Effects Of Cobalt Chloride On HSP90 Expression In Walker256 Cells And Effects Of 17 - AAG Inhibitor On Apoptosis And Growth

ObjectiveTo observe the effects of hypoxia on proliferation of hepatoma Walker256 cells. Hypoxia model was established by treatment with cobalt chloride (CoCl2). And then established a suitable invitro model of hepatoma Walker256 cells. To observe the effects of hypoxia on apoptosis and hepatoma cells Walker256 ability of HSP90 expression, then to explore the mechanism.Methods1.Inverted microscope was used to observe different concentrations of CoCl2 (0,50,100,200,300,500μmol/L) and different concentrations of 17-AAG (0,0.1,0.25,0.5,1,2μmol/L) were treated liver Walker256 48h after cell change, the cell morphology was observed。2.Different concentrations of COC12 (0,50,100,200,300,500μmol/L)and different concentrations of inhibitor 17-AAG (0,0.1,0.25,0.5,1,2μmol/L) were treated with Walker256 cells for different hours. Then undergo MTT to find their cellular toxicities. 3.Five different concentrations of CoCl2 (0,50,100,200,300,500μmol/L) and 0.5μmol/L 17-AAG were respectively co-cultivated with Walker256 cells. Western blot was used to assess the protein expression of HSP90 and Bcl-2 in Walker256 cells.4.Annexin-FITC/PI double staining flow cytometry was used to detect 17-AAG on apoptosis of hepatoma cells Walker256。5.Established animal models to observe the effects of 17-AAG on rats tumor growth. The Walker256 cells suspension were seeded in the neck of rats.Divide the tumor rats into two groups, one group was given 17-AAG inhibitor, another not (15 rats in each group). Observe tumor’s growth, compared the tumor volume of two groups.Results1.A certain concentration of CoCl2 could induce cell damage. When CoCl2 concentrations reached 300 and 500μmol/L, morphological changes were observed by inverted microscope,cell membranes apparent shrinkage and a large number of cell debris;Different concentrations of CoCl2 could inhibit the activity of Walker256cells. After Walker256 cells were treated by different concentrations of CoCl2 for 48 hours, the results of Western Blot shows that the expression of HSP90 increased, and this effect shows dose-dependent.2.Different concentrations of the HSP90 inhibitor 17-AAG on Walker256 cell proliferation in a dose-and time-dependent changes.17-AAG also have an impact on cell morphology, after 48h Walker256 cell full round, abundant cytoplasm, uniform size, encapsulated in control group, and by the effect of 17-AAG Walker256 cells significantly reduced, cell edge not the whole, poor shape, some cells capsule rupture. Analysised the apoptosis in normoxia, hypoxia,17-AAG+ normoxic group and 17-AAG+hypoxia group by Annexin-FITC/PI staining method,the results showed that: ① apoptosis ratio in hypoxia group was no significant difference compared with normoxic group (P> 0.05); ② apoptosis rate increased in 17-AAG+normoxic group compared with normoxic group (P<0.05);③poptosis ratio increased in 17-AAG +hypoxia group compared with hypoxia group (P<0.05); ④ apoptosis rate increased in 17-AAG+ hypoxia group compared to 17-AAG+ normoxic group (P<0.05).Detected the expression of HSP90 and Bcl-2 in normoxia, hypoxia、17-AAG+ normoxi、 17-AAG+ hypoxia group by Western Blot, and the results showed:① compared with normoxic group, HSP90 and Bcl-2 expression increased in hypoxia group (P<0.05)② compared with normoxic group, HSP90 and Bcl-2 expression was inhibited in 17-AAG+ normoxic group (P <0.05)③Compared with the hypoxia group, HSP90 and Bcl-2 expression also inhibited in the 17-AAG+hypoxic group (P<0.05)④he differences of Bcl-2 and HSP90 expression between 17-AAG+hypoxia group and 17-AAG+ normoxic group were not statistically significance (P> 0.05).3.17-AAG inhibitors can inhibit rats implanted tumor growth, with delivery time extended, we found that rats implanted tumor growth was significantly slower than the control group, while the average volume of tumor in the control group were always increasing.Conclusion1.Different concentrations of CoCl2 could induce HSP90 expression, the induction was more significant when CoCl2 was 200μmol/L.2.HSP90 inhibitor 17-AAG could inhibit rats implanted tumor growth.3.HSP90 inhibitor 17-AAG could significantly promote cell apoptosis, and thus the mechanism presumably may be that after 17-AAG combined with HSP90,the combination inhibited the expression of anti-apoptotic Bcl-2 protein, which significantly increased apoptosis in the tumor.