RESUMO
In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression.
Assuntos
Chalcona/análogos & derivados , Células Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Quinonas/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor Von Hippel-Lindau/biossíntese , Carthamus tinctorius/química , Hipóxia Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Chalcona/isolamento & purificação , Chalcona/farmacologia , Células Endoteliais/citologia , Flores/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Plantas Medicinais/química , Quinonas/isolamento & purificação , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Veias Umbilicais/citologia , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
AIM: To develop a cell-based model by stable transfection of SH-SY5Y with mutant A53T human alpha-synuclein, recapitulating neurotoxicity of alpha -synuclein overexpression. METHODS: The overexpression of mutant alpha -synuclein was analyzed by Western blotting, immunocytochemistry, and RT-PCR. Cell viability was processed when treated with different concentrations of 1-methyl-4-phenylpyridinium (MPP+) and exogenous dopamine (DA) for 24, 48, and 72 h by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Early apoptosis and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide double staining, respectively. DNA was isolated and applied to agarose gel for electrophoresis; the typical DNA "ladder"represented severe apoptosis. We also used this model to screen 99 compounds with therapeutic potential by MTT assay. RESULTS: One of the stably-transfected clones overexpressed exogenous genes on both the protein level and the transcriptive level. Significant differences in cytotoxicity were found between the pcDNA3.1(+) group and the pcDNA3.1(+)-hm alpha-synuclein group in the presence of the same concentration of MPP+ and DA within the same incubation time. The level of either early apoptosis or late apoptosis/necrosis was remarkably increased in transfected cells compared with the control after treatment with 100 micromol/L MPP+ for 24 h. In addition, the presence of the typical DNA "ladder" was observed in the pcDNA3.1(+)-hm alpha-synuclein group when treated with 200 micromol/L MPP+ for 48 h. After the screening experiment, 12 of the 99 compounds were found to decrease DA-induced cytotoxicity on cell viability. CONCLUSION: We established a cell-based model which is useful for studying the function of alpha-synuclein and screening compounds with therapeutic potential. In addition, it was identified that cells overexpressing A53T mutant alpha-synuclein were significantly vulnerable against MPP+ or dopamine exposures.