精品原癌基因pim3在胰腺癌发生中的作用机制以及分子靶向治课件

原癌基因原癌基因Pim-3Pim-3在胰腺癌发生中的作在胰腺癌发生中的作 用机制以及分子靶向治疗药物的开发用机制以及分子靶向治疗药物的开发 复旦大学附属肿瘤医院实验研究中心 复旦大学上海医学院肿瘤学系 李 影奕 1. Pim-3激酶在胰腺癌发生中的作用机制 2. 以Pim-3为靶向治疗分子的新型有效的 抗肿瘤药物的开发 报告内容报告内容 1. Pim-3激酶在胰腺癌发生中的作用机制 p34 p34 p34 p37 p40 Kinase Domain Pim-1 Pim-2 Pim-3 Deneen B. et al. Mol Cell Biol 2003;23:3897 Mikkers H. et al. Mol Cell Biol 2004;24:6104 Pim-3 与恶性转化相关Pim基因敲出的小鼠生 命力旺盛且可繁殖后代 Pim-3是原癌基因。 研究背景研究背景 Pim-3 is a proto-oncogene with serine/threonine kinase activity. Fujii C et al. Int J Cancer. 2005 Mar 20;114(2):209-18. Pim-3激酶是否与肝脏以外的其它脏器肿瘤发生有关？ 我们聚焦在同是内胚层来源的器官，胰腺。 Normal Tissue Cancer Tissues Pim-3 Pim-3 蛋白在人胰腺癌组织和胰腺蛋白在人胰腺癌组织和胰腺 癌细胞株中高表达癌细胞株中高表达 PCI55 PCI35 -Pim-3 Actin 36 43 kDa WCL Pim-3 shRNA Non Scramble Pim-3 PCI55 Pim-3 siRNA Pim-3 siRNA 干扰干扰Pim-3Pim-3蛋白表达，导致人胰腺蛋白表达，导致人胰腺 癌细胞株细胞凋亡，从而降低细胞活力癌细胞株细胞凋亡，从而降低细胞活力 Scramble shRNAPim-3 shRNA DNA Content No shRNA SubG1 Annexin V Pim-3 shRNA Scramble shRNA Pim-3 shRNA Scramble shRNA No-shRNA Scramble shRNA Pim3 shRNA 0 1 2 3 4 5 8 7 6 5 4 3 2 1 0 Cell viability ratio Time after passage (days) * * * * Pim-1, Pim-2 Pim-3 ? Bad Ser136 Survival Factor PI3K AKT Bad 14-3-3 Survival Ser112 Pim-1 and Pim-2 Can Inactiavte Pro-apoptotic Molecule, Bad, by Phosphorylating Its Serine Residues Apoptosis Bad Bcl-XL + Bcl-XL Pim-3BadPim-3/Bad PCI55 20mm Pim-3 Pim-3 和和 Bad Bad 的相互作用的相互作用 a-Pim3 HeLa a-Bad a-IgG IP: PCI35 PCI55 PCI55 25 26 kDa Bad IgG Blot: anti-Pim3 Blot: anti-Bad 50 36 Pim-3 IgG Immunoprecipitation with anti-Pim-3 Western blotting with anti-Bad Washing with Stripping buffer Western blotting with anti-Pim-3 Pim-3pBad112Pim-3/pBad112 PCI35 PCI55 20mm Pim-3Pim-3和磷酸化和磷酸化BadBad在细胞质内共定位在细胞质内共定位 Actin Bad pBad112 pBad136 pBad155 Pim-3 26 26 26 26 43 KDa 36 shRNA Non Scramble Pim-3 PCI55 降低降低Pim-3 Pim-3 蛋白表达可以减少蛋白表达可以减少 Bad Bad112 112磷酸化 磷酸化 小结（1） Pim-3 Pim-3 对于难治性胰腺癌来说是对于难治性胰腺癌来说是 一个很好的靶向治疗分子一个很好的靶向治疗分子 2. 以Pim-3为靶向治疗分子的新型有效 的抗肿瘤药物开发 胰腺癌是我国十大恶性肿瘤之一 早期诊断困难 晚期患者手术治疗5年生存率低于5%，发病率等于死亡率 传统的化学治疗存在抗肿瘤能力低，杀伤指数过小，副作用大等缺点 分子靶向治疗具有特异性强，效果显著，正常组织损伤小等优点 现有的抑制增殖因子受体活性的分子靶向治疗缺乏肿瘤治疗的特异性，而且容 易产生耐药性 研究背景研究背景 Pim-3Pim-3作为药物靶点，特异性更强。作为药物靶点，特异性更强。 Pim-3与Pim-1具有高度的同源性（70%）和类似的生物功能 推测Pim-3的立体空间结构 低分子化合物-Pim-3激酶复合物立体空间结构 从DrugBank和PubChem数据库中筛选了23种类似低分子化合物 低分子化合物的筛选低分子化合物的筛选 选择并合成了对Pim-3有高亲和性的小分子化合物-蔓生百部 碱（stemonamide）合成中间体 蔓生百部碱是百部属生物碱中的一种，其在中蔓生百部碱是百部属生物碱中的一种，其在中 国和日本民间医学中作为镇咳药。国和日本民间医学中作为镇咳药。 合成顺序：合成顺序：T-10 T-6, T-10 T-4 T-5, T-10 T-2 T-3T-10 T-6, T-10 T-4 T-5, T-10 T-2 T-3 蔓生百部碱合成中间体结构蔓生百部碱合成中间体结构 Pim-3 Pim-1 Pim-2 T2, T5T2, T5体外抑制体外抑制PimPim 激酶活性激酶活性 IC50 (mmol/L) Pim-3 Pim-1 Pim-2 AKT-1 AKT-2 T2 T5 T10 AKT1/2 78 92 2000 nd 90 56 2000 nd 33 50 2000 nd 1934 2000 2000 2000 31.58 72.47 345 462 T-2T-2和和T-5T-5抑制抑制PimPim激酶活性，却不抑制激酶活性，却不抑制AKT/PKBAKT/PKB的活性的活性 Times after treatment with inhibitor (days) Cell proliferation ratio T-2T-2T-5T-5 L3.6plL3.6pl T-10T-10 Miapaca-2Miapaca-2 T-2T-2， T-5 T-5体外抑制人胰腺癌细胞株的细胞增殖体外抑制人胰腺癌细胞株的细胞增殖 Cell line name T-2T-5 L3.6pl6.626.1 MiaPaca-2 5.922.9 IC50 (mmol/L) 过表达过表达Pim-3Pim-3可以部分逆转由可以部分逆转由T-2T-2引起的细胞活力下降引起的细胞活力下降 Relative OD450 MiaPaca-2 T-2 T-2 引起人胰腺癌细胞产生细胞凋亡引起人胰腺癌细胞产生细胞凋亡 T-2T-2抑制抑制Pim-3Pim-3激酶活性，阻止激酶活性，阻止pBadpBad112 112, , 减少 减少Bcl-XBcl-X L L 的表达，的表达， 依赖细胞色素依赖细胞色素C C和和Caspase-3Caspase-3途径导致细胞凋亡。途径导致细胞凋亡。 -DMSO T-2 +12 h +48 h +24 h pBad112 Bad Bcl-XL Cytochrome C Cleaved-caspase-3 L3.6pl Actin pBad136 Caspase-3 Cdc25A PIM-3 pBad155 5X105 L3.6pl Subcutaneous injection 36 15days Sacrify : Intraperitoneal injection 15 16 17 18 1929 30 31 32 3322 23 24 25 26(days) T-2 (20mg/kg) T-2T-2抑制裸鼠皮下胰腺肿瘤的生长抑制裸鼠皮下胰腺肿瘤的生长 T-2T-2引起裸鼠皮下胰腺肿瘤细胞凋亡和抑制细胞增殖引起裸鼠皮下胰腺肿瘤细胞凋亡和抑制细胞增殖 T-2 T-2 抑制肿瘤新生血管形成抑制肿瘤新生血管形成 * T-2 T-2 对裸鼠血清中的一些生化指标没有影响对裸鼠血清中的一些生化指标没有影响 DMSO (N=5) T2 (N=5) 20 DMSOT2 0 5 10 15 ALT (U/l) NS Na (mEq/l) 144 148 152 DMSOT2 156 160 NS 0 2 4 6 8 DMSOT2 K(mEq/l) 10 NS DMSOT2 Cl (mEq/l) 0 108 112 116 120 NS DMSO T2 (n=5) (n=6) Hematological parameters 9.30.07 8.080.33 49.883.06 46.721.90 30.043.84 9.91.70 84.21.90 81.171.10 00 0.170.07 00 00 13.401.83 16.671.00 2.400.46 2.000.15 RBC Platelets WBC Neutrophil Eosinophils Basophil Lymphocytes Monocytes 106/ml 104/ml 103/ml % % % % % BALB/C裸鼠全血中白细胞正常值 3.841.64 103/ml T-2T-2抑制荷瘤小鼠全血中白细胞的数量增多，抑制荷瘤小鼠全血中白细胞的数量增多， 使其恢复到正常使其恢复到正常 蔓生百部碱可以作为设计蔓生百部碱可以作为设计Pim-3Pim-3激酶抑制剂的先导化合物激酶抑制剂的先导化合物 开发新药，应用临床，为难治性胰腺癌提供新的治疗方法开发新药，应用临床，为难治性胰腺癌提供新的治疗方法 小结（2） 线粒体 谢谢！ zyq4ww)!8FaQhFIlBb9nM3qBu-5IAL)mJWRpBbbv!t%lxlQK(Dof6#vPG-T#dH3tFy+2zld1YPR3OXzW!WNHL)IOmEDq8$U%7hZ#bJeSfxt)0xfchccqYO2(D%apJ5LG4)fOo2Mb6dQWC+kBTkMRqz-SPTNT3KKahtWnYizvzu&85&PXSuQEL3debk8O&PC*54-Bv8ytN&#J9vIGi+h7Mub$CBpG9XY46Qvn)vvJ#Q%2+UvBk0)tf5rbG$uF+lXyhg*gzn#h0R6J)QzWWYb+RUG0Cb0(xpa2LER-G21AC4uof*gep&PWLlc!JpF9VpplKZntv7fj0ES4AD!rkBC!Knl$8X&hVr3G%$)c9BW8f-dOJPJ-yKo14t5%*cQrbJ3OXWqvUMIt4-ie1LMW%- 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