The Effects Of HSP90 Inhibitor 17-DMAG On The Colon Cancer Cell Line HCT116

Objective: To study the effects of HSP90 inhibitor 17-dimethylaminoethylam- ino-17- demethoxygeldanamycin(17-DMAG) on the proliferation and apoptosis of human colon cancer cell line HCT116 and explore its mechanism.Methods: The human colon cancer HCT116 cells in exponential phase were treated with different concentrations of 17-DMAG. The morphorlogic changes of HCT116 cells treated with 17-DMAG were observed by the means of the inverted phase-contrast microscope and the laser scanning confocal microscopy. The absorbance(OD) value was detected through MTT assay and the inhibition rate was calculated. The cell cycle of HCT116 cells treated with 17-DMAG was analyzed by flow cytometry(FCM). The apoptosis of HCT116 cells induced by 17-DMAG was measured by Annexin V-FITC/PI assay. The cytoplasmic level of the signaling molecule Akt and Survivin were evaluated through Western Blot assay, and the level of Akt-mRNA and Survivin-mRNA were detected by reverse transcriplase polymerase chain reaction(RT-PCR).Results: Under the inverted phase-contrast microscope, shrunk and misshaped HCT116 cells treated by 17-DMAG were observed, and the cell density decreased compared with the control group. As the concentration of 17-DMAG increased, the number of the cells significantly reduced. Aoptotic manifestation such as the chromatin condensation was observed under the laser scanning confocal microscopy. MTT assay showed that 17-DMAG inhibit the proliferation of human colon cancer cell line HCT116 in a dose- and time-dependent manner(P<0.05). The cell cycle analysis by FCM showed little evidence for an increase in mitotic figures or an accumulation of adherent cells in either G2-M or G1-S following 17-DMAG treatment. Annexin V-FITC/PI assay revealed that cell apoptotic ratio of 1.0μmol/L,2.5μmol/L,5.0μmol/L, density was 6.0%,13.6%, and 40.4%. The difference was significant between the disposal group and the control group(1.6%). After the HCT116 cells were treated with 17-DMAG, the decrease of cytoplasmic level of Akt and Survivin were proved by Western Blot assay(P<0.05). The expression of Akt-mRNA and Survivin-mRNA were down-regulated through ladder strap as the increase of 17-DMAG density in RT-PCR test.Conclusions: 17-DMAG can inhibit the proliferation of the human colon cancer cell line HCT116 in a dose-and time-dependent manner, and induce its apoptosis. The mechanism of the effect of 17-DMAG on HCT116 may be related to the depletion of Akt protein and Survivin protein in the cells.