上皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是EGFR突变非小细胞肺癌(NSCLC)的一线治疗方法；然而，产生抗药性的速度非常快。因此，本研究的目标是建立和表征一个Gefitinib耐药的NSCLC细胞系（HCC827GR），并评估天然产物结合第三代EGFR-TKI AZD9291的治疗效果。HCC827GR中Gefitinib和AZD9291的IC50显著高于HCC827 (p<0.05)。此外，无界附着集落实验表明，HCC827GR细胞比其前体更具侵袭性。这反映在HCC827GR与HCC827的基因/蛋白质表达变化上，经由癌症药物抗性实时聚合酶链反应（RT-PCR）和Western blot分析。筛选了三种天然产物，咖啡酸苄酯（CAPE）与AZD9291的综合细胞毒作用最显著。具体而言，流式细胞术显示AZD9291+CAPE显著增加了细胞前G1的比例，触发了细胞周期和Caspase-Glo3/7检测中细胞凋亡的显著增加，与单独AZD9291相比。此外，Western blot显示，与AZD9291相比，HCC827GR细胞经AZD9291+CAPE处理后，p-EGFR/p-AKT显著下调。此外，明显可见的是，AZD9291+CAPE特异地导致了细胞增殖相关基因p21、cyclin D1和survivin蛋白表达的显著降低。最后，经过修订的RT-PCR/Western blot数据表明，AZD9291+CAPE至少部分通过PLK2途径发挥其协同作用。综上，这些结果表明CAPE是一个临床相关化合物，可以辅助AZD9291治疗EGFR-TKI耐药细胞，调控与癌症抗性/治疗相关的关键基因/蛋白质。© 2023国际生物化学和分子生物学联合会。

Epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for EGFR mutated non-small cell lung cancer (NSCLC); however, resistance rapidly develops. The objective of this study was therefore to establish and characterize a gefitinib resistant NSCLC line (HCC827GR) and evaluate the therapeutic effects of natural products in combination with third-generation EGFR-TKI, AZD9291. The IC50 of gefitinib and AZD9291 in HCC827GR were significantly higher than those of HCC827 (p < 0.05). Furthermore, anchorage-independent colony assay indicated that HCC827GR cells were more aggressive than their predecessors. This was reflected by the gene/protein expression changes observed in HCC827GR versus HCC827 profiled by cancer drug resistance real-time polymerase chain reaction (RT-PCR) array and Western blot. Three natural products were screened and caffeic acid phenethyl ester (CAPE) exhibited the most significant combinative cytotoxic effect with AZD9291. Specifically, flow cytometry revealed that AZD9291 + CAPE considerably increased the fraction of cell in pre-G1 of the cell cycle and caspase-Glo3/7 assay showed a dramatic increase in apoptosis when compared to AZD9291 alone. Furthermore, Western blot showed significant downregulation of p-EGFR/p-AKT in HCC827GR cells treated with AZD9291 + CAPE as compared to AZD9291. Moreover, it is evident that AZD9291 + CAPE specifically resulted in a marked reduction in the protein expressions of the cell-proliferation-related genes p21, cyclin D1, and survivin. Finally, refined RT-PCR/Western blot data indicated that AZD9291 + CAPE may at least partially exert its synergistic effects via the PLK2 pathway. Together, these results suggest that CAPE is a clinically relevant compound to aid AZD9291 in treating EGFR-TKI resistant cells through modulating critical genes/proteins involved in cancer resistance/therapy.© 2023 International Union of Biochemistry and Molecular Biology.