Effects Of Gossypol Acetic Acid On Proliferation And Protein Expressions Of E-cadherin And P16Gene In Human Adenoid Cystic Carcinoma ACC-M Cell

Objective1. To investigate the effects on proliferation and apoptosis in human adenoid cystic carcinoma with lung metastasis cell line ACC-M treated by different concentrations of GAA in vitro;2. To detect the effects on protein expressions of E-cadherin and P16gene in cell line ACC-M treated by different concentrations of GAA in vitro;3. To investigate the effects on protein and mRNA expressions of DNMT1gene in cell line ACC-M treated by different concentrations of GAA. To discuss the mechanism of tumor suppression of GAA and provide the theory and experimental base for prevention and therapy on carcinoma cells treated by GAA.Methods1. ACC-M cells and HFL1(human lung fibroblasts cell linel) cells were cultivated in vitro and treated by different concentrations of GAA,and their pre-and-post treated morphology changing were observed through Light Microscope.2. To detect the effects on proliferation inhibition in ACC-M cells and HFL1cells treated by different concentrations of GAA through CCK-8assay, and then calculate theIC50of GAA for24、48、72h.3. To observe apoptosis changing in ACC-M cells before or after being treated by different concentrations of GAA through cell flow cytometry (FCM).4. To investigate the effects on the protein expressions of E-cadherin P16and DNMT1gene in ACC-M cells treated by different concentrations of GAA for24h、48、72h through Western-blot;5. To investigate the effects on the mRNA expressions ofDNMT1gene in ACC-M cells treated by different concentrations of GAA for24h、48、72h through real-time fluorescence quantitative PCR.Results1. CCK-8assay showed that cell line ACC-M and cell line HFL1were both inhibited by different concentrations of GAA for different time. The inhibitary effects have a dose-dependent and time-dependent fashion in a certain range. Cell line ACC-M:IC50of GAA for24h、48h、72h was33.20μmol/L、15.50μmol/L、8.60μmol/L respectively;Cell line HFL1:IC50of GAA for241、48h、72h was35.13μmol/L、28.00μmol/Ls、26.80μmol/L respectively.2. After the treatment with GAA for10μmol/L,20μmol/L,30μmol/L and40μ mol/L,the result of FCM showed that the apoptosis rate of ACC-M cells was positively correlated with dose-dependent of GAA.3. Compared with the control group, the result of Western-blot indicated that the protein expressions of24h:E-cadherin and p16gene were increased. DNMT1gene was decreased; the protein expressions of48h:E-cadherin and p16gene were increased, DNMT1gene was no obvious changed; the protein expressions of72h:E-cadherin gene was increased, p16and DNMT1gene were no obvious changed.4. The result of real-time fluorescence quantitative RT-PCR showed that the mRNA expressions of DNMT1gene in cell line ACC-M was reduced after GAA treatment in a dose-dependent way (P<0.05)Conclusion1. GAA can suppress proliferation and induce apoptosis of humanadenoid cystic carcinoma with lung metastasis cell line ACC-M in a dose-dependent and time-dependent fashion.2. GAA can increase the protein expressions of E-cadherin and p16gene in human adenoid cystic carcinoma cell line ACC-M. It means that the two kinds of gene may be the antitumor target of GAA.3. GAA can reduce the protein and mRNA expression of DNMT1gene in human adenoid cystic carcinoma cell line ACC-M. It is suggested that GAA can make the down-regulation of DNMT1activity and thus play a tumor suppressor role, this is probably one of the antitumor mechanism of GAA...