【作者】 聂秀青；

【导师】 沈坤炜； 柳光宇；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 目的：(1)探讨BH3-Only蛋白在拉帕替尼诱导Her2阳性乳腺癌细胞凋亡中的作用及其与FOXO3a蛋白的关系；(2)研究ABT-737和拉帕替尼联合应用与Her2阳性的SK-BR3细胞的联合作用及其机制。方法：(1)流式细胞仪Annexin V染色的方法检测细胞凋亡,Real TimePCR和Western blot检测Her2信号通路相关蛋白、BH3-Only家族蛋白和FOXO3a蛋白的表达在拉帕替尼处理后的差异,SiRNA技术抑制Bim和FOXO3a蛋白的表达,确定它们在拉帕替尼诱导的细胞凋亡中的作用以及FOXO3a蛋白对Bim蛋白表达的调控。(2)CCK-8法研究拉帕替尼和ABT-737单用和联合应用的细胞效应,利用中位效应原理并使用Calcusyn软件分析两药的联合作用。Western blot检测BCL-2家族蛋白在拉帕替尼单用和联合应用ABT-737后的变化,免疫共沉淀法检测ABT-737对BCL-2蛋白拮抗Bim蛋白促凋亡活性的抑制作用。结果：(1)拉帕替尼作用于HER阳性乳腺癌细胞株BT-474和SK-BR3,诱导细胞发生凋亡并伴有Caspase 3和BAX蛋白的激活,促凋亡的BH3-Only家族蛋白之一Bim蛋白和FOXO3a的表达增多,SiRNA抑制Bim和FOXO3a的表达,能够抑制拉帕替尼诱导的细胞凋亡,抑制FOXO3a表达的同时,Bim蛋白表达降低。(2)ABT-737和拉帕替尼联合应用于SK-BR3细胞能够产生协同作用；拉帕替尼诱导SK-BR3细胞Bim蛋白表达增高,抗凋亡的MCL-1蛋白表达降低；拉帕替尼诱导Bim蛋白表达增多同时与BCL-2蛋白结合也增多,ABT-737和拉帕替尼联合应用未改变拉帕替尼对BCL-2家族蛋白表达变化的影响,但是能够抑制BCL-2蛋白和Bim蛋白的结合。结论：(1)拉帕替尼能够诱导HER2阳性乳腺癌细胞BT474和SK-BR3细胞发生凋亡,拉帕替尼诱导细胞凋亡可能通过转录激活激活Bim蛋白的表达,激活线粒体细胞凋亡途径促进细胞发生凋亡,Bim蛋白的表达受FOXO3a蛋白的调控,因此,拉帕替尼可能通过抑制PI3K/AKT信号通路,促进FOXO3a蛋白的表达增多,转录激活Bim蛋白的表达,激活线粒体细胞凋亡途径是细胞发生凋亡。(2)ABT-737和拉帕替尼联合应用于Her2阳性的乳腺癌细胞SK-BR3能够产生协同作用,其协同作用的机制可能为一方面拉帕替尼抑制了抗凋亡蛋白MCL-1蛋白的表达,促进了ABT-737的抗肿瘤效应,另一方面ABT-737抑制了拉帕替尼诱导Bim蛋白表达增多同时发生的与BCL-2蛋白的结合增多,促进了Bim蛋白的促凋亡活性。更多还原

【Abstract】 Objective:(1) To explore the role of BH3-Only family proteins in the apoptosis induced by Lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 cells and whether regulated by FOXO3a protein. (2) To study the synergy effect between BH3 mimitic compound, ABT-737 and Lapatinib in Her2 positive breast cancer cell SK-BR3 and explore the mechanism of the synergy effect.Methods:(1) The extent of apoptosis was evaluated by flow cytometric analysis utilizing annexin V-fluorescein isothiocyanate staining method. The Her2 pathway protein, BH3-Only family protein and protein FOXO3a were measured by Real-time PCR and Western blot methods. RNA interference method inhibiting the express of Bim and FOXO3a protein to ascertain the role of them in the apoptosis induced by lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 and whether Bim expression regulated by FOXO3a protein. (2). Utilize CCK8 method to study the toxic effect of BH3 mimitic compound, ABT-737 and Lapatinib in the Her2 positive breast cancer cell SK-BR3. According median-effect principal (Chou-Talalay combination index method), utilize the Calcusyn software analysis the combination index (CI) to ascertain the synergistic effective between them in SK-BR3 cell. The difference of BCL-2 family proteins induced by Lapatinib alone or combined with ABT-737 in SK-BR3 cell was detected by Western blot method. The association of BCL-2 and Bim protein was detected by Coimmunoprecipitation method.Result:(1). The apoptosis induced by Lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 cells accompanied by a pronounced increase of Caspase 3 cleavage and the conformational change of BAX. The expression of one of the BH3-only family protein Bim and FOXO3a increase after treatment of Lapatinib. RNA interference of Bim and FOXO3a inhibit the apoptosis induced by Lapatinib in SK-BR3 cell, and the increase of Bim protein induced by Lapatinib was inhibited by RNA interference of FOXO3a. (2) The BH3-mimitci compound ABT-737 synergizes the antitumor activity of Lapatinib in SK-BR3 cell. With the increase of Bim protein induced by Lapatinib the expression of MCL-1 protein was decreased. Lapatinib-induced Bim is primarily sequestered by Bcl-2 rather than Mcl-1 and Bcl-xL; these associations are disrupted by ABT-737.Conclusion:(1) Lapatinib induced apoptosis require Bim through mitochondrial pathway and regulated by FOXO3a in Her2 positive breast cancer cells. The AKT-FOXO3a-Bim axis may be the important role in lapatinib induced apoptosis. (2) The BH3 mimetic compound ABT-737 synergize the antitumor activity of Lapatinib for Her2 positive breast cancer cell SK-BR3. The mechanism may be the Lapatinib induced decrease of MCL-1 protein enhancing the activity of ABT-737, on the other hand ABT-737 disrupted the associations in Lapatinib-induced Bim sequestered by Bcl-2.更多还原