RESUMO
<p><b>OBJECTIVE</b>To investigate the mechanism of norcantharidin (NCTD)-induced SMMC-7721 hepatoma cell apoptosis.</p><p><b>METHODS</b>SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin V/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression.</p><p><b>RESULTS</b>NCTD inhibited SMMC-7721 cell growth in a time- and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD.</p><p><b>CONCLUSIONS</b>NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.</p>
Assuntos
Humanos , Apoptose , Fator de Indução de Apoptose , Metabolismo , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes , Farmacologia , Caspase 3 , Metabolismo , Linhagem Celular Tumoral , Citocromos c , Metabolismo , Citometria de Fluxo , Neoplasias Hepáticas , Metabolismo , Patologia , Potenciais da Membrana , Mitocôndrias , Metabolismo , Proteína X Associada a bcl-2 , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To approach the associated mechanism by which alpha-synuclein (alpha-Syn) might regulate the metabolism of dopamine.</p><p><b>METHODS</b>A DNA fragment, located at -495 to +25 of the human tyrosine hydroxylase (TH) gene, was amplified by PCR and inserted into the pGL(3)-Basic luciferase reporter vector. The recombinant plasmid pGL(3)-THprom was transfected into a dopaminergic cell line MES23.5 or a alpha-Syn over-expressed MES23.5 (named MES23.5/halpha-Syn(+)). The promoter activity was detected by the Dual Luciferase Assay System.</p><p><b>RESULTS</b>The luciferase activities in the MES23.5 cells transfected with pGL(3)-Basic, pGL(3)-THprom, and pGL(3)-Control vectors were 5.60+/-0.67, 26.80+/-4.11, and 32.90+/-4.75, respectively. On the other hand, the luciferase activity of pGL(3)-THprom in the MES23.5 (26.80+/-4.11) was significantly higher than that in the MES23.5/halpha-Syn(+) (14.40+/-0.61) (P<0.01).</p><p><b>CONCLUSION</b>These results indicate that the - 495 to +25 region in the TH gene possesses promoter activity for controlling the gene expression, and that alpha-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.</p>
Assuntos
Animais , Camundongos , Ratos , Linhagem Celular Tumoral , Dopamina , Regulação para Baixo , Genética , Regulação Enzimológica da Expressão Gênica , Genética , Genes Reporter , Genética , Vetores Genéticos , Genética , Hibridomas , Luciferases , Genética , Neurônios , Metabolismo , Doença de Parkinson , Genética , Metabolismo , Regiões Promotoras Genéticas , Genética , Elementos Reguladores de Transcrição , Genética , Substância Negra , Metabolismo , Transfecção , Tirosina 3-Mono-Oxigenase , Genética , alfa-Sinucleína , GenéticaRESUMO
<p><b>OBJECTIVE</b>To construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.</p><p><b>METHODS</b>Eukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.</p><p><b>RESULTS</b>We found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.</p><p><b>CONCLUSION</b>The apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.</p>