【作者】 张敏；

【导师】 叶敏；

【作者基本信息】 福建医科大学 ， 肿瘤药理， 2014， 硕士

【摘要】 目的：研究FW-04-806对HER2阳性胃癌细胞的体内外抗肿瘤活性及其机制，探讨其与Lapatinib的联合作用，为大环双内酯化合物FW-04-806这种潜在的Hsp90抑制剂的开发应用提供理论依据。方法：（1）四甲基偶氮唑蓝(MTT)法体外检测FW-04-806对HER2阳性胃癌细胞NCI-N87、OE19的增殖抑制作用；（2）集落形成法检测FW-04-806对HER2阳性胃癌细胞的集落形成抑制能力；（3）通过FITC/PI双染法检测FW-04-806对HER2阳性胃癌细胞的凋亡作用；（4）通过PI染色法检测FW-04-806对胃癌细胞周期的影响；（5）Western blot法检测对胃癌细胞蛋白的影响；（6）免疫共沉淀法检测蛋白间相互作用；（7）免疫组化观察Hsp90客户蛋白的表达变化；（8）体内异种移植瘤模型检测FW-04-806的抑瘤率。结果：（1）FW-04-806呈剂量依赖性抑制胃癌细胞NCI-N87、OE19、AGS、SGC-7901的增殖，对应半数抑制率分别为24.17、29.61、58.13、60.59μmol·L-1，结果显示FW-04-806对HER2阳性胃癌细胞（NCI-N87、OE19）更敏感；（2）长期集落形成实验结果显示，FW-04-806对HER2阳性胃癌细胞NCI-N87、OE19的集落形成能力有明显的抑制作用；（3）随着FW-04-806作用NCI-N87和OE19细胞药物浓度的增加，流式细胞仪检测到的细胞凋亡率逐渐增加；（4）FW-04-806明显改变细胞周期，减少S期，阻滞胃癌细胞周期于G2-M期；（5）FW-04-806阻滞HER2下游细胞信号通路，抑制HER2、Akt和ERK的磷酸化，增加凋亡蛋白cleaved caspase3、cleaved parp的表达；（6）免疫共沉淀结果显示，FW-04-806作用胃癌细胞OE19，诱使Hsp90/CDC37复合物解离，减少CDC37与Hsp90结合（；7）免疫组化切片图可以观察到，FW-04-806明显减少Hsp90客户蛋白HER2、Akt的表达；（8）FW-04-806在异种移植瘤模型中有效抑制肿瘤生长，与对照组相比，200mg/kg实验组抑瘤率为48.0%，P<0.01，差异具有统计学意义；（9）FW-04-806联合Lapatinib具有协同作用，使增殖抑制和凋亡诱导增强。结论：（1）FW-04-806对HER2阳性的胃癌细胞具有良好的体内外抗肿瘤作用。（2）FW-04-806可能是一种潜在的Hsp90抑制剂，通过干扰CDC37与Hsp90的结合，抑制Hsp90功能，从而降解Hsp90客户蛋白HER2和Akt，阻滞HER2下游细胞信号通路。（3）FW-04-806与Lapatinib联合用药具有协同抑制作用。该协同作用可能是通过增强抑制增殖和诱导凋亡这两个途径来实现。更多还原

【Abstract】 Objective: To investigate the efficacy of FW-04-806against HER2-positive gastric cancer cell lines in vivo and in vitro, and the combination effect of FW-04-806with Lapatinib.Methods:(1) MTT assay was used to assess cell proliferous inhibition of FW-04-806in vitro.(2) The inhibitory effect of colony formation in HER2-positive gastric cancer cells was tested by colony formation. The apoptotic induction of FW-04-806in HER2-positive gastric cancer cells was detected by FITC-PI double staining.(4) Cell cycle was analyzed by PI staining.(5) Western blot was applied to reveal the expression of related protein level.(6) Co-immunoprecipitation was used to investigate protein-protein interactions.(7) The expression of Hsp90client proteins was showed by Immunohistochemistry.(8) The tumor growth inhibition of FW-04-806was evaluated in tumor xenograft model.Results:(1) FW-04-806inhibited the cell proliferation of gastric cancer cell lines NCI-N87、OE19、AGS and SGC-7901in dose-dependent manner with IC50of24.17、29.61、58.13、60.59μmol·L-1, respectively, showing more sensitivity in HER2-positive gastric cancer cell lines.(2) FW-04-806significantly suppressed the colony formation of HER2-positive gastric cancer cells NCI-N87and OE19.(3) With the increase of drug concentration, the apoptosis rate increased, after the treatment of FW-04-806in NCI-N87and OE19cells.(4) FW-04-806blocked HER2-positive gastric cancer cells in the G2-M phase of the cell cycle.(5) FW-04-806blocked HER2downstream signaling pathway, inhibiting the phosphorylation of HER2、Akt and ERK and increasing the expression of apoptotic proteins, such as cleaved caspase3and cleaved parp.(6) FW-04-806dissociated Hsp90/CDC37complex, interrupting the binding of Hsp90and CDC37.(7) FW-04-806obviously degraded the expression of Hsp90client proteins HER2and Akt.(8)FW-04-806significantly inhibited tumor growth in vivo with inhibition of tumor growth of48.0%, P<0.01, the difference of which has statistically significance.Conclusion:(1) FW-04-806shows potent efficacy against HER2-positive gastric cancer in vitro and in vivo.(2) FW-04-806may be a potential Hsp90inhibitor, inhibiting the Hsp90function by interrupting the binding of Hsp90and CDC37, resulting in the degradation of Hsp90client proteins HER2、Akt, and the blockage of HER2downstream signaling pathways.(3) FW-04-806is synergistic with Lapatinib, the mechanism of which may be based on the enhancement of proliferous inhibition and apoptotic induction.更多还原