赫赛汀对乳腺癌SK-BR3细胞Notch-1信号通路的影响及意义

摘要

Objective: To investigate the effects and mechanism of action of herceptin on Notch-1 protein in breast cancer SK-BR3 cells, and to explore the significance of Notch-1 signaling pathway in breast cancer cell resistance to herceptin.Methods: The breast cancer cells SK-BR3 with HER2 overexpression and MDA-MB-231 overexpression with HER2 non-overexpression were selected.Immunocytochemistry and Western-blot analysis were used to detect the expression of Notch-1 protein and activate Notch-1 ( Notch-1IC ) in the SK-BR3 and MDA-MB-231 cells, respectively.SK-BR3 cells were treated with herceptin.Western blot analysis was used to detect the expression of Notch-1IC and HER2 proteins.Reverse transcriptase polymerase chain reaction was used to assay HES-1 mRNA expression.Co-immunoprecipitation detected the interaction between HER2 and Notch-1 proteins in the SK-BR3 cells.Results: The level of Notch-1IC nucleic localization and the expression of Notch-1IC protein in SK-BR3 cells ( 9.37 ± 0.64 ) were significantly lower than in the MDA-MB-231 cells ( 21.665 ± 1.11 ) ( P ＜ 0.01 ).SK-BR3 cells were treated with herceptin.Compared with the controls, the expression of Notch-1IC protein and HES-1 mRNA was significantly increased ( P ＜ 0.01 ).There was no apparent change in HER2 protein expression after herceptin treatment ( F = 0.973, P ＞ 0.05 ).Co-immunoprecipitation showed coprecipitation between the Notch-1 and HER2 proteins.Conclusion: The activity of Notch-1 protein was decreased in the breast cancer cells with HER2 overexpression.HER2 could combine with Notch-1, thereby negatively regulating Notch-1 activity.Herceptin could increase the activity of Notch-1, which could be associated with the cell resistance.%目的:探讨赫赛汀对乳腺癌SK-BR3细胞Notch-1蛋白的影响及其作用机制,认识Notch-1信号通路在乳腺癌细胞形成赫赛汀耐药中的意义.方法:选用HER-2过表达乳腺癌SK-BR3细胞及HER-2非过表达乳腺癌MDA-MB-231细胞,免疫细胞化学法和Western blot 法检测两细胞株中Notch-1 蛋白的表达;应用赫赛汀处理SK-BR3 细胞,Western blot 法检测HER-2、Notch-1通路活性分子Notch-1IC的蛋白表达;RT-PCR法检测Notch-1靶基因HES-1 mRNA的表达;免疫共沉淀法检测SK-BR3细胞中Notch-1与HER-2是否存在相互作用.结果:Notch-1IC核定位水平及Notch-1IC蛋白表达水平在HER-2过表达乳腺癌细胞(9.37±0.64)中明显低于HER-2非过表达乳腺癌细胞(21.665±1.11),P＜0.01;赫赛汀处理SK-BR3细胞后,与未处理组比较,细胞内Notch-1IC蛋白及HES-1 mRNA水平均明显增高(P＜0.01,P＜0.01),HER-2蛋白表达水平在处理前后未发生明显变化(F=0.973,P＞0.05);免疫共沉淀结果显示Notch-1与HER-2蛋白之间存在共沉淀.结论:Notch-1蛋白在HER-2过表达乳腺癌细胞中活性下降;HER-2与Notch-1结合,可能负性调控Notch-1;赫赛汀活化的Notch-1信号通路,可能与细胞耐药发生有关.