RESUMO
MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1 , p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.
Assuntos
Humanos , Apoptose/efeitos dos fármacos , Benzofuranos/administração & dosagem , Endófitos/química , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Xylariales/química , Proteínas Reguladoras de Apoptose/genética , Benzofuranos/isolamento & purificação , Proteínas de Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , /efeitos dos fármacos , /efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Citometria de Fluxo , Fatores de Transcrição Forkhead/efeitos dos fármacos , Cycadopsida , /efeitos dos fármacos , Células HeLa , Proteínas Nucleares/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , Fatores de Transcrição/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacosRESUMO
The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60 percent and reduced the migration to about 30 percent. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.