Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells
Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells
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DOI：
10.1007/s00280-006-0219-4
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发表时间：
2006-11-01
影响因子：
3
通讯作者：
Nakano, Shuji
中科院分区：
文献类型：
--
作者：
Qin, Baoli;Ariyama, Hiroshi;Nakano, Shuji
目的：虽然吉非替尼是一种选择性表皮生长因子受体（EGFR）酪氨酸激酶抑制剂，已被证明通过阻断 EGF 受体发挥抗肿瘤活性，但 EGFR 下游信号通路在吉非替尼敏感性中的作用仍不清楚。在这项研究中，我们研究了 Src 和 Ras 这两种主要癌基因产物在吉非替尼敏感性中与许多人类癌症发病机制有关的机制作用。方法：使用亲本和 v-src 或 c-H-ras 转染的 HAG-1 人胆囊腺癌细胞系，分别通过 WST-1 测定、流式细胞术和蛋白质印迹测定吉非替尼对细胞毒性、细胞周期扰动和凋亡以及 EGFR、Akt 和 Erk 酪氨酸磷酸化的影响。结果：激活的 Ras 和 Src 分别赋予吉非替尼近 30 倍和 200 倍的强耐药性。吉非替尼诱导 24 小时细胞周期 G0/G1 期的细胞积累，亲代 HAG-1 细胞中凋亡细胞群逐渐扩大，但这些效应在 v-src 或 c-H-ras 转染的细胞系中完全消失。吉非替尼治疗后，HAG-1 细胞中 EGFR 激活以及随后通过 Erk 和 Akt 进行的下游激活均受到显着抑制。相比之下，吉非替尼未能抑制 v-src 转染细胞中 Akt 和 Erk 的激活，以及 Erk 的激活，但不能抑制 c-H-ras 转染细胞中 Akt 的激活，尽管在这些细胞系中阻断了 EGFR 激活。用除草霉素 A（一种 Src 酪氨酸激酶抑制剂）处理 v-src 转染的细胞，部分逆转了吉非替尼耐药性，同时抑制了 Akt 和 Erk。结论：我们的结果表明，激活的 Ras 和 Src 可以通过激活 Akt 和 Erk 信号通路中的一个或两个来诱导吉非替尼耐药，从而为评估 EGFR 下游的这些特定信号分子以定制治疗提供了战略依据。
Purpose: Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Methods: Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively. Results: Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk. Conclusion: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.