PNRC多肽通过影响Ras信号途径抑制BT-474细胞的增殖  

A PNRC-derived peptidimer inhibits proliferation of BT-474 cells by interfering with Ras signaling

作　　者：陈彬[1] 王越[1] 胡晨[2] 阳玉鹏[1] 李渝萍[1] 娄桂予[1] 张艳[1] 何凤田[1] 周度金[1] 

机构地区：[1]第三军医大学生物化学与分子生物学教研室,重庆400038 [2]第三军医大学学员旅8队,重庆400038

出　　处：《中国癌症杂志》2009年第3期161-165,共5页China Oncology

基　　金：国家自然科学基金(No.30672055);重庆市自然科学基金(CSTC.2006BB5352)

摘　　要：背景与目的:根据富含脯氨酸的核受体辅调节蛋白(proline-rich nuclear receptor coregulatory protein,PNRC)与Grb2相互作用位点资料和其他基于干扰Grb2/SH3与SOS的相互作用的富含脯氨酸短肽的设计原则,我们设计、合成了基于PNRC的抗Ras、PTD融合多肽(PTD-PARP),研究该多肽在BT474乳腺癌细胞中对Ras信号途径的影响及其抗肿瘤增殖活性。方法:用多肽合成仪合成PNRC的抗Ras PTD融合多肽PTD-PARP和其单独的PTD多肽。以PTD多肽为对照,将增加浓度的PTD-PARP与分离的、EGF刺激的BT474细胞裂解物共温育,采用免疫共沉淀结合Western blot检测Grb2免疫共沉淀复合物中SOS和PNRC的含量情况。用PTD-PARP-琼脂糖凝胶4B活化偶联磁珠免疫共沉淀BT474细胞裂解物,采用PI3K,Nck和Grb2的抗体检测沉淀复合物中这些含SH3结构域的蛋白与PTD-PARP的结合能力。Western blot检测PTD-PARP对EGF刺激的BT474细胞内Ras和MAPK活化的影响。采用软琼脂克隆形成实验考察PTD-PARP对BT474细胞体外增殖的影响。结果:PTD-PARP能以剂量依赖方式抑制SOS、PNRC与Grb2的结合,它与Grb2的结合具有特异性,PTD-PARP能降低BT474细胞中EGF刺激的Ras和MAKP活化,并能显著抑制BT474细胞的体外软琼脂克隆形成。结论:PNRC抗Ras PTD融合多肽可通过影响Ras信号途径抑制乳腺癌细胞的增殖。Background and purpose： Based on the interaction between PNRC and Grb2, and the designed principle of other proline-rich peptides interferring with the interaction of Grb2/SH3 and SOS, an anti-Ras PTD fused PNRC-derived peptidimer, PTD-PARP, had been designed and synthesized. This study was done to investigate its effect on Ras signaling and the anti-proliferation activity in BT474 breast cancer cells. Methods： Two polypeptides, PTD-PARP and PTD alone were synthesized by peptide synthesizer. The lysates of EGF-stimulated BT-474 cells after treatment with either PTD control or different concentration of peptidimer were isolated and immunoprecipitated with a Grb2 antibody. The expression of PNRC and SOS in the immunoprecipitates was detected by western blot analysis. The peptidimer affinity for the proteins containing SH3 domain such as PI3K,Nck and Grb2 which were affinity- precipitated by the peptidimer coupled with EAH Sepharose 4B from BT474 lyses was detected by western blot. EGF- induced Ras and MAPK activation in BT474 cells treated with the peptidimer was observed by western blot. Finally, in vitro anti-proliferative activity of the peptidimer on BT474 cells was determined by the efficiency of colony formation in soft agar. Results： The binding of Grb2 to SOS or PNRC could be inhibited by the peptidimer in a dose-dependent manner. The peptidimer specifically recognized Grb2 and did not interact with PI3K or Nck. EGF-induced Ras and MAPK activation, and the efficiency of colony formation in soft agar in BT474 cells was blocked by the peptidimer. Conclusion： An anti-Ras PTD fused PNRC-derived peptidimer, PTD-PARP, can inhibit the proliferation of BT474 cells by interferring with Ras signaling.

关 键 词：PNRC 多肽 PTD RAS 信号传导 

分 类 号：R512.62[医药卫生—内科学] R394.8[医药卫生—临床医学]