The Effects Of HSP90 Inhibitor DMAG On The Leukemia Cell Line HL-60

Objective: To study the effect of growth inhibiting and its related mechanism of the new heat shock protein 90 (HSP90) inhibitor 17-dimethylaminoethylamino-17- demethoxygeldanamycin (DMAG) on the cultured leukemia cell line, HL-60 cells. To explore the synergistic characteristics of inhibiting cell growth after HL-60 cells were treated with DMAG and daunorubicin (DNR) simultaneouly.Methods:①The morphorlogic changes of HL-60 cells treated with DMAG were observed by the means of light microscope and electron microscope.②The growth curve of HL-60 cells treated with DMAG and DNR was done to evaluate the inhibiting effects of the two drugs through MTT assay.③T he apoptosis of HL-60 cells induced by DMAG and DNR was examined by flow cytometry (FCM).④C ell cycle of HL-60 cells treated with DMAG were analyzed by FCM.⑤In order to approach the mechanism of DMAG, the cytoplasmic level of the signaling molecule Raf was evaluated through FCM and Westen Blot assay, and the level of raf-mRNA was detected through RT-PCR.Results:①U nder light microscope, shrunk and misshaped HL-60 cells treated by DMAG were observed, and the cell density decreased compared with the control group. Apoptotic manifestation such as the chromatic marginalization and the formation of crescent chromatin was observed under electron microscope.②MTT tests show that the proliferation of HL-60 cells were inhibited in a dose and time dependent manner by DMAG. The 50% inhibiting concentration (IC50) of 72h was 0.488μM.③Synergistic effects caused by DMAG and DNR on inhibiting the proliferation of the HL-60 cells were observed through MTT tests. The coefficient of drug interaction (CDI) of the two drugs was less than 1.0, and FCM tests demonstrated that more apoptotic cells were induced by the coadministration of the two drugs.④The cell cycle analysis by FCM showed that the G0/G1 phase ratio increased significantly and the S phase ratio decreased accordingly after HL-60 cells were treated with DMAG.⑤After the HL-60 cells were treated with DMAG, the decrease of cytoplasmic level of Raf was found through FCM and proved by Westen Blot assay while the level of raf-mRNA didn't change in RT-PCR test.Conclusions: DMAG can induce the apoptosis and inhibit the growth of HL-60 cells, and the combination of DMAG with DNR produced a synergistic effect. The mechanism of the effect of DMAG on HL-60 cells may be related to the depletion of Raf protein in the cells.