Fleming Jason B, Shen Guo-Liang, Holloway Shane E, Davis Mishel, Brekken Rolf A
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593, USA
Mol Cancer Res. 2005 Jul;3(7):413-23. doi: 10.1158/1541-7786.MCR-04-0206.
Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.
K-ras基因的突变是胰腺腺癌发生发展过程中的早期事件，因此，针对突变型K-ras的RNA干扰（RNAi）可能代表一种新的治疗方法。在本研究中，我们检测了胰腺肿瘤细胞暴露于突变特异性K-ras小干扰RNA后的表型和分子变化。通过RNAi特异性降低Panc-1和MiaPaca-2细胞中活化的K-ras，导致细胞发生变化，与形成恶性肿瘤的能力降低一致。这些变化通过不同机制发生，但可能反映了每个细胞系对癌基因刺激的依赖性。两种细胞系在K-ras RNAi后增殖均减少，但只有MiaPaca-2细胞凋亡增加。两种细胞系在K-ras敲低后迁移均减少，但细胞系之间整合素水平的变化不一致。两种细胞系均显示c-myc下游的代谢相关基因GLUT-1水平发生改变，Panc-1细胞GLUT-1水平降低，而MiaPaca-2细胞在K-ras敲低后表达水平升高。此外，K-ras RNAi后，Panc-1和MiaPaca-2细胞的血管生成潜能均降低。Panc-1细胞增加了血管生成内源性抑制剂血小板反应蛋白-1的表达水平，而MiaPaca-2细胞减少了血管内皮生长因子的产生，血管内皮生长因子是胰腺肿瘤血管生成的主要刺激因子。我们发现，通过RNAi沉默突变型K-ras会导致体外肿瘤细胞行为的改变，并表明特异性靶向突变型K-ras可能对体内胰腺癌有效。
