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1.
ACS Appl Mater Interfaces ; 11(11): 10578-10588, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30802029

RESUMO

Hepatocellular carcinoma (HCC) poses a great threat to human health. The elegant combination of gene therapy and chemotherapy by nanocarriers has been repeatedly highlighted to realize enhanced therapeutic efficacy relative to monotreatment. However, the leading strategy to achieve the efficient codelivery of the gene and drug remains the electrostatic condensation with the nucleic acid and the hydrophobic encapsulation of drug molecules by the nanocarriers, which suffers substantially from premature drug leakage during circulation and severe off-target-associated side effects. To address these issues, we reported in this study the codelivery of liver-specific miRNA-122 and anti-cancer drug 5-fluorouracil (5-Fu) using a macromolecular prodrug approach, that is, electrostatic condensation with miRNA-122 using galactosylated-chitosan-5-fluorouracil (GC-FU). The delivery efficacy was evaluated comprehensively in vitro and in vivo. Specifically, the biocompatibility of GC-FU/miR-122 nanoparticles (NPs) was assessed by hemolysis activity analysis, BSA adsorption test, and cell viability assay in both normal liver cells (L02 cells) and endothelial cells. The resulting codelivery systems showed enhanced blood and salt stability, efficient proliferation inhibition of HCC cells, and further induction apoptosis of HCC cells, as well as downregulated expression of ADAM17 and Bcl-2. The strategy developed herein is thus a highly promising platform for an effective codelivery of miRNA-122 and 5-Fu with facile fabrication and great potential for the clinical translation toward HCC synergistic therapy.


Assuntos
Materiais Biocompatíveis/química , MicroRNAs/metabolismo , Pró-Fármacos/química , Proteína ADAM17/metabolismo , Animais , Apoptose/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular , Quitosana/química , Regulação para Baixo/efeitos dos fármacos , Portadores de Fármacos/química , Sinergismo Farmacológico , Fluoruracila/química , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Hemólise/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/química , Nanopartículas/química , Nanopartículas/toxicidade , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico
2.
World J Gastroenterol ; 17(32): 3700-8, 2011 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-21990951

RESUMO

AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by polymerase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfected into these two cell lines in vitro. mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migration and invasion of these cells were examined separately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing and Transwell chambers assay. RESULTS: The siRNA directed against PIK3CA effectively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA resulted in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells. CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.


Assuntos
Invasividade Neoplásica/fisiopatologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
3.
Acta Pharmacol Sin ; 29(10): 1233-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18817629

RESUMO

AIM: To investigate the effect of diallyl disulfide (DADS), a component of garlic, on apoptosis in human mammary cancer cell line (MCF-7) and its mechanisms. METHODS: Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays. Morphology of apoptotic cells was detected by acridine orange and ethidium bromide staining. Apoptotic cells stained with propidium iodide were examined using flow cytometry. Protein levels were detected by Western blot analysis. RESULTS: DADS inhibited the proliferation of MCF-7 cells and induced the apoptotic ratio to increase rapidly. Cleavage of the caspase-3 and caspase-3 substrate poly(ADP-ribose) polymerase was observed in MCF-7 cells after 24 h of treatment with DADS. When the MCF-7 cells were treated with 200 micromol x L DADS, the stress-activated protein kinase extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase, was inhibited after 6 h; c-Jun N-terminal kinase (JNK), that is stress-activated protein kinase (SAPK), and p38 mitogen-activated protein kinase were activated after 6 h. CONCLUSION: These results suggest that DADS both inhibits the proliferation of MCF-7 cells and induces apoptosis of MCF-7 cells. The mechanisms may include the inhibition of ERK and the activation of the SAPK/JNK and p38 pathways.


Assuntos
Compostos Alílicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Sulfetos/farmacologia , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Clin Exp Pharmacol Physiol ; 34(5-6): 450-6, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17439414

RESUMO

1. The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors. 2. Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase-polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry. 3. The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT. 4. Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.


Assuntos
Antineoplásicos/farmacologia , Inativação Gênica , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/genética , Proteína bcl-X/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Proteína bcl-X/metabolismo
5.
Acta Biochim Biophys Sin (Shanghai) ; 38(10): 704-10, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17033717

RESUMO

Changes in drug sensitivity in Bcl-XL small interfering RNA (siRNA) transfected Hepg2 hepatocellular carcinoma cells were investigated in this study. Bcl-XL siRNA and negative siRNA expression vector were constructed and stably transfected into Hepg2 cells. Reverse transcription (RT)-PCR, western blot and immunofluorescence were used to detect the target gene expression at mRNA and protein levels. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and hydroxycamptothecin (HCPT) were evaluated with MTT. The Bcl-XL mRNA and protein expression levels in Bcl-XL siRNA transfectants were reduced compared with negative siRNA transfectants or mock cells. MTT results showed that Bcl-XL siRNA transfected cells have a higher cell inhibition rate than negative vector transfected cells or untreated cells after treatment with 13, 130, 1300 and 13,000 mg/L of 5-FU. Bcl-XL siRNA transfected cells also showed increased drug-sensitivity compared with negative vector transfected cells or untreated cells after treatment with 0.18, 0.36, 0.72 and 1.44 mg/L HCPT. Flow cytometry (FCM) results demonstrated that the sub-G1 population increased in the Bcl-XL siRNA group, compared with the negative siRNA group and untreated control group, after the addition of 5-FU (1300 mg/L) and HCPT (0.72 mg/L). siRNA targeting Bcl-XL gene can specifically down-regulate Bcl-XL expression in Hepg2 cells, and can increase spontaneous cell apoptosis and sensitize cells to 5-FU or HCPT.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Fluoruracila/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , RNA Interferente Pequeno/fisiologia , Proteína bcl-X/genética , Antimetabólitos Antineoplásicos/administração & dosagem , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Fluoruracila/administração & dosagem , Humanos , RNA Mensageiro/metabolismo , Proteína bcl-X/biossíntese
6.
J Drug Target ; 14(1): 21-6, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16603448

RESUMO

To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/análogos & derivados , Fluoruracila/farmacologia , Genes bcl-2/genética , RNA Interferente Pequeno/farmacologia , Antimetabólitos Antineoplásicos/administração & dosagem , Western Blotting , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Caspase 3 , Caspases/biossíntese , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Citometria de Fluxo , Fluoruracila/administração & dosagem , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Tiazóis , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genética
7.
Acta Biochim Biophys Sin (Shanghai) ; 37(8): 555-60, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16077903

RESUMO

To investigate the inhibitory effect of the Bcl-XL small interfering RNA (siRNA) on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein (GFP) siRNA was constructed and transfected into MGC-803 cells, together with GFP expression vector pTrace SV40. GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange (AO) and flow cytometry. Results were as follows: (1) 48 h after GFP expression vector and GFP siRNA co-transfection, the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group, according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. (2) Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis (21.17%+/-1.26% vs. 1.19%+/-0.18% and 1.56%+/-0.15% respectively, P < 0.05 vs. negative siRNA or untreated control). siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , RNA Interferente Pequeno/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Adenocarcinoma/genética , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Marcação de Genes/métodos , Terapia Genética/métodos , Humanos , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias Gástricas/genética , Transfecção/métodos
8.
Zhonghua Xue Ye Xue Za Zhi ; 25(5): 273-6, 2004 May.
Artigo em Chinês | MEDLINE | ID: mdl-15182534

RESUMO

OBJECTIVE: To explore the molecular mechanisms of G(2)/M checkpoint initiated by diallyl disulfide (DADS) in HL-60 cells. METHODS: Cell viability was determined by MTT assay. Cell cycle was assayed by flow cytometry. The expression of phospho-p38, Cdc25B and Cdc2, and p38 mRNA were measured by Western blotting and RT-PCR, respectively. RESULTS: After treatment with DADS at 5 - 160 micro mol/L for 0 - 72 h, the growth of HL-60 cells were suppressed in a concentration-dependent manner and the inhibitory effect of DADS (20 micro mol/L) was similar to that of ATRA (10 nmol/L) (P > 0.05). Incubation of HL-60 cells with DADS (20 micro mol/L) for 12 h could activate G(2)/M checkpoint and increase the expression of phospho-p38 MAPK, followed by the expression of phospho-Cdc25B and phospho-Cdc2 (P < 0.05). SB202190, a specific inhibitor of p38 MAPK, markedly blocked the phosphorylation of p38 MAPK, Cdc25B and Cdc2 (P < 0.05). CONCLUSION: DADS could induce the G(2)/M arrest in HL-60 cells which may be involved in the activation of p38 MAP kinase.


Assuntos
Compostos Alílicos/farmacologia , Divisão Celular/efeitos dos fármacos , Dissulfetos/farmacologia , Fase G2/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 20(1): 34-7, 2004 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-15182617

RESUMO

AIM: To screen the active peptides which can suppress adhesion of monocytes to endothelial cells(ECs) from a random peptide phage library. METHODS: The ECs injured by oxidized low density lipoprotein (ox-LDL) 100 mg/L was used as target cells and polypeptides specifically binding to the injured ECs were isolated from the phage-displayed peptide library. The positive clones were characterized by ELISA, and the amino acid sequences of the peptides were deduced by DNA sequencing. Cell counting was used to evaluate the adhesion rate between ECs and monocytes. RESULTS: Six positive clones specifically binding to injured EC were isolated. Four of them contained a repeated sequence of leucine-leucine. Among the four clones, two had an identical sequence, which could reduce the adhesion rate of monocytes to ECs by 17%. CONCLUSION: One peptide which can suppress adhesion of monocytes to ECs is isolated from a random peptide phage library.


Assuntos
Adesão Celular/efeitos dos fármacos , Biblioteca de Peptídeos , Peptídeos/farmacologia , Sequência de Aminoácidos , Células Endoteliais/fisiologia , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Análise de Sequência de DNA
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