Label-free cell phenotypic assessment of the molecular mechanism of action of epidermal growth factor receptor inhibitors
Label-free cell phenotypic assessment of the molecular mechanism of action of epidermal growth factor receptor inhibitors
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DOI：
10.1039/c3ra40426a
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发表时间：
2013-01-01
期刊：
影响因子：
3.9
通讯作者：
Fang, Ye
中科院分区：
文献类型：
--
作者：
Deng, Huayun;Wang, Chaoming;Fang, Ye
表皮生长因子受体（EGFR）是几种临床批准的酪氨酸激酶抑制剂（TKI）药物（包括吉非替尼和厄洛替尼）治疗癌症的靶点。这些药物的临床特征涉及多种机制。然而，人们对这些药物在整个细胞水平上的分子作用机制知之甚少。在这里，我们应用无标记生物传感器使能的动态质量再分布（DMR）测定来评估三种EGFR抑制剂吉非替尼、厄洛替尼和AG 1478改变A431和HT-29（两种表达EGFR的天然癌细胞系）中EGFR信号传导的分子作用机制。使用持续性抑制剂处理的全细胞DMR测定显示，所有抑制剂在两种细胞系中剂量依赖性地抑制EGFR信号传导，但通常在A431中显示出比HT-29细胞更高的效力。具有抑制剂洗脱的DMR测定显示，洗脱意外地增加了吉非替尼和AG-1478抑制A431中EGFR信号传导的效力，但略微降低了所有三种抑制剂在HT 29中的效力。微流控下的DMR测定表明，使用缓冲液灌注去除抑制剂导致EGF信号的时间依赖性恢复，A431细胞中的恢复速度比HT-29细胞慢。相比之下，微流体下的DMR测定显示，去除可逆竞争性拮抗剂导致两种不同的G蛋白偶联受体（GPCR）的信号传导完全恢复，这两种不同的G蛋白偶联受体是A431中的β（2）-肾上腺素能受体和HT-29细胞中的GPR 35。总之，我们的研究结果表明，对于EGFR抑制剂，其摄取和保留，而不是结合动力学，主导其无标记细胞表型功效;然而，对于GPCR拮抗剂，结合特性对抑制作用至关重要。本研究还暗示了DMR测定在不同模拟条件下用于阐明激酶抑制剂药物的细胞表型药理学，特别是转运蛋白相关耐药性的潜力。
Epidermal growth factor receptor (EGFR) is the target of several clinically approved tyrosine kinase inhibitor (TKI) drugs including gefitinib and erlotinib in the treatment of cancer. Multiple mechanisms have been implicated in the clinical features of these drugs. However, little is known about the molecular mechanism of action of these drugs at the whole cell level. Here we applied a label-free biosensor-enabled dynamic mass redistribution (DMR) assay to assess the molecular mechanism of action of three EGFR inhibitors, gefitinib, erlotinib and AG1478, to alter the EGFR signaling in A431 and HT-29, two native cancer cell lines expressing the EGFR. The whole-cell DMR assays with the persistent inhibitor treatment showed that all inhibitors dose-dependently inhibited the EGFR signaling in both cell lines, but generally displayed higher potency in A431 than HT-29 cells. The DMR assays with the inhibitor washout showed that the washout unexpectedly increased the potency of gefitinib and AG-1478 to inhibit the EGFR signaling in A431, but slightly decreased the potency of all three inhibitors in HT29. The DMR assays under microfluidics showed that the removal of the inhibitors using buffer perfusion resulted in a timedependent recovery of EGF signaling that is slower in A431 than HT-29 cells. In contrast, DMR assays under microfluidics showed that the removal of reversible competitive antagonists led to the full recovery of the signalling of two distinct G protein-coupled receptors (GPCRs), the beta(2)-adrenergic receptor in A431 and the GPR35 in HT-29 cells. Together, our results suggest that for EGFR inhibitors their uptake and retention, rather than binding kinetics, dominate their label-free cell phenotypic efficacy; however, for GPCR antagonists the binding characteristics are critical to the inhibitory effects. This study also implicates the potential of DMR assays under different simulation conditions for elucidating the cell phenotypic pharmacology, in particular transporter-related drug resistance, of kinase inhibitor drugs.