Regulatory effect of 17-AAG on VEGF expression in gastric cancer cells via STAT3 signaling pathway
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摘要:
目的 观察17-烯丙胺-17-脱甲氧格尔德霉素(17-allylamino-17-desmethoxy-geldanamycin, 17-AAG)对人胃癌MKN-45细胞信号转导和转录活化蛋白3(signal transducer and activator of transcriptions 3, STAT3)、血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白表达水平的影响, 并探寻STAT3通路所发挥的作用。 方法 体外培养人胃癌MKN-45细胞, 分别给予不同剂量及不同作用时间17-AAG进行作用, 四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞增殖能力; RT-PCR法和Western Blotting法检测各组细胞STAT3和VEGF mRNA和蛋白的表达水平。 结果 0.165~10 mg/L的17-AAG作用24、48 h后对MKN-45细胞有显著的抑制作用, 且有明显的时间、剂量依赖性; 1.0、2.0、3.0、5.0 mg/L的17-AAG作用48 h后, 各组细胞STAT3和VEGF mRNA和蛋白表达均下调, 且具有浓度依赖性; 3.0 mg/L 17-AAG作用12、24、48h后, 各组细胞STAT3和VEGF mRNA和蛋白表达均下调, 且具有时间依赖性。 结论 17-AAG对人胃癌MKN-45细胞的增殖具有明显的抑制作用, 并能抑制STAT3和VEGF mRNA和蛋白的表达, 17-AAG可能通过对STAT3通路的负性调控作用而抑制VEGF表达。 Abstract:Objective This study aims to investigate the effects of 17-allylamino-17-desmethoxy-geldanamycin(17-AAG) on the gene and protein expressions of VEGF and STAT3 pathways in MKN-45 human gastric cancer cells, and explore the potential mechanisms of the STAT3 signaling pathway that mediate these effects. Methods MKN-45 cells were seeded in Dulbecco's Modified Eagle Medium.The thiazolyl blue method, i.e., methyl thiazolyl tetrazolium assay, was performed to evaluate the inhibitory effects of 17-AAG on the proliferation of MKN-45 cells at different times, with different doses.The gene and protein expression of VEGF and STAT3 were determined by reverse transcription polymerase chain reaction(RT–PCR) and Western blot analysis. Results MKN-45 cells were treated with 17-AAG at 0.165 mg/L to 10 mg/L for 24 and 48 h, respectively.17-AAG significantly inhibited MKN-45 cell proliferation in a time-and dose-dependent manner.The results of the RT–PCR and Western blot assays showed that the gene and protein expressions of VEGF and STAT3 in MKN-45 cells induced by 17-AAG were significantly down-regulated in a dose-dependent manner when the cells were treated with 17-AAG at 1.0, 2.0, 3.0, and 5.0 mg/L for 48 h.The gene and protein expressions of VEGF and STAT3 in the MKN-45 cells were significantly down-regulated in a time-dependent manner when the cells were treated with 17-AAG at 3.0 mg/L for 12, 24, and 48 h. Conclusion The drug 17-AAG can inhibit cell proliferation and down-regulate the gene and protein expressions of VEGF and STAT3 in MKN-45 human gastric cancer cells in vitro; the down-regulation of VEGF expression may be associated with the STAT3 signaling pathway. -
Key words:
- 17-AAG /
- cell proliferation /
- VEGF /
- STAT3
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图 1 17-AAG体外不同作用浓度对人胃癌MKN-45细胞抑制率
Figure 1. Inhibition ratio of various concentrations of 17-AAG in vitro on MKN-45 human gastric cancer cells
图 2 不同作用浓度17-AAG对MKN-45细胞中STAT3、VEGF m RNA表达的影响
Figure 2. Effect of various concentrations of 17-AAG on STAT3 andVEGF m RAN expressions in MKN-45 cells
图 3 不同作用时间17-AAG对MKN-45细胞中VEGF、STAT3 m RNA表达的影响
Figure 3. Effect of 17-AAG at various action times on VEGF and STAT3m RNA expressions in MKN-45 cells
图 4 不同浓度17-AAG对MKN-45细胞中STAT3、VEGF蛋白表达的影响
Figure 4. Effect of various concentrations of 17-AAG on STAT3 andVEGF protein expressions in MKN-45 cells
图 5 不同时间17-AAG对MKN-45细胞中STAT3、VEGF蛋白表达的影响
Figure 5. Effect of various concentrations of 17-AAG on STAT3 andVEGF protein expressions in MKN-45 cells
表 1 RT-PCR引物序列和产物大小及反应条件
Table 1. Primer sequences, products size and conditions for RT-PCR
表 2 17-AAG体外不同作用时间对MKN-45细胞抑制率（n=5，x±s，%）
Table 2. Inhibition ratio of 17-AAG at various action times in vitro on MKN-45 cells
表 3 不同作用浓度17- AAG对MKN-45细胞系VEGF、STAT3基因mRNA表达的影响（x±s）
Table 3. Effect of various concentrations of 17-AAG on STAT3 and VEGF gene mRAN expressions in MKN-45 cells
表 4 不同时间17-AAG对人MKN-45细胞STAT3、VEGF mRNA表达的影响（x±s，n=5）
Table 4. Effect of various concentrations of 17-AAG on STAT3 and VEGF protein expressions in MKN-45 human cells
表 5 不同浓度17-AAG对人MKN-45细胞系STAT3、VEGF蛋白表达的影响（x±s，n=5）
Table 5. Effect of 17-AAG at various action times on STAT3 and VEGF mRNA expressions in MKN-45 human cells
表 6 不同时间17-AAG对人MKN-45细胞系STAT3、VEGF蛋白表达的影响（x±s，n=5）
Table 6. Effect of 17-AAG at various action times on STAT3 and VEGF protein expressions in MKN-45 human cells
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