Chang Gee-Chen, Hsu Shih-Lan, Tsai Jia-Rong, Liang Fong-Pin, Lin Sheng-Yi, Sheu Gwo-Tarng, Chen Chih-Yi
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, ROC.
Biochem Pharmacol. 2004 Oct 1;68(7):1453-64. doi: 10.1016/j.bcp.2004.06.006.
Epithelial growth factor receptor (EGFR) has been proposed as a target for anticancer therapy. ZD1839 (Iressa) is a quinazoline derivative that selectively inhibits the EGFR tyrosine kinase activity and is under clinical use in cancer patients. However, the molecular mechanisms involved in ZD1839-mediated anticancer effects remain largely uncharacterized. In this study, exposure of human lung adenocarcinoma A549 cells to ZD1839 caused G1 arrest, and subsequently induced apoptosis. Moreover, ZD1839 increased the protein levels of p27(KIP1) and retinoblastoma-related Rb2/p130 while decreased the expression of cyclin-dependent kinase-2 (CDK2), CDK4, CDK6 and cyclin-D1, cyclin-D3. In vitro kinase assay showed that ZD1839 decreased these CDKs expression in A549 cells, leading to significantly reduce their kinase activities. In addition, ZD1839-induced death of A549 cells with characteristics of apoptosis including apoptotic morphological changes, DNA fragmentation and enhancement of TUNEL-positive cell. These events were accompanied by a marked increase of Fas protein expression, and activation of caspase-2, -3, -8. Co-treatment of cells with Fas antagonist antibody significantly blocked ZD1839-induced apoptosis. Caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, were also capable of restoring cell viability. Our results indicate that downregulation of the expression and function of CDK2, CDK4, CDK6, cyclin-D1 and cyclin-D3, as well as upregulation of p27(KIP1) and pRb2/p130, are strong candidates for the cell cycle regulator that arrests ZD1839-treated A549 cells at G1 phase. Furthermore, upregulation of Fas appears to play a major role in the initiation of ZD1839-induced apoptosis, activation of caspase-8/caspase-3 cascade is involved in the execution phase of this death program.
表皮生长因子受体（EGFR）已被提议作为抗癌治疗的靶点。ZD1839（易瑞沙）是一种喹唑啉衍生物，可选择性抑制EGFR酪氨酸激酶活性，目前正在癌症患者中进行临床应用。然而，ZD1839介导的抗癌作用所涉及的分子机制在很大程度上仍未明确。在本研究中，人肺腺癌A549细胞暴露于ZD1839会导致G1期阻滞，并随后诱导细胞凋亡。此外，ZD1839增加了p27（KIP1）和视网膜母细胞瘤相关蛋白Rb2/p130的蛋白水平，同时降低了细胞周期蛋白依赖性激酶-2（CDK2）、CDK4、CDK6以及细胞周期蛋白-D1、细胞周期蛋白-D3的表达。体外激酶分析表明，ZD1839降低了A549细胞中这些CDK的表达，导致其激酶活性显著降低。此外，ZD1839诱导A549细胞死亡，具有凋亡特征，包括凋亡形态变化、DNA片段化以及TUNEL阳性细胞增加。这些事件伴随着Fas蛋白表达的显著增加，以及半胱天冬酶-2、-3、-8的激活。用Fas拮抗剂抗体共同处理细胞可显著阻断ZD1839诱导的细胞凋亡。半胱天冬酶-8和半胱天冬酶-3抑制剂，但不是半胱天冬酶-9抑制剂，也能够恢复细胞活力。我们的结果表明，CDK2、CDK4、CDK6、细胞周期蛋白-D1和细胞周期蛋白-D3的表达和功能下调，以及p27（KIP1）和pRb2/p130的上调，是使ZD1839处理的A549细胞在G1期阻滞的细胞周期调节因子的有力候选者。此外，Fas的上调似乎在ZD1839诱导的细胞凋亡起始中起主要作用，半胱天冬酶-8/半胱天冬酶-3级联反应的激活参与了该死亡程序的执行阶段。
