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达沙替尼抑制前列腺癌细胞雄激素受体磷酸化机制研究 被引量:3

Mechanism study of dasatinib inhibiting phosphorylation of androgen receptor in prostatic cancer cells
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摘要 目的探讨酪氨酸激酶抑制剂达沙替尼抑制前列腺癌细胞雄激素受体(AR)磷酸化的机制。方法以HEK-293T及COS7细胞株为研究对象,野生型AR(WT-AR)载体及其突变载体ARY267F、ARY534F分别与Ack1或Src载体共转染细胞,Western blot方法检测AR磷酸化位点。以LNCaP细胞株为研究对象,在无雄激素条件下,用表皮生长因子(EGF)或内皮样生长因子家族成员heregulin处理后,Western blot法检测AR磷酸化状态,再加入达沙替尼或用siRNA转染法沉默Ack1或Src基因后,Western blot法观察AR磷酸化变化;反转录-实时定量聚合酶链反应(RT-PCR)法检测不同处理的LNCaP细胞前列腺特异抗原(PSA)以及人激肽释放酶2(hK2)mRNA的表达。结果转染载体后,HEK-293T细胞中Ack1激酶介导ARTyr267特异位点磷酸化,COS7细胞中Src介导AR Tyr534特异位点磷酸化。heregulin处理LNCaP细胞后,AR Tyr267位点磷酸化;加入达沙替尼后,该位点磷酸化被抑制;Ack1被沉默后,heregulin诱导的AR Tyr267位点磷酸化亦被抑制。EGF处理LNCaP细胞后,AR Tyr534位点磷酸化;加入达沙替尼后,该位点磷酸化被抑制;Src被沉默后,EGF诱导的AR Tyr534位点磷酸化亦被抑制。EGF或heregulin可上调LNCaP细胞内源性AR靶基因PSA和hK2 mRNA水平(P〈0.05),给予达沙替尼后,抑制了heregulin诱导的PSA和hK2 mRNA水平(P〈0.05),但EGF所诱导的PSA mRNA及hK2 mRNA的表达水平无明显变化(P〉0.05)。结论达沙替尼抑制ARTyr267、ARTyr534位点磷酸化,其通过抑制heregulin诱导的前列腺癌细胞AR Tyr267特异位点磷酸化及前列腺癌标志物PSA及hK2 mRNA的表达而发挥抗前列腺癌效应。 Objective To investigate the mechanism of dasatinib, tyrosine kinase inhibitor, inhibiting androgen receptor (AR) phosphorylation in prostate cancer cells. Methods HEK-293T and COS7 cell lines were cotransfected by wild-type (WT)-AR, ARY267F or ARY534F with Ackl or Src, respectively, and Western blot was used to detect the AR phosphorylation sites. LNCaP cells were treated by EGF or heregulin without androgen, then Western blot was used to detect AR phosphorylation. After these LNCaP cells were treated by dasatinib or transfection with siRNA to silence Ackl or Src gene, Western blot was used to observe the effect on AR phosphorylation, and quantitative real-time reverse transcription polymerase chain (RT-PCR) was applied to detect PSA mRNA and hk2 mRNA. Results After transfection, Ackl kinase mediated the phosphorylation of AR Tyr267 in HEK-293T cells, and Src mediated AR Tyr534 phosphorylation in COS7 cells. When LNCaP cells were treated by heregulin, AR Tyr267 was phospho@ated, but its phosphorylation was inhibited after these cells were treated by dasatinib or ackl gene was silenced. When LNCaP ceils were treated by EGF, AR Tyr534 was phosphorylated, but its phosphorylation was inhibited after these cells were treated by dasatinib or Src gene was silenced. EGF or heregulin raised endogenous AR target gene, PSA and hK2, mRNA levels in LNCaP cells (P 〈 0.05). However, after these cells were treated by dasatinib, PSA and hK2 mRNA levels induced by heregulin were decreased (P 〈 0.05), but those induced by EGF PSA were no significant changes (P 〉 0.05). Conclusion Dasatinib can inhibit AR Tyr267 and AR Tyr 534 phosphorylation, and it may play a significant role in anti-prostate cancer cells by inhibiting Ackl-mediated AR Tyr-267 phosphorylation and the expression of PSA mRNA and hk2 mRNA induced by heregulin.
出处 《肿瘤研究与临床》 CAS 2016年第6期361-365,共5页 Cancer Research and Clinic
基金 国家自然科学基金(81272842)
关键词 达沙替尼 雄激素受体 前列腺肿瘤 磷酸化 Ack1激酶 SRC激酶 Dasatinib Androgen receptor Prostatic neoplasms Phosphorylation Ackl kinase Src kinase
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