Zhang Yize, Han Chae Young, Duan Fu Gang, Fan Xing-Xing, Yao Xiao-Jun, Parks Robin J, Tang Yi-Jun, Wang Mei-Fang, Liu Liang, Tsang Benjamin K, Leung Elaine Lai-Han
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macao, China.
2Departments of Obstetrics & Gynecology and Cellular & Molecular Medicine, Ottawa Hospital Research Institute, University of Ottawa and Chronic Disease Program, Ottawa, ON K1H 8L6 Canada.
Cancer Cell Int. 2019 Jul 19;19:188. doi: 10.1186/s12935-019-0910-2. eCollection 2019.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths primarily due to chemoresistance. Somatic mutation of 3 (36%) and epidermal growth factor receptor (; > 30%) are major contributors to cisplatin (CDDP) resistance. Substantial evidence suggests the elevated levels of reactive oxygen species (ROS) is a key determinant in cancer. The elevated ROS can affect the cellular responses to chemotherapeutic treatments. Although the role of EGFR in PI3K/Akt signaling cascade in NSCLC is extensively studied, the molecular link between EGFR and p53 and the role of ROS in pathogenesis of NSCLC are limitedly addressed. In this study, we investigated the role of p53 in regulation of ROS production and EGFR signaling, and the chemosensitivity of NSCLC.
In multiple NSCLC cell lines with varied p53 and EGFR status, we compared and examined the protein contents involved in EGFR-Akt-P53 signaling loop (EGFR, P-EGFR, Akt, P-Akt, p53, P-p53) by Western blot. Apoptosis was determined based on nuclear morphological assessment using Hoechst 33258 staining. Cellular ROS levels were measured by dichlorofluorescin diacetate (DCFDA) staining followed by flow cytometry analysis.
We have demonstrated for the first time that activation of p53 sensitizes chemoresistant NSCLC cells to CDDP by down-regulating EGFR signaling pathway and promoting intracellular ROS production. Likewise, blocking EGFR/PI3K/AKT signaling with PI3K inhibitor elicited a similar response. Our findings suggest that CDDP-induced apoptosis in chemosensitive NSCLC cells involves p53 activation, leading to suppressed EGFR signaling and ROS production. In contrast, in chemoresistant NSCLC, activated Akt promotes EGFR signaling by the positive feedback loop and suppresses CDDP-induced ROS production and apoptosis.
Collectively, our study reveals that the interaction of the p53 and Akt feedback loops determine the fate of NSCLC cells and their CDDP sensitivity.
非小细胞肺癌（NSCLC）是癌症死亡的主要原因，主要是由于化疗耐药。3（36%）和表皮生长因子受体（; > 30%）的体细胞突变是顺铂（CDDP）耐药的主要因素。大量证据表明活性氧（ROS）水平升高是癌症的关键决定因素。ROS升高可影响细胞对化疗治疗的反应。尽管EGFR在NSCLC的PI3K/Akt信号级联中的作用已得到广泛研究，但EGFR与p53之间的分子联系以及ROS在NSCLC发病机制中的作用研究较少。在本研究中，我们研究了p53在调节ROS产生和EGFR信号传导中的作用，以及NSCLC的化疗敏感性。
在多种具有不同p53和EGFR状态的NSCLC细胞系中，我们通过蛋白质印迹法比较并检测了参与EGFR-Akt-P53信号环（EGFR、P-EGFR、Akt、P-Akt、p53、P-p53）的蛋白质含量。使用Hoechst 33258染色基于核形态学评估确定细胞凋亡。通过二氯荧光素二乙酸酯（DCFDA）染色，然后进行流式细胞术分析来测量细胞ROS水平。
我们首次证明，p53的激活通过下调EGFR信号通路和促进细胞内ROS产生，使化疗耐药的NSCLC细胞对CDDP敏感。同样，用PI3K抑制剂阻断EGFR/PI3K/AKT信号传导也引发了类似的反应。我们的研究结果表明，CDDP诱导的化疗敏感NSCLC细胞凋亡涉及p53激活，导致EGFR信号传导和ROS产生受到抑制。相反，在化疗耐药的NSCLC中，激活的Akt通过正反馈环促进EGFR信号传导，并抑制CDDP诱导的ROS产生和细胞凋亡。
总体而言，我们的研究表明p53和Akt反馈环的相互作用决定了NSCLC细胞的命运及其对CDDP的敏感性。
