Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, 17 Yongwai Main St, Nanchang, 330006, Jiangxi, China.
Health Management Centre, The First Affiliated Hospital, Chengdu Medical College, 278 Baoguang St, Xindu Distr, Chengdu, 610500, Sichuan, China.
BMC Cancer. 2020 Jul 8;20(1):634. doi: 10.1186/s12885-020-07111-w.
Drug resistance is a major cause of therapeutic failure that is often associated with elevated autophagy and apurinic/apyrimidinic endonuclease 1 (APE1) expression. Herein, we investigated the role of APE1 and autophagy in A549 cells treated with cisplatin.
SILAC proteomics was applied to obtain a panoramic view of cisplatin treatment in KRAS-mutant A549 cells. Quantity analysis of cellular apoptosis and autophagy was based on flow cytometry. Western blotting was used to examine the expression levels of apoptosis- and autophagy-related proteins, as well as those of APE1. Knockdown of APE1 was achieved by RNA interference. Immunoprecipitation was further employed to reveal the molecular interaction of APE1, p53, and LC3 when A549 cells were exposed to cisplatin.
SILAC proteomics revealed that 72 canonical pathways, including base excision repair (BER) and autophagy signalling pathways, were regulated after cisplatin treatment in A549 cells. Cisplatin markedly induced autophagy and apoptosis in A549 cells, accompanied by remarkable APE1 increase. Suppression of autophagy enhanced the inhibition effect of cisplatin on cell growth, proliferation, and colony formation; however, APE1 inhibition enhanced the expression of LC3-I/II, suggesting that APE1 and autophagy are compensatory for cell survival to evade the anticancer action of cisplatin. Immunoprecipitation results revealed the triple complex of APE1-p53-LC3 in response to cisplatin plus CQ in A549 cells. Dual inhibition of APE1 and autophagy significantly enhanced cisplatin-induced apoptosis, which eventually overcame drug resistance in cisplatin-resistant A549 cells.
Dual inhibition of APE1 and autophagy greatly enhances apoptosis in parental KRAS-mutant A549 cells and cisplatin-resistant A549 cells via regulation of APE1-p53-LC3 complex assembly, providing therapeutic vulnerability to overcome cisplatin resistance in the context of KRAS-mutant lung cancer.
耐药性是治疗失败的主要原因，通常与自噬和脱嘌呤/脱嘧啶内切酶 1（APE1）表达升高有关。在此，我们研究了 APE1 和自噬在顺铂处理的 A549 细胞中的作用。
使用 SILAC 蛋白质组学获得 KRAS 突变 A549 细胞中顺铂处理的全景图。基于流式细胞术对细胞凋亡和自噬的数量进行分析。Western blot 用于检测凋亡和自噬相关蛋白以及 APE1 的表达水平。通过 RNA 干扰敲低 APE1。免疫沉淀进一步揭示了 A549 细胞暴露于顺铂时 APE1、p53 和 LC3 的分子相互作用。
SILAC 蛋白质组学揭示了 72 条经典途径，包括碱基切除修复（BER）和自噬信号通路，在 A549 细胞中顺铂处理后被调节。顺铂显著诱导 A549 细胞自噬和凋亡，同时 APE1 显著增加。抑制自噬增强了顺铂对细胞生长、增殖和集落形成的抑制作用；然而，APE1 抑制增强了 LC3-I/II 的表达，表明 APE1 和自噬是细胞存活的补偿，以逃避顺铂的抗癌作用。免疫沉淀结果显示，在 A549 细胞中，顺铂加 CQ 反应产生 APE1-p53-LC3 三聚体。双重抑制 APE1 和自噬显著增强了顺铂诱导的凋亡，最终克服了顺铂耐药的 A549 细胞的耐药性。
双重抑制 APE1 和自噬通过调节 APE1-p53-LC3 复合物组装，极大地增强了 KRAS 突变 A549 细胞和顺铂耐药 A549 细胞中的凋亡，为克服 KRAS 突变肺癌中顺铂耐药提供了治疗弱点。
