RESUMO
BACKGROUND: Our previous studies demonstrated that p53-induced gene 11 (PIG11) was involved in arsenic trioxide (As(2)O(3))-induced apoptosis in human gastric cancer MGC-803 cells. Here, we studied further PIG11 expression in human hepatocellular carcinoma (HCC) tissues and cell lines and compared the sensitivity to As(2)O(3)-induced cell apoptosis in HepG2 and L-02 cells. METHODS: PIG11 expression in human normal liver tissues, HCC tissues, and cell lines was determined by immunohistochemistry and immunocytochemistry methods, using an anti-human PIG11 antibody. Cell viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diplenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytometry. Reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to analyze PIG11 mRNA and protein expression in cells. Protein intensity was calculated by comparison with the intensity of beta-actin, using densitometry. PIG11 was knocked down using small interfering RNA (siRNA). RESULTS: We found that PIG11 expression was significantly downregulated in HCC tissue and the cell lines (Bel-7402, SMMC-7721, HepG2 cells). Further, HepG2 cells were more sensitive to As(2)O(3)-induced apoptosis than L-02 cells. The expression of PIG11 mRNA and protein was upregulated to a greater extent in HepG2 than in L-02 cells. In the presence of actinomycin D or cycloheximide, the amount of PIG11 protein expression did not increase. Likewise, the inhibition of PIG11 by siRNA decreased As(2)O(3)-induced PIG11 protein expression by more than 85% and partially prevented As(2)O(3)-induced apoptosis in both HepG2 and L-02 cells. CONCLUSION: The above results demonstrated that the PIG11 gene may be involved in As(2)O(3)-induced apoptosis in HepG2 cells and suggested that the adaptive response of PIG11 expression is one of the important factors in enhancing cell sensitivity to As(2)O(3)-induced apoptosis.
Assuntos
Apoptose/genética , Arsenicais/farmacologia , Carcinoma Hepatocelular/genética , Genes Supressores de Tumor/fisiologia , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Óxidos/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Criança , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação para Baixo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
PIG11 (p53-induced protein 11), one of early transcriptional targets of tumor suppressor p53, was up-regulated in the induction of apoptosis or cell growth inhibition by multiple chemopreventive agents. However, its biological role remains unclear. Here, we expressed His(6)-tagged PIG11 protein in Escherichia coli and demonstrated the recombinant His(6)-tagged PIG11 protein could bind to supercoiled and relaxed closed circular plasmid DNA or linear DNA with different length using gel retardation assays in vitro. The interaction between DNA and PIG11 protein was sequence-independent and related to charge effect. The reducing thiol group in PIG11 protein was involved in the binding activity of PIG11 to DNA. Furthermore, the images of atomic force microscopy directly confirmed the binding of DNA and PIG11 protein and showed the PIG11-DNA complex formed a beads-on-a-string appearance in which PIG11 protein associated with DNA as polymer. These findings suggest that PIG11 protein may play an important role by interaction with other biological molecules in the regulation of apoptosis and provided us a novel angel of view to explore the possible function of PIG11 in vivo.
