Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Curr Cancer Drug Targets. 2010 May;10(3):319-31. doi: 10.2174/156800910791190229.
The present study is the first to show in pancreatic cancer (PC) the growth inhibition and apoptosis by novel MDM2 inhibitors (MI-319 & 219) through reactivation of p53 pathway. Our results highlight two new secondary targets of MDM2 inhibitor 'SIRT1' and Ku70. SIRT1 which has a role in ageing and cancer and is known to regulate p53 signaling through acetylation. Ku70 is a key component of non-homologous end joining machinery in the DNA damage pathway and is known to regulate apoptosis by blocking Bax entry into mitochondria. Growth inhibition and apoptosis by MI-219, MI-319 was accompanied by increase in levels of p53 along with p21(WAF1) and the proapoptotic Puma. SiRNA against p21(WAF1) abrogated the growth inhibition of PC cells confirming p21(WAF1) as a key player downstream of activated p53. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 interaction in drug exposed PC cells. In combination studies, the inhibitors synergistically augmented anti-tumor effects of therapeutic drug gemcitabine both in terms of cell growth inhibition as well as apoptosis. Surface plasmon resonance studies confirmed strong binding between MI-319 and Ku70 (K(D) 170 nM). Western blot revealed suppression of SIRT1 and Ku70 with simultaneous upregulation of acetyl-p53 (Lys379) and Bax. Co-Immunoprecipitation studies confirmed that MI-319 could disrupt Ku70-Bax and SIRT1-Bax interaction. Further, using wt-p53 xenograft of Capan-2, we found that oral administration of MI-319 at 300 mg/kg for 14 days resulted in significant tumor growth inhibition without any observed toxicity to the animals. No tumor inhibition was found in mut-p53 BxPC-3 xenografts. In light of our results, the inhibitors of MDM2 warrant clinical investigation as new agents for PC treatment.
本研究首次在胰腺癌（PC）中通过重新激活 p53 通路，展示了新型 MDM2 抑制剂（MI-319 和 MI-219）的生长抑制和凋亡作用。我们的结果突出了 MDM2 抑制剂的两个新的次要靶标“SIRT1”和 Ku70。SIRT1 在衰老和癌症中起作用，并且已知通过乙酰化调节 p53 信号。Ku70 是 DNA 损伤途径中非同源末端连接机制的关键组成部分，并且通过阻止 Bax 进入线粒体来调节凋亡。MI-219 和 MI-319 的生长抑制和凋亡伴随着 p53 水平的增加，以及 p21(WAF1)和促凋亡的 Puma。针对 p21(WAF1)的 siRNA 消除了 PC 细胞的生长抑制，证实了 p21(WAF1)是激活的 p53 下游的关键参与者。免疫沉淀-蛋白质印迹分析显示，药物暴露的 PC 细胞中 MDM2-p53 相互作用减少。在联合研究中，抑制剂协同增强了治疗药物吉西他滨的抗肿瘤作用，无论是在细胞生长抑制还是凋亡方面。表面等离子体共振研究证实了 MI-319 与 Ku70 之间的强结合（K(D)为 170 nM）。蛋白质印迹显示 SIRT1 和 Ku70 受到抑制，同时乙酰化 p53（Lys379）和 Bax 上调。共免疫沉淀研究证实，MI-319 可以破坏 Ku70-Bax 和 SIRT1-Bax 相互作用。此外，使用 Capan-2 的 wt-p53 异种移植，我们发现，在 14 天内口服给予 MI-319 300 mg/kg，导致肿瘤生长显著抑制，而对动物没有观察到毒性。在 mut-p53 BxPC-3 异种移植中未发现肿瘤抑制。鉴于我们的结果，MDM2 的抑制剂值得作为治疗 PC 的新药物进行临床研究。
