The Apoptotic Effect Of17-DMAG On Colon Cancer HT-29Cells And Proteomic Study

BackgroundColon cancer is the malignant lesions of intestinal epithelial occurring under the environment of genetic and other carcinogenic factors. In recent years, the incidence of colon cancer have increased gradually. Colon cancer usually occult onset, and it often have no obvious clinical manifestations. Because the progression of the disease is slow, it is mostly at middle or late stage of cancer when obvious symptoms show. In-depth study of the molecular mechanisms of cancer development and development of the new cancer drugs are the important frontier of basic biomedical research now.17-DMAG is HSP90inhibitor, and it is expected to become a new treatment drug of advanced colon cancer.ObjectveStudying the effect of HSP90inhibitor17-DMAG (17-dimethylamine ethylamine-17-go to the methoxy Jige Stewart ADM) in the apoptosis of human colon cancer HT-29cells. And using proteomics methods futher studying its regulation of protein.Methods1Detecting the changes of the rate of apoptosis of HT-29cells by flow cytometry using Annexin VFITC/PI double staining.2Detecting the changes of plasma membrane proteins after24hours of17-DMAG stimulating by immunoblotting experiments verify the Na+-K+-ATPase and Prohibitin.3Extracting0.5pM17-DMAG stimulating group and the control group plasma membrane proteins and use two-dimensional gel electrophoresis separation of proteins. Quick silver staining method staining gel, and futher analysis per group differences in protein spots using ImageMaster2D Platinum6.0image analysis software.4Extracting0.5μM17-DMAG stimulating group and the control group secreting proteins and use two-dimensional gel electrophoresis separation of proteins. Quick silver staining method staining gel, and futher analysis per group differences in protein spots using ImageMaster2D Platinum6.0image analysis software.Results1Annexin V-FITC/PI double staining flow cytometry results show that natural early apoptosis rate of normal control group are (0.87±0.02)%, early apoptosis rate of0.25μmol/L、0.5μmol/L、1.0μmol/L and2.5μmol/L17-DMAG are (2.32±1.78)%、(4.93±0.46)%.(6.86±1.36)%与(7.59±1.28)%; normal control group total apoptosis rate are (6.3±0.75)%, total apoptosis rate of0.25μmol/L、0.5μmol/L,1.0μmol/L and2.5μmol/L17-DMAG are (8.51±1.91)%、(20.88±13.97)%、(25.11±1)%和(32.11±3.13)%. Compare to the control group, the apoptosis rate difference was statistically significant. The apoptosis rate of12h,24h,36h of drug group were higher than the corresponding control group, and the difference was statistically significant (p<0.05).2After24h of effect of0.5μmol/L17-DMAG, the purity of Na+-K+-ATPaseincreased and the purity of Prohibitin decreased.329difference protein points were found.48difference protein points were found.ConclusionsHeat shock protein90inhibitor17-DMAG presents time-dose-dependent inhibiton of cell proliferation of human colon cancer HT-29. With the increase of the concentration of the drug and the extension of the duration of action, the inducedthe apoptosis capacity of human colon cancer cell increase. The application of proteomic analysis found29difference plasma membrane protein and8secreting proteins points between drug action group and the control group.