Chen Hui, Jia Bin, Zhang Qiang, Zhang Yu
Department of Lung Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Lung Cancer Center, Tianjin, China.
Front Oncol. 2022 Apr 21;12:870636. doi: 10.3389/fonc.2022.870636. eCollection 2022.
Gefitinib (GE) is a first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for patients with advanced non-small cell lung cancer (NSCLC) carrying EGFR activating mutations. However, drug resistance limits the clinical efficacy of gefitinib and ultimately leads to extremely poor clinical benefit. Meclofenamic acid (MA) is a non-steroidal anti-inflammatory drug (NSAID) that relieves moderate and severe pain. In the present study, we aim to determine the MA sensibilization of GE in NSCLC.
MTT assay was conducted to determine the synergistic effect of MA with GE in GE-sensitive and -resistant cell lines based on the Chou-Talalay method. The Annexin V-PI flow cytometry analysis was conducted to evaluate apoptosis. Western blot assay was used to detect alterations of EGFR downstream molecules. Tritium-labeled GE accumulation analysis was used to determine the efflux activity of GE. Dot blot assays were conducted to determine m6A levels after the MA and GE co-administration. Western blot evaluated the expression of FTO, c-Myc, MRP7, BCRP, and apoptotic proteins.
MA showed a significant synergistic effect with GE in GE-resistant NSCLC cells; co-administration of MA with GE induced caspase-related apoptosis in resistant NSCLC cells. Moreover, EGFR downstream molecules, including Akt and MAPKs pathways, were significantly inhibited by the MA-GE combination. Short-term incubation of MA did not alter the efflux of GE; however, after incubation for 24 h, the accumulation of tritium-labeled GE significantly increased. A mechanism study showed that co-administration of MA and GE significantly downregulated BCRP and MRP7 expression in GE-resistant cells; increased N6-methylation was also observed after co-administration. The FTO/c-Myc was determined as target pathways on MA and GE co-administration mechanisms.
Our findings provide novel therapeutic approaches for GE-resistant NSCLC by combination use with MA through FTO-mediated N6-demethylation.
吉非替尼（GE）是用于携带EGFR激活突变的晚期非小细胞肺癌（NSCLC）患者的一线表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI）。然而，耐药性限制了吉非替尼的临床疗效，最终导致临床获益极差。甲氯芬那酸（MA）是一种非甾体抗炎药（NSAID），可缓解中度和重度疼痛。在本研究中，我们旨在确定MA对NSCLC中GE的增敏作用。
基于Chou-Talalay方法，采用MTT法测定MA与GE在GE敏感和耐药细胞系中的协同作用。采用Annexin V-PI流式细胞术分析评估细胞凋亡。采用蛋白质免疫印迹法检测EGFR下游分子的变化。采用氚标记的GE蓄积分析来确定GE的外排活性。采用斑点印迹法测定MA与GE联合给药后的m6A水平。蛋白质免疫印迹法评估FTO、c-Myc、MRP7、BCRP和凋亡蛋白的表达。
MA在GE耐药的NSCLC细胞中与GE显示出显著的协同作用；MA与GE联合给药诱导耐药NSCLC细胞中与半胱天冬酶相关的细胞凋亡。此外，MA-GE组合显著抑制了包括Akt和MAPKs通路在内的EGFR下游分子。MA短期孵育未改变GE的外排；然而，孵育24小时后，氚标记的GE蓄积显著增加。机制研究表明，MA与GE联合给药显著下调了GE耐药细胞中BCRP和MRP7的表达；联合给药后还观察到N6-甲基化增加。FTO/c-Myc被确定为MA与GE联合给药机制的靶标通路。
我们的研究结果通过FTO介导的N6-去甲基化与MA联合使用，为GE耐药的NSCLC提供了新的治疗方法。
