Effects Of17-AAG On Proliferation And Apoptosis Of Human Gastric Carcinoma Cell Line BGC-823and The Underlying Mechanisms

Gastric cancer is one of the most frequent digestive tract tumors whichgreatly endangers human health and life. It is necessary to find an effectivetreatment to improve the survival rate and the quality of life of patients withgastric carcinoma. Hsp90is a molecular chaperone of heat shock proteinfamily and can regulate and maintain the conformation and function ofproteins. Hsp90inhibitor affects the progression of tumor through regulatingthe expression of proteins. In this experiment, the human gastric carcinomacell line BGC-823was used to study the effects of17-AAG on theproliferation and apoptosis of cells.Part1The effects of17-AAG on growth and proliferation of BGC-823cellsObjective: To study the effects of17-AAG on growth and proliferationof BGC-823cells and the underlying mechanisms.Methods:1The human gastric carcinoma cell line BGC-823in exponential phasewas treated with different concentrations of17-AAG. The optical density (OD)value was detected at different time through MTT assay.2MTT method was used to make the growth curves of BGC-823cellstreated with17-AAG.3The effect of17-AAG on proliferation of the single cell was detectedby clonogenic assay.4Cell-cycle distribution of BGC-82cells was determined by flowcytometry after the cells had been treated with1000nmol/L17-AAG for48h.5Wester Blot was used to study the mechanisms underlying proliferationinduced by17-AAG. Results:1The result of MTT assay showed that17-AAG inhibited BGC-823cellgrowth in concentration-and time-dependent manners.2The result of growth curves showed that17-AAG inhibited the growthof BGC-823cells for a long time.3The clonogenic assay results indicated that the colony formation rate ofBGC-823cells was57.29%in control group, which was decreased to53.38%and8.08%by17-AAG100nmol/L and1000nmol/L, respectively (P<0.01).4Treatment of BGC-823cells with17-AAG1000nmol/L for48h led tothe accumulation of the S-phase cells (P<0.05), which was accompanied bythe reduction of the ratio of cells in G1-phase compared with the controlgroup(P<0.01).5The expression of CDK4was significantly increased by17-AAG andthe expression of PCNA was down-regulated by100nmol/L and1000nmol/Lof17-AAG. Meanwhile, the expression of Akt1and MIF was significantlyreduced.Conclusions:1In a concentration-and time-dependent manner,17-AAG inhibited theproliferation of BGC-823cells in vitro. The inhibition lasted for a long time.2The colony formation of BGC-823cells was reduced by17-AAG.3The cell cycle of BGC-823cells was interfered by17-AAG.4The inhibitory effect of17-AAG on proliferation of BGC-823cells wasrelated to cell cycle arrest and the effect on cell cycle was associated withincreasing expression of CDK4and decreasing expression of PCNA.5The inhibitory effect of17-AAG on proliferation of BGC-823cells,was also related to downregulating the expression of MIF and suppressing theactivation of Akt pathway.Part2The effect of17-AAG on apoptosis of BGC-823cellsObjective: To study the effect of17-AAG on apoptosis of BGC-823cells and the underlying mechanisms. Methods:1The morphological changes of BGC-823cells treated with17-AAGwere observed by means of the optical microscope.2The fluorescence microscope was used to observe the apoptoticmorphological changes in BGC-823cells induced by17-AAG.3Western Blot was used to study the mechanisms underlying apoptosisinduced by17-AAG.Results:1BGC-823cells treated by different concentrations of17-AAG becameshrunk and misshaped in different degrees.2The morphology of BGC-823cells was observed by fluorescencemicroscopy following DAPI staining. Along with the concentration of17-AAG increased, the degree of cell apoptosis enhanced. Morphologicalchanges such as nucleuspyknosis, chromatin condensation, blue fluorescenceenhancement and apoptotic body formation were found in the BGC-823cellstreated with17-AAG.3The results of Western Blot showed that17-AAG caused a markeddown-regulation of Bcl-2protein expression and the expression ofpro-apoptotic proteins Bax and p53was significantly increased by17-AAG. Inaddition, the ratio of Bax/Bcl-2was increased in a concentration-dependentmanner.Conclusions:117-AAG caused morphologic changes of BGC-823cells.217-AAG induced apoptosis of BGC-823cells.3The effect of17-AAG on apoptosis of BGC-823cells could beassociated with increasing p53protein expression and the ratio of Bax/Bcl-2,followed by activation of mitochondrial pathways.