| ObjectiveOsteosarcoma is the most common solid malignant diseases of childhood; however, to the knowledge to date, the cause of osteosarcoma has remained mostly unknown. Genetic alterations of genes that are specific for osteosarcoma have not been identified. Genetic alternations in the status of DNA methylation, known as epigenetic alterations, are the most common molecular alterations in human neoplasia. Aberrant methylation in the promoter region of tumor-related genes is associated closely with epigenetically mediated gene silencing, which is a common feature in human tumors. APAF-1 gene, located at chromosome locus 12q23, is a key factor in the mitochondrial apoptotic pathway downstream of p53, and is a potential tumor suppressor gene. APAF-1 gene methylation is found in various types of cancer and tumor cell lines. Previously, we found a DNA methylation of APAF-1 gene in osteosarcoma cell line Hs888T, but the role of this pathway in regulating apoptosis in this tumor is unknown. In this study, we analyze the role and mechanism of 5-aza-CdR in the occurrence and development of Osteosarcoma cell line Hs888T.Methods1,Main reagentOsteosarcoma cell line Hs888T were purchased from Shanghai Cell laboratory, 5-Aza-CdR was purchased from Sigma company, DMEM medium purchased from Hyclone company,APAF-1-IgG was purchased from Santa Cruz Company and other reagents from Sigma company.2,Cell Culture and treat with 5-Aza-CdRThe cells were cultured in DMEM medium at 37℃in a 5% CO2 atmosphere. After 24h Hs888T cells were treated with different concentrations of 5-aza-CdR (0, 0.1 and 0.3mmol/L) in media for 48 h,96h, then harvested with trypsinization for in vitro experiments.3,MTT was used to detect the growth of Hs888T cellsThe concentration of cells was adjusted to 1×103cell/100μl.1, and then reloaded to 96-well plates by 200μL/well. Both the experimental group and the control had six replied wells for the measurement by MTT assay. The cell were incubated for 48h,96h respectively,added with 5g/L MTT and incubated for 4h. The absorbance value at 570nm (A490) was determined by a microplate reader using the value of blank control for zero adjustment.4,Flow cytometry was used to detect the apoptosis of Hs888T cellsCells of each group were harvested at different time points. RNA enzyme was added at 37℃and reacted for 1 h after cells were fixed in 70% alcohol at 4℃for 24 h (final concentration 50μg/mL). After 30 min of PI solution (concentration 100μg/mL) staining, cells were counted by monochromatic fluorescence flow cytometry to observe the apoptosis rate.5,The protein level of APAF-1 was assayed by Western blotCells were lysed in lysis buffer. The protein was quantified with Coomassie. Equal amount of proteins (100μg) were size fractionated on SDS-PAGE. Proteins were then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% defatted milk powder at 37℃for 2h. The blocked membrane was then incubated with primary antibodies at 4℃overnight. After washing the membrane with TTBS for twice, the membrane was incubated with secondary antibody at 37℃for 1h. The membrane visualized by ECL chemiluminescence reagent. The films were scaned using gelimaging system.6,Statistical analysisStudent's t test was applied to analyze the differences between treatment groups. All statistical calculations were performed by Spss 16.0 for windows software. P values less than 0.05 were considered statistically significant. Results5-aza-CdR significantly inhibited the cell growth and increased the apoptosis of Hs888T cells in dose and time dependent manners. After treatment with 5-aza-CdR, cells were arrested in G0/G1-phase. The APAF-1 expressed significantly in Hs888T cells after treatment with 5-aza-CdR.Conclusion5-aza-CdR significantly inhibited Hs888T cells growth, which probably results from the demethylation of some genes.
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