Edwards Lincoln A, Woo Janet, Huxham Lynsey A, Verreault Maite, Dragowska Wieslawa H, Chiu Gigi, Rajput Ashish, Kyle Alastair H, Kalra Jessica, Yapp Donald, Yan Hong, Minchinton Andrew I, Huntsman David, Daynard Tim, Waterhouse Dawn N, Thiessen B, Dedhar Shoukat, Bally Marcel B
Department of Advanced Therapeutics, BC Cancer Agency, Vancouver, British Columbia, Canada.
Mol Cancer Ther. 2008 Jan;7(1):59-70. doi: 10.1158/1535-7163.MCT-07-0329.
Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1alpha). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser473), secreted VEGF, G2-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm3 versus control 549 mm3). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1alpha expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas.
在胶质母细胞瘤异种移植模型中，通过多个终点评估整合素连接激酶（ILK）作为治疗靶点，这些终点包括肿瘤微环境中与治疗相关的变化。在体外测试胶质母细胞瘤细胞系对小分子抑制剂QLT0254和QLT0267的敏感性。使用MTT法、流式细胞术、半胱天冬酶激活和DAPI染色评估细胞活力、细胞周期和细胞凋亡。蛋白质印迹法和酶联免疫吸附测定法用于蛋白质分析（ILK、蛋白激酶B/蛋白激酶B（PKB/Akt）、血管内皮生长因子（VEGF）和缺氧诱导因子-1α（HIF-1α））。使用QLT0267（200毫克/千克，每日一次）口服治疗RAG2-M小鼠的U87MG胶质母细胞瘤异种移植模型，对生长速率、细胞增殖、5-溴脱氧尿苷（BrdUrd）、血管质量（CD31标记）、血管灌注（Hoechst 33342）和缺氧（EF-5）进行体内评估。用QLT0254和QLT0267在体外抑制ILK导致磷酸化PKB/Akt（Ser473）水平降低、分泌的VEGF水平降低、G2-M期阻滞和诱导细胞凋亡。用QLT0267治疗的小鼠肿瘤生长显著延迟（治疗组为213立方毫米，对照组为549立方毫米）。与未治疗的小鼠相比，QLT0267治疗的小鼠U87MG肿瘤细胞增殖的原位分析显著降低。重要的是，与对照肿瘤相比，QLT0267治疗的肿瘤中VEGF和HIF-1α表达降低，血管质量百分比和Hoechst 33342灌注的肿瘤血管数量也降低（35%对83%）。用新型小分子抑制剂抑制ILK会导致与治疗相关的肿瘤生长延迟、肿瘤血管生成减少以及肿瘤脉管系统功能改变。应在临床上对表现出ILK失调的癌症（如PTEN缺失的胶质母细胞瘤）中确定所选ILK抑制剂（QLT0267）的治疗效果。
