Rusnak D W, Lackey K, Affleck K, Wood E R, Alligood K J, Rhodes N, Keith B R, Murray D M, Knight W B, Mullin R J, Gilmer T M
Department of Cancer Biology, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
Mol Cancer Ther. 2001 Dec;1(2):85-94.
The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.
表皮生长因子受体（EGFR）和ErbB-2跨膜酪氨酸激酶目前在癌症治疗中是多种机制的作用靶点。GW2016是一种强效的ErbB-2和EGFR酪氨酸激酶结构域抑制剂，对纯化的EGFR和ErbB-2的IC50值分别为10.2 nM和9.8 nM。本报告描述了GW2016在细胞生长试验中对过表达EGFR或ErbB-2的人肿瘤细胞系的疗效：HN5（头颈癌）、A-431（外阴癌）、BT474（乳腺癌）、CaLu-3（肺癌）和N87（胃癌）。正常人类包皮成纤维细胞、非致瘤性上皮细胞（HB4a）和未过表达的肿瘤细胞（MCF-7和T47D）作为阴性对照进行检测。化合物暴露3天后，过表达EGFR和ErbB-2的肿瘤细胞系中生长抑制的平均IC50值<0.16 microM。肿瘤细胞与人包皮成纤维细胞系的平均选择性为100倍。通过蛋白质印迹分析在BT474和HN5细胞系中验证了EGFR和ErbB-2受体自身磷酸化以及下游调节因子AKT的磷酸化受到抑制。作为细胞毒性与生长停滞的衡量指标，在短暂暴露于GW2016后，通过体外生长试验对HN5和BT474细胞进行评估。细胞用五种浓度的GW2016处理3天，在去除化合物后再监测12天的细胞生长情况。在这些肿瘤细胞系中的每一种中，都达到了GW2016的浓度，此时不再有体外生长。此外，通过溴脱氧尿苷掺入和碘化丙啶染色确定，在平行实验中观察到了生长停滞和细胞死亡。GW2016治疗以剂量反应方式抑制HN5和BT474细胞的肿瘤异种移植生长，口服剂量为30和100 mg/kg，每日两次，高剂量时完全抑制肿瘤生长。总之，这些结果表明GW2016对肿瘤细胞具有优异的效力，对肿瘤细胞与正常细胞具有选择性，提示GW2016对过表达EGFR或ErbB-2的肿瘤患者具有治疗价值。
