with a strong response to anti-tumour agents, such as 5-FU and CDDP, and whose significance has been confirmed in a mouse model in vivo. The present study showed that 5mC methylation levels are inversely correlated with gene expression in a chemotherapy-resistant stem cell model of gastrointestinal cancer. This mathematical method can be used to simultaneously quantify and identify chemoresistant potential targets in gastrointestinal cancer stem cells.
Assuntos
5-Metilcitosina/metabolismo , Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Gastrointestinais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Fluoruracila/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Bone morphogenetic protein-6 (BMP-6) enhances aldosterone production by upregulating angiotensin II (Ang II)-to-MAPK pathway. Here we investigated effects of Ang II and potassium on the BMP system in human adrenocortical H295R cells. BMP-6 transcription was transiently downregulated by treatments with Ang II and potassium. Aldosterone also decreased BMP-6 expression at a high concentration. Chemical inhibitions of transcription and translation abolished the transient reduction of BMP-6, suggesting that destabilization of BMP-6 mRNA was hardly involved while new protein synthesis was possibly mediated in this mechanism. However, BMP-6 protein was stably detected during the exposures of Ang II and potassium. Notably, Ang II, potassium and aldosterone decreased mRNA levels of follistatin that extracellularly neutralizes bioactivities of activins and BMPs although the BMP-6 receptor expression was unaffected. Given the maintenance of bioavailable BMP-6 protein and the receptor expression in adrenocortical cells, endogenous BMP-6 may be a key autocrine modulator for aldosterone production.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Aldosterona/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Angiotensina II/farmacologia , Proteína Morfogenética Óssea 6 , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Folistatina/genética , Folistatina/metabolismo , Humanos , Immunoblotting , Análise de Sequência com Séries de Oligonucleotídeos , Potássio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aldosterone production is modified by several growth factors that reside in the adrenal. We have recently reported the existence of a bone morphogenetic protein (BMP) system in human adrenocortical cells, in which BMP-6 augments aldosterone synthesis. Here, we investigated functional roles of BMP-6, focusing on the differential regulation of aldosterone synthesis induced by angiotensin (Ang) II and potassium (K). In human adrenocortical H295R cells, BMP-6 augmented Ang II-induced CYP11B2 transcription and mRNA and aldosterone production but had no effect on K-induced aldosterone production. Inhibition of endogenous BMP-6 action by neutralizing antibodies impaired aldosterone production induced by Ang II but not that induced by K. Blockage of ligand-receptor binding using extracellular domain (ECD) of BMP type I receptors revealed that ECDs to activin receptor-like kinase (ALK)-2 and ALK-3 significantly reduced the aldosterone production induced by Ang II. None of the type I-receptor ECDs tested had any effect on K-induced aldosterone levels. Overexpression of a dominant negative-activin type II receptor construct selectively decreased Ang II-induced aldosterone production without having any effect on K-induced aldosterone production. BMP type II receptor-dominant negative had no effect on aldosterone induced by either Ang II or K. These results infer that BMP-6 acts through ALK-2, ALK-3, and activin type II receptor receptors in adrenocortical cells. BMP-6 pretreatment extends the induction of ERK1/2 phosphorylation by Ang II and treatment with ECDs to ALK-2 and ALK-3 impaired Ang II-induced ERK phosphorylation. The specific inhibitor of ERK activation, U0126, suppressed the activation of CYP11B2 transcription induced by BMP-6 without affecting Smad phosphorylation and Tlx2-Luc activity. Collectively, the endogenous BMP-6 system plays critical roles in aldosterone production between Ang II and K through ERK signaling pathway.