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1.
Drug Des Devel Ther ; 14: 1117-1125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214797

RESUMO

A method for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagle plasma by UPLC-MS/MS was developed and validated. After the plasma was extracted by acetonitrile precipitation, the analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) using acetonitrile-formic acid as the mobile phase in gradient mode. The analytes were monitored by multiple reaction monitoring (MRM) in electrospray negative ion mode. The mass transfer pairs were m/z 368.97→119.01 for parecoxib, m/z 312.89→118.02 for valdecoxib, and m/z 379.98→316.02 for celecoxib (internal standard, IS). The correlation coefficients of parecoxib and valdecoxib ranged from 5 to 4000 ng/mL were greater than 0.9998. The recovery of parecoxib and valdecoxib was greater than 82.54%. The inter- and intra-day precision RSD values were 1.36~3.65% and 2.28~5.91%, respectively. The accuracy of RE values were -1.38%~1.96%. Finally, the matrix effect (ME) and stability were also within acceptable criteria. This method had been successfully applied to the pharmacokinetics of parecoxib and valdecoxib in beagle plasma after injection of parecoxib (1.33 mg/kg, intramuscular injection).

2.
J Pharm Biomed Anal ; 177: 112850, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31499430

RESUMO

In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5-200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats.


Assuntos
Ftalazinas/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/sangue , Animais , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Masculino , Modelos Animais , Nitrilos/administração & dosagem , Nitrilos/sangue , Ftalazinas/administração & dosagem , Ftalazinas/sangue , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/sangue , Quinolinas/administração & dosagem , Quinolinas/sangue , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
3.
Drug Des Devel Ther ; 13: 3343-3355, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571835

RESUMO

Objective: To investigate the effects of Chinese herb Danzhi Xiaoyao pills on the pharmacokinetics of venlafaxine and its metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in beagles by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods: Six beagles (half male, half female) were chosen to test, being fasted before the experiment but having free access to drinking water 1 day before being fed drugs. After oral administration of venlafaxine hydrochloride tablets (10.28 mg/kg), the blood samples were collected in succession at different points in time. After 1-week washout period, Danzhi Xiaoyao pills (0.6g/kg) were given through oral administration to the six beagles every morning until the 7th day, venlafaxine hydrochloride tablets (10.28 mg/kg) were given after feeding Danzhi Xiaoyao pills (0.6g/kg) half an hour and blood samples were collected continuously at different points. All samples were analyzed by UPLC-MS/MS, and the main pharmacokinetic parameters of venlafaxine, ODV and NDV were computed by DAS 2.0. Results: The Cmax of the venlafaxine group (control group) and the combination group (experimental group) were (2267.26±252.89) ng/mL and (1542.64±190.73) ng/mL, respectively. The AUC(0-∞) of the two groups were (13,934.79±3609.23) ng·h/mL and (8001.91±2167.58) ng·h/mL, respectively. The ODV Cmax of the two groups were (2253.80±215.81) ng/mL and (2721.37±118.20) ng/mL, and AUC(0-∞) were (13,974.99±2784.04) ng·h/mL and (17,539.44±1894.29) ng·h/mL, respectively. The NDV Cmax of the two groups were (50.98±5.76) ng/mL and (58.74±12.33) ng/mL, and AUC(0-∞) were (179.26±34.94) ng·h/mL and (220.68±51.41) ng·h/mL, respectively. After administration of Danzhi Xiaoyao pills, the Cmax and AUC(0-∞) of venlafaxine decreased significantly, indicating that the plasma exposure of venlafaxine decreased. The increase of Cmax and AUC(0-∞) of ODV and NDV indicated a rise in plasma exposure. Conclusion: Danzhi Xiaoyao pills can accelerate the metabolism of venlafaxine in beagles. In clinical, when venlafaxine was co-administrated with Danzhi Xiaoyao pills, dose adjustment of venlafaxine should be taken into account.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Cloridrato de Venlafaxina/farmacocinética , Animais , Área Sob a Curva , Cromatografia Líquida , Cicloexanóis/sangue , Cicloexanóis/farmacocinética , Succinato de Desvenlafaxina/sangue , Cães , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Masculino , Comprimidos , Espectrometria de Massas em Tandem
4.
Sci Rep ; 5: 12230, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26178819

RESUMO

Pancreatic cancer is one of the more common cancers with a poor prognosis. Some varieties of cancer are related to virus infection. As a virus-induced protein, APOBEC3G (A3G) presents extensive anti-virus ability, but the role of A3G in pancreatic cancer was previously unknown. The expression of A3G in pancreatic cancer was examined using TaqMan real-time qPCR, immunohistochemical and immunofluorescent staining. Subsequently, the role of A3G in pancreatic cancer was evaluated in vivo using the tumor xenograft model. Anoikis was detected by colony formation assay and flow cytometry in vitro. The Akt kinase activity and target protein PTEN were examined by co-immunoprecipitation and immunoblot. The virus-induced protein A3G was significantly up-regulated in pancreatic cancer, and the up-regulation of A3G promoted xenograft tumor formation. A3G inactivated PTEN by binding to the C2 tensin-type and PDZ domains, thereby inducing anoikis resistance through Akt activation. Our results demonstrate that the up-regulation of A3G in pancreatic cancer cells induces anoikis resistance, and they provide novel insight into the mechanism by which A3G affects the malignant behavior of pancreatic cancer cells.


Assuntos
Anoikis/fisiologia , Citidina Desaminase/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Desaminase APOBEC-3G , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Camundongos , Camundongos Nus , PTEN Fosfo-Hidrolase/fisiologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Regulação para Cima
5.
J Chem Inf Model ; 54(2): 462-9, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24432790

RESUMO

Water is the natural medium of molecules in the cell and plays an important role in protein structure, function and interaction with small molecule ligands. However, the widely used molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method for binding energy calculation does not explicitly take account of water molecules that mediate key protein-ligand interactions. We have developed a protocol to include water molecules that mediate ligand-protein interactions as part of the protein structure in calculation of MM/PBSA binding energies (a method we refer to as water-MM/PBSA) for a series of JNK3 kinase inhibitors. Improved correlation between water-MM/PBSA binding energies and experimental IC50 values was obtained compared to that obtained from classical MM/PBSA binding energy. This improved correlation was further validated using sets of neuraminidase and avidin inhibitors. The observed improvement, however, appears to be limited to systems in which there are water-mediated ligand-protein hydrogen bond interactions. We conclude that the water-MM/PBSA method performs better than classical MM/PBSA in predicting binding affinities when water molecules play a direct role in mediating ligand-protein hydrogen bond interactions.


Assuntos
Proteína Quinase 10 Ativada por Mitógeno/química , Simulação de Dinâmica Molecular , Água/química , Ligantes , Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Termodinâmica
7.
World J Gastroenterol ; 19(45): 8219-26, 2013 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-24363512

RESUMO

Helicobacter pylori (H. pylori) infection might initiate and contribute to the progression of lymphoma from gastric mucosa-associated lymphoid tissue (MALT). Increasing evidence shows that eradication of H. pylori with antibiotic therapy can lead to regression of gastric MALT lymphoma and can result in a 10-year sustained remission. The eradication of H. pylori is the standard care for patients with gastric MALT lymphoma. Cytotoxin-associated gene A (CagA) protein, one of the most extensively studied H. pylori virulence factors, is strongly associated with the gastric MALT lymphoma. CagA possesses polymorphisms according to its C-terminal structure and displays different functions among areas and races. After being translocated into B lymphocytes via type IV secretion system, CagA deregulates intracellular signaling pathways in both tyrosine phosphorylation-dependent and -independent manners and/or some other pathways, and thereby promotes lymphomagenesis. A variety of proteins including p53 and protein tyrosine phosphatases-2 are involved in the malignant transformation induced by CagA. Mucosal inflammation is the foundational mechanism underlying the occurrence and development of gastric MALT lymphoma.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Linfoma de Zona Marginal Tipo Células B/microbiologia , Neoplasias Gástricas/microbiologia , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Transformação Celular Neoplásica/metabolismo , Genótipo , Infecções por Helicobacter/complicações , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/metabolismo , Fenótipo , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Fatores de Virulência/genética
8.
Arch Environ Contam Toxicol ; 65(3): 357-67, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23712771

RESUMO

Endocrine-disrupting chemicals have attracted great concern. As major metabolites of polychlorinated biphenyls (PCBs), hydroxylated polychlorinated biphenyls (HO-PCBs) may disrupt estrogen hormone status because of their structural similarity to estrogen endogenous compounds. However, interactions between HO-PCBs and estrogen receptors (ERs) are not fully understood. In the present work, a molecular modeling study combining molecular docking, molecular dynamics simulations, and binding free energy calculations was performed to characterize the interactions of three HO-PCBs (4'-HO-PCB50, 2'-HO-PCB65, and 4'-HO-PCB69) having much different estrogenic activities with ERß. Docking results showed that binding between ligands and ERß was stabilized by hydrogen bond and hydrophobic interactions. The binding free energies of three ligands with ERß were calculated, and further binding free energy decomposition analysis indicated that the dominating driving force of the binding between the ligands and ERß was the van der Waals interaction. Some key residues, such as Leu298, Phe356, Gly472, His475, and Leu476, played important roles in ligand-receptor interactions by forming hydrophobic and hydrogen bond interactions with ligands. The results may be beneficial to increase understanding of the interactions between HO-PCBs and ERß.


Assuntos
Disruptores Endócrinos/química , Poluentes Ambientais/química , Estradiol/metabolismo , Receptor beta de Estrogênio/química , Modelos Moleculares , Bifenilos Policlorados/química , Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/farmacologia , Poluentes Ambientais/metabolismo , Poluentes Ambientais/farmacologia , Receptor beta de Estrogênio/metabolismo , Humanos , Ligações de Hidrogênio , Hidroxilação , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Bifenilos Policlorados/metabolismo , Bifenilos Policlorados/farmacologia
9.
J Zhejiang Univ Sci B ; 13(11): 884-93, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23125081

RESUMO

BACKGROUND AND OBJECTIVE: ST13, is the gene encoding the HSP70 interacting protein (HIP). Previous research has shown that ST13 mRNA and protein levels are down-regulated in colorectal cancer (CRC) tissues compared with adjacent normal tissues. This study aims at the role of ST13 in the proliferation and migration of CRC cells. METHODS: The transcript level of ST13 in different CRC cell lines was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). ST13-overexpressed and ST13-knockdown CRC cells were constructed respectively by lentiviral transduction, followed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, plate colony formation, cell-cycle analysis, and migration assays to evaluate the influence of ST13 on proliferation and migration in vitro. Moreover, a mouse xenograft study was performed to test in vivo tumorigenicity of ST13-knockdown CRC cells. RESULTS: Lentivirus-mediated overexpression of ST13 in CRC cells inhibited cell proliferation, colony formation, and cell migration in vitro. In contrast, down-regulation of ST13 by lentiviral-based short hairpin RNA (shRNA) interference in CRC cells significantly increased cell proliferation and cloning efficiency in vitro. In addition, down-regulation of ST13 expression significantly increased the tumorigenicity of CRC cells in vivo. CONCLUSIONS: ST13 gene is a proliferation regulator that inhibits tumor growth in CRC and may affect cell migration.


Assuntos
Proteínas de Transporte/genética , Movimento Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas Supressoras de Tumor/genética , Animais , Western Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células HT29 , Humanos , Camundongos , Camundongos Nus , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/metabolismo
10.
J Zhejiang Univ Sci B ; 13(3): 159-67, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22374607

RESUMO

OBJECTIVE: Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. METHODS: Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. RESULTS: Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin ß-4-like protein 3, and tight junction-associated protein 1. CONCLUSIONS: Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.


Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cultura de Células , Separação Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Análise Serial de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas , Microambiente Tumoral/fisiologia
11.
J Zhejiang Univ Sci B ; 13(1): 11-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22205615

RESUMO

OBJECTIVE: We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes. METHODS: We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity. RESULTS: A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2. CONCLUSIONS: These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Feminino , Células HT29 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Nus , Proteína Homeobox Nanog , Transplante de Neoplasias , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Projetos Piloto , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
12.
Mol Biol Rep ; 39(5): 5459-64, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22207169

RESUMO

Anti-tumor activity and mechanism of matrine is evaluated and investigated. MTT assay showed that the matrine was able to inhibit gastric cancer cell line MNK45 in a dose-dependent manner. The concentration required for 50% inhibition (IC50) was found to be 540 µg/ml. This anti-tumor function was achieved through modulation of the NF-κB, XIAP, CIAP, and p-ERK proteins expression in cell line MNK45. By western blot analysis, we found that expression of NF-κB, XIAP, CIAP, and p-ERK proteins in cell line MNK45 would vary with varying concentration of matrine. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyzes are need. These results overall indicate that matrine can be used as an effective anti-tumor agent in therapy of gastric cancer.


Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Quinolizinas/farmacologia , Neoplasias Gástricas/patologia , Alcaloides/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Quinolizinas/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
13.
Radiother Oncol ; 101(1): 66-72, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21641068

RESUMO

BACKGROUND: Tumor radioresistance often leads to treatment failure during radiotherapy. New strategies like developing radiosensitizer are clinically important. Intervention with DNA double-strand break repair is an effective way to modulate tumor cell radiosensitivity. This study focused on the mutant Artemis fragment-enhanced radiosensitivity of human cervical cancer cells. MATERIAL AND METHODS: We constructed two pEGFP-C1-based eukaryotic expression vectors encoding full-length and the mutant Artemis fragment (D37N-413aa), respectively. HeLa cells were stably transfected with these plasmids or vector. Cell survival was measured by the clonogenic assay. The γH2AX foci assay was used to monitor DNA repair after irradiation. Co-immunoprecipitation and Western blot analysis were performed to study protein interaction and phosphorylation of Artemis. RESULTS: Expression of the mutant Artemis fragment (D37N-413aa) delayed DNA DSB rejoining after irradiation, thereby enhanced radiosensitivity of HeLa cell. Further experiments indicate that this mutant Artemis fragment bind to DNA-PKcs and ATM, inhibited phosphorylation of endogenous Artemis, the key molecule for DNA repair and cell radiosensitivity. CONCLUSIONS: The dominant negative mutant Artemis fragment (D37N-413aa) enhanced tumor cell radiosensitivity through blocking activity of endogenous Artemis and DNA repair. It is the first time to modulate tumor cell radiosensitivity via targeting Artemis. This novel mechanism of radiosensitivity strongly suggests the potential role of Artemis in cancer therapy.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteínas Mutantes Quiméricas/farmacologia , Proteínas Nucleares/genética , Tolerância a Radiação/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Endonucleases , Feminino , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Proteínas Mutantes Quiméricas/genética , Fosforilação , Tolerância a Radiação/genética , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/farmacologia
14.
Int J Nanomedicine ; 6: 3207-18, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22238509

RESUMO

PURPOSE: The failure of cancer treatments is partly due to the enrichment of cancer stem-like cells (CSLCs) that are resistant to conventional chemotherapy. A novel micelle formulation of oxaliplatin (OXA) encapsulated in chitosan vesicle was developed. The authors postulate that micelle encapsulation of OXA would eliminate both CSLCs and bulk cancer cells in colorectal cancer (CRC). EXPERIMENTAL DESIGN: In this study, using stearic acid-g-chitosan oligosaccharide (CSO-SA) polymeric micelles as a drug-delivery system, OXA-loaded CSO-SA micelles (CSO-SA/OXA) were prepared. Intracellular uptake of CSO-SA/OXA micelles was assessed by confocal microscope. The effects of free OXA, the empty carrier, and CSO-SA/OXA micelles were tested using human CRC cell lines in vitro and in vivo. RESULTS: The micelles showed excellent internalization ability that increased OXA accumulation both in CRC cells and tissues. Furthermore, CSO-SA/OXA micelles could either increase the cytotoxicity of OXA against the bulk cancer cells or reverse chemoresistance of CSLC subpopulations in vitro. Intravenous administration of CSO-SA/OXA micelles effectively suppressed the tumor growth and reduced CD133+/CD24+ cell (putative CRC CSLC markers) compared with free OXA treatment, which caused CSLC enrichment in xenograft tumors (P < 0.05). CONCLUSION: The results of this study indicate that CSO-SA micelle as a drug-delivery carrier is effective for eradicating CSLCs and may act as a new option for CRC therapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Portadores de Fármacos/farmacologia , Micelas , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Análise de Variância , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Feminino , Células HT29 , Humanos , Camundongos , Camundongos Nus , Microscopia Confocal , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacocinética , Oxaliplatina , Esferoides Celulares , Ácidos Esteáricos/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Biomed Environ Sci ; 21(4): 339-44, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18837299

RESUMO

OBJECTIVE: To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells. METHODS: D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair. RESULTS: D6-R cells showed higher and broader initial shoulder (D0=2.08 Gy, Dq=1.64 Gy, N=2.20) than the parent D6 cells (D0=1.84 Gy, Dq=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF2 (63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of cells in G1 (44.1% vs 57.2%) and G2-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells. CONCLUSIONS: D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/patologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Citometria de Fluxo , Humanos , Microscopia de Fluorescência
16.
Proc Natl Acad Sci U S A ; 105(8): 3041-6, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18287029

RESUMO

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.


Assuntos
Apoptose/efeitos dos fármacos , Indóis/química , Melanoma/tratamento farmacológico , Modelos Moleculares , Oncogenes/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/química , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Escherichia coli , Humanos , Indóis/uso terapêutico , Concentração Inibidora 50 , Camundongos , Camundongos SCID , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/uso terapêutico
17.
World J Gastroenterol ; 14(4): 554-62, 2008 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-18203287

RESUMO

AIM: To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells. METHODS: Human AGS cells transfected with cagA and blank vector were treated with specific mitogen-activated protein kinase kinase (MEK) inhibitor. Total cell proteins were combined by strong anion exchange (SAX2) and weak cation exchange (CM10) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information. RESULTS: When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching. CONCLUSION: Biomarkers with m/z 4229, 8162 and 9084 are ERK1/2 phosphorylation dependent, and therefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.


Assuntos
Adenocarcinoma/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Análise Serial de Proteínas , Neoplasias Gástricas/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção
18.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 36(5): 465-9, 2007 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-17924465

RESUMO

OBJECTIVE: To screen metronidazole (MTZ) -resistance associated gene fragments of Helicobacter pylori (H.pylori) by suppression subtractive hybridization(SSH). METHODS: The suppression subtractive hybridization (SSH) was used to screen the different DNA fragments between MTZ-resistant and -susceptible clinical strains of H.pylori. The resistant strains specific gene fragments were obtained by SSH and identified by dot blotting. RESULT: Among the 120 subtractive colonies which were randomly chosen, 37 DNA fragments were different (>or=2 times) in DNA-copy numbers between resistant and susceptible strains and 17 of them were significantly different (>or=3 times). These 17 DNA fragments were sequenced subsequently. Ten of them were new sequences and the other 7 were duplicated sequences. These sequences represented respectively: depeptide ABC transporter periplasmic dipeptide-binding protein (dppA), permease protein (dppB), et al. CONCLUSION: Gene fragments specific to MTZ-resistant H. pylori strains were obtained by SSH and these genes may associated with MTZ-resistance of H.pylori.


Assuntos
Farmacorresistência Bacteriana/genética , Helicobacter pylori/genética , Metronidazol/farmacologia , Hibridização de Ácido Nucleico/métodos , Anti-Infecciosos/farmacologia , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Helicobacter pylori/efeitos dos fármacos , Humanos , Análise de Sequência de DNA
19.
World J Gastroenterol ; 13(12): 1847-50, 2007 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-17465479

RESUMO

AIM: To screen for metronidazole (MTZ)-resistance associated gene fragments of H pylori by suppression subtractive hybridization (SSH). METHODS: Five MTZ-resistant (tester, T) and 1 MTZ-susceptible (driver, D) clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS: Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies (> or = 2 times) in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies (> or = 3 times). Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein (dppA), permease protein (dppB), ribosomal protein S4 (rps4), ribonuclease III (rnc), protease (pqqE), diaminopimelate epimerase (dapF), acetatekinase (ackA), H pylori plasmid pHP51 and H pylori gene 1334. CONCLUSION: Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant H pylori.


Assuntos
Anti-Infecciosos/farmacologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Helicobacter pylori/genética , Metronidazol/farmacologia , Hibridização de Ácido Nucleico/métodos , Fragmentação do DNA , Dosagem de Genes , Testes Genéticos , Infecções por Helicobacter/tratamento farmacológico , Humanos , Immunoblotting , Análise de Sequência de DNA
20.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 35(1): 91-8, 2006 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-16470928

RESUMO

OBJECTIVE: To explore the relationship between mutant p53 and multidrug resistance in gastric cancer. METHODS: Mutant p53 (mp53) and mp53+sv40Tag were transferred to gastric cancer cell line SGC-7901. The MDR-1 mRNA was examined using RT-PCR, and the difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 was compared with those with mp53+sv40Tag and controls by MTT method. RESULTS: SGC-7901 cells with mutant p53 showed higher MDR-1 mRNA than that of other two groups. SGC-7901 cells with mutant p53 showed higher chemotherapeutic sensitivity to 5-Fu than that with mp53+sv40Tag and control (P<0.05), but no difference between those with mp53+sv40Tag and control (P>0.05). SGC-7901 cells with mutant p53 and those with mp53+sv40Tag showed higher chemotherapeutic sensitivity to ADM than control (P<0.05), but no difference between those with mp53 and with mp53+sv40Tag (P>0.05). There was no difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 compared with those with mp53+sv40Tag and control to CDDP (P>0.05). CONCLUSION: Mutante p53 genes relates to multidrug resistance of gastric cancer.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Resistência a Múltiplos Medicamentos/genética , Mutação , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenocarcinoma/genética , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais Cultivadas
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