Zhang Yun-Gang, Du Qiang, Fang Wei-Gang, Jin Mu-Lan, Tian Xin-Xia
Department of Pathology, Peking University Health Science Center, Beijing 100083, P R China.
Int J Oncol. 2008 Sep;33(3):595-602.
Inhibition of epidermal growth factor receptor (EGFR) signaling is a promising treatment strategy for malignant tumors. In this study, we evaluated the effectiveness of tyrphostin AG1478, a potent and specific inhibitor of EGFR tyrosine kinase, on the growth, apoptosis and invasion of breast cancer cells. Western blotting demonstrated that AG1478 inhibited the phosphorylation of EGFR, ERK1/2 and AKT in a dose-dependent manner. Three proliferation analyses, MTT, cell counting, and clone formation assay, consistently showed that AG1478 significantly inhibited cell proliferation in a dose-dependent manner. FACS analysis demonstrated that AG1478 promoted cell apoptosis. In addition, TRAP assay exhibited that AG1478 significantly suppressed telomerase activity of tumor cells, which was parallel with growth inhibition. Semi-qantitative RT-PCR revealed that the suppression of telomerase activity was correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. These data suggest that AG1478 suppressed cellular growth by inhibiting cellular proliferation, inducing apoptosis and inhibiting telomerase activity. Furthermore, we also examined the effects of AG1478 on cellular invasion. Boyden chamber invasion assay showed that AG1478 significantly inhibited cell invasion in a dose-dependent manner. Western blotting revealed that AG1478 could down-regulate the expression of MMP-9, which may be one of the mechanisms by which AG1478 suppressed cellular invasion. In conclusion, this study demonstrated that Tyrphostin AG1478 effectively inhibited the proliferation and invasion of breast cancer cells. Tyrphostin AG1478 may be a potential EGFR-targeted therapeutic agent for breast cancer.
抑制表皮生长因子受体（EGFR）信号传导是一种很有前景的恶性肿瘤治疗策略。在本研究中，我们评估了EGFR酪氨酸激酶的强效特异性抑制剂 tyrphostin AG1478 对乳腺癌细胞生长、凋亡和侵袭的影响。蛋白质免疫印迹法表明，AG1478 以剂量依赖性方式抑制 EGFR、ERK1/2 和 AKT 的磷酸化。MTT、细胞计数和克隆形成试验这三种增殖分析一致表明，AG1478 以剂量依赖性方式显著抑制细胞增殖。流式细胞术分析表明，AG1478 促进细胞凋亡。此外，端粒重复序列扩增法（TRAP 法）显示，AG1478 显著抑制肿瘤细胞的端粒酶活性，这与生长抑制情况平行。半定量逆转录聚合酶链反应（RT-PCR）显示，端粒酶活性的抑制与人类端粒酶催化亚基（hTERT）mRNA 表达的降低相关，hTERT mRNA 是其酶活性的限速决定因素。这些数据表明，AG1478 通过抑制细胞增殖、诱导凋亡和抑制端粒酶活性来抑制细胞生长。此外，我们还研究了 AG1478 对细胞侵袭的影响。Boyden 小室侵袭试验表明，AG1478 以剂量依赖性方式显著抑制细胞侵袭。蛋白质免疫印迹法显示，AG1478 可下调基质金属蛋白酶-9（MMP-9）的表达，这可能是 AG1478 抑制细胞侵袭的机制之一。总之，本研究表明 tyrphostin AG1478 有效抑制乳腺癌细胞的增殖和侵袭。tyrphostin AG1478 可能是一种潜在的针对 EGFR 的乳腺癌治疗药物。
