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1.
Nagoya J Med Sci ; 81(3): 415-425, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31579332

RESUMO

Eye problems are an adverse reaction sometimes found in chemotherapy. Although not life-threatening, they can reduce patients' quality of life. The highest incidence of eye problems is reported for the combination anticancer drug S-1 (tegafur-gimeracil-oteracil), and methods to prevent or treat the eye problems caused by this drug are presently lacking. To determine early detection methods and treatment for adverse ocular reactions, we measured changes in tear volume and levels of tegafur (FT) and 5-fluorouracil (5-FU), an active metabolite of FT, in the tears of patients with long-term use of S-1. A total of 11 patients receiving S-1 monotherapy as adjuvant chemotherapy after gastric cancer surgery were included. Tear volume and FT and 5-FU levels in tears were measured by liquid chromatography with tandem mass spectrometry during a maximum of 8 treatment cycles (48 weeks). For analysis, patients were divided into two groups: "watering eyes" (n=6, complaints of watering eyes at least once during the treatment period) and "no watering eyes" (n=5, no complaints of watering eyes). Both groups exhibited increased FT and 5-FU levels in tears upon initiation of S-1 treatment, and levels rapidly decreased upon discontinuation. Our findings suggest a relationship between FT level in tears and tear volume in patients with long-term S-1 use. The symptom of watering eyes may thus be linked to FT level in tears.

2.
Anticancer Res ; 39(9): 4711-4720, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519570

RESUMO

BACKGROUND/AIM: Dynamics of circulating tumor cells (CTCs) after molecular targeting therapy remain unclear. MATERIALS AND METHODS: We examined changes in CTC numbers and morphology early after targeting therapy in EGFR-mutated PC-9 human lung cancer and HER2-gene amplified GLM-1 gastric cancer mouse CTC models using a cytology-based semi-automated CTC detection platform. RESULTS: Erlotinib and T-DM1 inhibited cell growth mainly by induction of apoptosis in vitro. The number of CTCs detected 5-10 days after targeting therapy in mice was significantly increased compared to CTC numbers before therapy. The increased CTCs after therapy consisted of apoptotic CTCs and viable CTCs. This heterogeneous population of CTCs reflects well the cell population of the primary tumor disrupted by therapy. CONCLUSION: CTCs can be mobilized from the primary tumor due to tissue disruption in acute response to targeting therapy, suggesting potential usefulness of CTC monitoring as a predictor of therapeutic response in the clinical settings.


Assuntos
Amplificação de Genes , Mutação , Células Neoplásicas Circulantes/metabolismo , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Animais , Antineoplásicos/farmacologia , Biomarcadores , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Células Neoplásicas Circulantes/patologia , Neoplasias Gástricas/tratamento farmacológico
3.
J Biol Chem ; 294(17): 6871-6887, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824540

RESUMO

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

4.
Biofactors ; 45(2): 253-258, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30537158

RESUMO

Human sirtuin 1 (hSIRT1) is a NAD+ -dependent deacetylase that regulates several cellular processes. Unlike resveratrol, natural polymeric phenolic compounds isolated from Vitaceae are mostly hSIRT1 inhibitors. The resveratrol tetramer, (+)-hopeaphenol ((+)-HP), and its geometric isomer, (-)-isohopeaphenol ((-)-iHP), were tested for inhibitory effects on purified hSIRT1 using a fluorescent derivative of peptide substrate p53-AMC (Fluor de Lys) and a cofactor NAD+ . The Lineweaver-Burk plots indicated that both (+)-HP and (-)-iHP were competitive inhibitors against NAD+ . Computer-assisted modeling of the binding of these molecules with hSIRT1 protein provided the most feasible conformation of the enzyme-inhibitor complex. © 2018 BioFactors, 45(2):253-258, 2019.


Assuntos
Polifenóis/farmacologia , Sirtuína 1/química , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Humanos , Fenóis/química , Fenóis/farmacologia , Polifenóis/química , Ligação Proteica , Resveratrol/química , Resveratrol/farmacologia , Sirtuína 1/antagonistas & inibidores , Estilbenos/química
5.
Technol Cancer Res Treat ; 17: 1533033818767936, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29649929

RESUMO

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)-labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma-xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression-dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

6.
FEBS Open Bio ; 8(3): 349-360, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29511612

RESUMO

Amyloid-ß (Aß), a primary component of amyloid plaques, has been widely associated with the pathogenesis of Alzheimer's disease. The Ca2+-binding protein regucalcin (RGN) plays multiple roles in maintaining cell functions by regulating intracellular calcium homeostasis, various signaling pathways, and gene expression systems. Here, we investigated the functional role of RGN against Aß-induced cytotoxicity in neuronally differentiated PC12 cells. Overexpression of RGN reduced Aß-induced apoptosis by reducing mitochondrial dysfunction and caspase activation. It also attenuated Aß-induced reactive oxygen species production and oxidative damage and decreased Aß-induced nitric oxide (NO) overproduction, upregulation of inducible NO synthase by nuclear factor-κB, and nitrosative damage. Interestingly, the genetic disruption of RGN increased the susceptibility of neuronally differentiated PC12 cells to Aß toxicity. Thus, RGN possesses antioxidant activity against Aß-induced oxidative and nitrosative stress and may play protective roles against Aß-induced neurotoxicity in Alzheimer's disease.

7.
Artigo em Inglês | MEDLINE | ID: mdl-29445512

RESUMO

Background: Most eye disorders are not fatal but may deteriorate the quality of life of a patient. The eye disorder that is most frequently reported in the cancer chemotherapy is associated with the combination of tegafur/gimeracil/potassium oxonate (S-1). However, preventive methods or treatment methods for the eye disorder have not yet been established. This study aimed to determine changes in tear volume and subjective ocular symptoms during the treatment period in patients receiving S-1 monotherapy for early detection of adverse effects in the eye and establishment of its treatment methods. Methods: This study included eleven patients receiving S-1 monotherapy as a postoperative adjuvant chemotherapy for gastric cancer. Six subjective ocular symptoms including watering eyes were evaluated and changes in tear volume measured by the Schirmer's test in patients receiving S-1 during the treatment period. In the present study, the patients were divided into "no watering eyes" (patients not experienced watering eyes) group and "watering eyes" (patients experienced watering eyes even once) group. Results: Six out of eleven patients developed watering eyes after receiving S-1 monotherapy. Among these, the earliest onset occurred on the 2nd week after oral administration. Watering eyes and eye discharge were highly related in patients having a trouble in daily life due to the decreased QOL. Changes in tear volume in the "watering eyes" group significantly increased compared to the "no watering eyes" group during the treatment period, especially when the patients had no subjective symptom of the increased tear volume. Conclusions: It is essential to prevent eye disorders including watering eyes as an adverse effect of S-1 administration. The present study recommends that the tear volume should be periodically measured using Schirmer's test, and the patient should be interviewed regarding the subjective ocular symptoms for the early detection of watering eyes caused by S-1 administration. If the tear volume can not be measured periodically, medical staffs should pay attention to the patient with eye discharge.

8.
J Nat Med ; 72(1): 260-266, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29151157

RESUMO

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.


Assuntos
Benzofuranos/química , Erythrina/química , Células HL-60/química , Isoflavonas/química , Leucemia/tratamento farmacológico , Apoptose , Humanos , Leucemia/patologia
9.
Cancer Sci ; 107(9): 1329-37, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27323954

RESUMO

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell-free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma-PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher-risk groups than that in lower-risk groups (P = 0.034). The concentration of plasma-/serum-PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160-bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM-DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB-2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.


Assuntos
Células Sanguíneas/metabolismo , DNA de Neoplasias/genética , Síndromes Mielodisplásicas/genética , Biomarcadores Tumorais , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Transformação Celular Neoplásica/genética , Análise Mutacional de DNA , DNA de Neoplasias/sangue , Progressão da Doença , Detecção Precoce de Câncer , Humanos , Mutação , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/diagnóstico , Reação em Cadeia da Polimerase , Prognóstico
10.
Nat Prod Commun ; 10(9): 1581-4, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26594764

RESUMO

It has been reported that many malignant human tissues, including breast, colon, and lung cancers, may show an elevated expression of glyoxalase I (GLO I). GLO I catalyzes the reaction to transform hemimercaptal, a compound formed from methylglyoxal (MG) and reduced glutathione, into S-D-lactoylglutathione, which is then converted to D-lactic acid by glyoxalase II. GLO I inhibitors are expected to be useful for inhibiting tumorigenesis through the accumulation of apoptosis-inducible MG in tumor cells. Here, we investigated the anti-proliferative activity of eight kinds of isoflavone isolated from Erythrina poeppigiana against the growth of HL-60 human leukemia cells from the viewpoint of GLO I inhibition. Of the compounds tested, the diprenyl isoflavone, isolupalbigenin, was shown to exhibit the highest anti-proliferative activity against HL-60 cells. Upon the treatment of HL-60 cells with isolupalbigenin, MG was significantly accumulated in the culture medium, and the caspase 3 activity of the cell lysate was elevated in a time-dependent manner. Thus, it is suggested that isolupalbigenin inhibits the enzyme GLO I, resulting in MG accumulation in the medium, and leading to cell apoptosis. Isolupalbigenin, with two prenyl groups in its A- and B-rings, might be expected to become a potent leading compound for the development of anticancer agents.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Erythrina/química , Isoflavonas/farmacologia , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/metabolismo , Antineoplásicos Fitogênicos/química , Sobrevivência Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Células HL-60 , Humanos , Isoflavonas/química , Lactoilglutationa Liase/genética , Estrutura Molecular
11.
J Nat Med ; 68(3): 636-42, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24756815

RESUMO

A novel isoflavone, erythgianin A (1), along with nine known compounds 2-10, was isolated from the stem bark of Erythrina poeppigiana (Leguminosae). The unusual isoflavone structure of 1, possessing a highly oxidized 3″,4″-dihydroxy-2″-hydroxymethyl-2″-methyl-2″,3″-dihydropyrano substituent, was determined on the basis of spectroscopic analyses. All of the isolated compounds were evaluated for their in vitro inhibitory activity toward human glyoxalase I. Among the isolates, isolupalbigenin (10) with two prenyl groups showed the highest inhibitory activity.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Erythrina/química , Lactoilglutationa Liase/antagonistas & inibidores , Fenóis/química , Fenóis/farmacologia , Humanos , Isoflavonas/química , Isoflavonas/farmacologia , Casca de Planta/química
12.
Gastric Cancer ; 17(3): 497-507, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24288123

RESUMO

BACKGROUND: Peritoneal metastasis is the most frequent pattern of recurrence after curative surgery for gastric cancer. However, such a recurrence is difficult to detect by conventional computed tomography (CT) and magnetic resonance imaging (MRI) at an early stage. To improve the sensitivity and specificity of diagnostic imaging for peritoneal metastasis, we developed a new type of multimodality imaging combining fluorescence imaging with near-infrared fluorophore (NIR)-labeled antibodies and MRI. METHODS: Dual optical imaging of peritoneal metastasis was carried out using luciferase-tagged gastric cancer cell lines and XenoLight CF750 or indocyanine green (ICG)-labeled anti-human epidermal growth factor receptor (EGFR) or CEA antibody as a probe in mice with Ivis in vivo imaging system. RESULTS: This whole-body fluorescent imaging system sensitively detected metastatic foci <1 mm in diameter in the peritoneal cavity noninvasively. Fluorescence imaging proved to be specific because the fluorescence signal was abolished by blocking with excess unlabeled antibody. Although this fluorescence imaging had higher sensitivity for detection of small-sized peritoneal metastases than MRI, it proved difficult to accurately determine organ distribution of the metastasis. We thus developed a multimodality imaging system by the fusion of the three-dimensional fluorescence image with the MRI image and demonstrated its improved diagnostic accuracy over either method alone. CONCLUSION: The present results suggest that multimodality imaging consisting of fluorescence imaging with NIR-labeled EGFR or CEA antibody and MRI allows sensitive, specific, and anatomically accurate detection of peritoneal metastasis noninvasively at an early stage.


Assuntos
Imagem por Ressonância Magnética/métodos , Imagem Óptica/métodos , Neoplasias Peritoneais/diagnóstico , Neoplasias Gástricas/patologia , Animais , Anticorpos Monoclonais , Antígeno Carcinoembrionário/imunologia , Receptores ErbB/imunologia , Humanos , Imagem Tridimensional/métodos , Verde de Indocianina , Masculino , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Neoplasias Peritoneais/secundário , Sensibilidade e Especificidade
13.
J Pharm Pharmacol ; 65(8): 1204-13, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23837588

RESUMO

OBJECTIVES: The aim of this study was to examine the mechanism underlying the inhibitory effect of our synthesized carbazolequinone derivatives on nitric oxide (NO) production in activated macrophages. METHODS: Lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated RAW264.7 macrophages were treated with carbazolequinone derivatives. The NO and prostaglandin E2 (PGE2 ) levels in cell culture supernatants fractions were measured by Greiss and ELISA assay, respectively. The expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was assessed by the real-time RT-PCR method. Nuclear factor kappa B (NF-κB) activation was detected by an NF-κB-dependent luciferase reporter assay. KEY FINDINGS: Our synthesized carbazolequinone derivatives (7-methoxy-2-methylcarbazole-1,4-quinone, 6-methoxy-2-methylcarbazole-1,4-quinone and 6-chloro-2-methylcarbazole-1,4-quinone) significantly inhibited LPS/IFN-γ-induced NO production and iNOS expression in RAW264.7 cells. They also inhibited the LPS/IFN-γ-mediated induction of COX-2 expression and PGE2 production. In addition, the LPS/IFN-γ-induced transcription activity of NF-κB was attenuated. Using the RAW264.7-tsAM5NE co-culture system, we found that these carbazolequinone derivatives protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. CONCLUSIONS: These results suggest that the three carbazolequinone derivatives inhibit LPS/IFN-γ-induced NO production via iNOS and COX-2 downregulation due to NF-κB inhibition. Therefore, these three carbazolequinone derivatives may be useful for developing a new drug against NO-mediated neurodegenerative diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Carbazóis/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Quinonas/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Carbazóis/química , Carbazóis/isolamento & purificação , Técnicas de Cultura de Células , Linhagem Celular , Ciclo-Oxigenase 2/genética , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Murraya/química , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Quinonas/química , Quinonas/isolamento & purificação
14.
Nat Prod Commun ; 7(11): 1479-82, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23285811

RESUMO

Study of the chemical constituents of the stems of Derris trifoliata Lour. (Leguminosae) collected in Singapore led to the isolation and identification of three known and two new rotenoid derivatives. The new derivatives, named derrisfolin A (1) and B (2), inhibited nitric oxide production in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma and lipopolysaccharide.


Assuntos
Derris/química , Óxido Nítrico/antagonistas & inibidores , Rotenona/análogos & derivados , Animais , Linhagem Celular , Camundongos , Estrutura Molecular
15.
J Nat Med ; 66(2): 357-61, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21879331

RESUMO

We carried out primary screening of 13 carbazole alkaloids isolated from the plant species Murraya euchrestifolia (Rutaceae) on cell growth inhibition of the human leukemia cell line HL-60. Among them, murrayafoline-A (1) and murrayazolinine (7) exhibited significant growth suppression due to apoptosis mediated by the activation of the caspase-9/caspase-3 pathway.


Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Murraya/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Alcaloides/química , Carbazóis/química , Carbazóis/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Células HL-60 , Humanos , Estrutura Molecular
16.
J Nat Med ; 65(2): 353-9, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21246298

RESUMO

It is well known that inflammation is associated with various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. An inflammatory mediator, nitric oxide (NO), is produced by inducible NO synthase (iNOS) in microglia and seems to be one of the possible causes of neurodegeneration. Several natural and synthetic compounds which exert anti-inflammatory effects by inhibiting NO production have been reported to date. The aim of this work was to investigate whether any of the 6 terpenoid coumarins (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin, and aurapten) isolated from Ferula szowitsiana DC. have inhibitory activity against NO production in RAW264.7 mouse macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Of the 6 terpenoid coumarins tested, methyl galbanate significantly decreased NO production in LPS/IFN-γ-stimulated RAW264.7 cells. In the presence of methyl galbanate, LPS/IFN-γ-induced iNOS mRNA expression was significantly decreased to 52% of the level found with LPS/IFN-γ stimulation alone. Methyl galbanate slightly attenuated COX-2 mRNA expression. Using the RAW264.7-tsAM5NE co-culture system, we showed that methyl galbanate protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. Our finding suggests that methyl galbanate may be useful for developing a new drug against neurodegenerative diseases.


Assuntos
Cumarínicos/química , Cumarínicos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Terpenos/química , Terpenos/farmacologia , Animais , Western Blotting , Linhagem Celular , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Umbeliferonas/química , Umbeliferonas/farmacologia
17.
Int J Hematol ; 93(2): 237-242, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21264552

RESUMO

BCR-ABL kinase domain mutations were sequentially analyzed in a patient with chronic myeloid leukemia (CML) who exhibited repeated B-lymphoid blast crisis (CML-BC) during treatment with imatinib and dasatinib. We first identified five mutant BCR-ABL clones: Y253H, G250E, F311L, F317L and K294RGG, which was generated by two-nucleotide mutations and six-nucleotide insertion, at the third BC during the imatinib treatment, and retrospectively found that three of them (Y253H, G250E, K294RGG) were already present at the second BC. The in vitro analysis using K294RGG mutant BCR-ABL-expressing 32D cells revealed that K294RGG mutation was imatinib resistant but dasatinib sensitive. Consistent with the in vitro data, the clone with K294RGG mutation was eliminated by the dasatinib treatment in this patient. During the imatinib treatment, several mutant clones emerged and expanded, while additional mutations on the same allele were not acquired. However, after the dasatinib treatment, wild-type BCR-ABL clone disappeared and T315I or F317L mutation was acquired in G250E and Y253H mutant clones on the same allele without the emergence of each sole mutant clone. Cytogenetic and immunoglobulin heavy chain gene rearrangement analysis revealed that all mutant clones that appeared in this patient might be derived from the same CML clone.


Assuntos
Crise Blástica/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mieloide de Fase Crônica/patologia , Mutagênese Insercional , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/genética , Pirimidinas/uso terapêutico , Antineoplásicos/uso terapêutico , Benzamidas , Crise Blástica/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Mesilato de Imatinib , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/genética , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores
18.
Cell Signal ; 23(4): 666-72, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21130871

RESUMO

Extracellular signal-regulated kinases (ERKs) play important physiological roles including proliferation, differentiation and gene expression. ERK5 contains kinase domain that shares homology with ERK1/2 and the T-E-Y activation motif at amino-terminal half, whereas the extended carboxy-terminal half is unique. Because the physiological role of ERK5 in glial cells remains unclear, we examined the involvement of ERK5 in expression of neurotrophic factors and cytokines in rat C6 glioma cells, comparing it with ERK1/2. Basic fibroblast growth factor (bFGF) induced both ERK5 and ERK1/2 phosphorylation in a time- and concentration-dependent manner. Among the neurotrophic factors and cytokines, bFGF induced significant gene expression of glial cell-derived neurotrophic factor (GDNF). The GDNF gene expression and protein synthesis induced by bFGF were blocked by BIX02189 and PD98059 that selectively inhibit ERK5 and ERK1/2 signaling, respectively. The effect was also blocked by overexpression of a dominant-negative MEK5 mutant, indicating that GDNF expression induced by bFGF requires both ERK5 and ERK1/2. Because GDNF gene expression is regulated by various transcription factors, we examined the activity of these factors. We demonstrated that phosphorylation of cAMP-response element-binding protein at Ser 133 was induced by bFGF, which was blocked by BIX02189 and PD98059. Expression of c-fos, a major component of activator protein-1, and early growth response-1 was enhanced by bFGF, and expression of these genes was blocked by BIX02189, PD98059 and overexpression of dominant-negative MEK5. Taking these results together, bFGF promotes GDNF expression accompanied by the activation of ERK5, ERK1/2 and their downstream transcription factors in C6 glioma cells.


Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neuroglia/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ativação Enzimática , Glioma , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Elemento de Resposta Sérica
19.
Cell Biol Int ; 35(4): 325-34, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21029049

RESUMO

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.


Assuntos
Glândulas Suprarrenais/citologia , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular Tumoral/citologia , Células Cromafins/citologia , Animais , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Células Cultivadas , Células Cromafins/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Neurogênese , Neurônios/citologia
20.
Neurosci Lett ; 438(1): 42-7, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18455310

RESUMO

We recently established adrenal medullary cell line tsAM5D, which was immortalized by use of a temperature-sensitive mutant of the oncogene simian virus 40 large T-antigen. In the present study, when co-treated with glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), tsAM5D cells proliferated at the permissive temperature (33 degrees C) for the T-antigen expression and differentiated into neuron-like cells at the nonpermissive temperature (39 degrees C). Interestingly, in GDNF/CNTF-treated cultures, the addition of pan-specific transforming growth factor (TGF)-beta-neutralizing antibody did not affect the cell proliferation at 33 degrees C, but significantly reduced the survival of neuronally differentiated cells at 39 degrees C. Using real-time RT-PCR for analysis of GDNF/CNTF-treated cells, we found that the expression of mRNAs for TGF-beta1, TGF-beta2, and TGF-beta3 was up-regulated by the temperature shift. These results suggest that autocrine TGF-beta signaling is necessary for the survival of GDNF/CNTF-differentiated tsAM5D cells upon the temperature shift.


Assuntos
Medula Suprarrenal/crescimento & desenvolvimento , Medula Suprarrenal/metabolismo , Comunicação Autócrina/fisiologia , Células Cromafins/metabolismo , Neurônios/metabolismo , Fator de Crescimento Transformador beta/genética , Medula Suprarrenal/citologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Fator Neurotrófico Ciliar/metabolismo , Fator Neurotrófico Ciliar/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Temperatura Ambiente , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/genética , Regulação para Cima/genética
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