Liu Yaming, Ge Xianming, Pang Jinlong, Zhang Yuhan, Zhang Hao, Wu Hongyan, Fan Fangtian, Liu Hao
Faculty of Pharmacy, Bengbu Medical College, Bengbu, China.
Department of Pharmacy, Bengbu Third People's Hospital, Bengbu, China.
Front Pharmacol. 2021 May 14;12:671328. doi: 10.3389/fphar.2021.671328. eCollection 2021.
The emergence of secondary resistance is the main failure cause of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a targeted therapy for non-small cell lung cancer (NSCLC). EGFR mutations of NSCLC cells can markedly increase glutamine transporter (SLC1A5) expression, thereby increasing glutamine metabolism. Glutamine metabolites can activate EGFR downstream signals, including mTOR, ERK1/2, STAT3, etc., which is an important cause for the decreased sensitivity of NSCLC to EGFR-TKIs. CCK8 and Annexin V/PI assays were conducted to detect the effects of Almonertinib and/or V9302 on the proliferation and apoptosis of NSCLC cells. Proteomics was used to determine the effect of Almonertinib on energy metabolism-related proteins in NSCLC. siRNA transfection was performed to study the effect of SLC1A5 down-regulation on cell proliferation. In addition, the effects of drugs on colony formation capacity were determined by colony formation assay. Immunofluorescence and Western blot were utilized to detect the apoptosis- and autophagy-related proteins expression. DAPI staining was utilized to detect the effect of drugs on the nucleus. Transmission electron microscope was used to observe the changes of submicroscopic structure such as autophagosomes and nucleus of cells. mCherry-GFP-LC3B tandem fluorescent protein was to used to detect the level of autophagy flux. Tumor-bearing nude mouse model was utilized to detect the effect of V9302 on the anti-tumor effect of Almonertinib . As a result, Almonertinib suppressed H1975 and A549 cell proliferation depended on its dosage and treatment duration, and it also induced apoptosis. A549 cells with wild-type EGFR had lower sensitivity to Almonertinib. The expression of SLC1A5 was up-regulated by stimulating with low concentration of Almonertinib in NSCLC cells. SLC1A5 was highly expressed in A549 cells with wild-type EGFR. Glutamine deletion or SLC1A5 inhibition/silencing inhibited the proliferation of NSCLC cells, and decreased cellular glutamine uptake. The combination of SLC1A5 inhibitor V9302 and Almonertinib had a synergistic inhibitory effect on the proliferation of NSCLC. V9302 enhanced the effect of Almonertinib in apoptosis-inducing in NSCLC cells. The combination of V9302 and Almonertinib might induce apoptosis by inhibiting autophagy.
继发性耐药的出现是表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）作为非小细胞肺癌（NSCLC）靶向治疗主要失败原因。NSCLC细胞的EGFR突变可显著增加谷氨酰胺转运体（SLC1A5）表达，从而增加谷氨酰胺代谢。谷氨酰胺代谢产物可激活EGFR下游信号，包括mTOR、ERK1/2、STAT3等，这是NSCLC对EGFR-TKIs敏感性降低的重要原因。进行CCK8和Annexin V/PI检测以检测阿美替尼和/或V9302对NSCLC细胞增殖和凋亡的影响。蛋白质组学用于确定阿美替尼对NSCLC中能量代谢相关蛋白的影响。进行siRNA转染以研究SLC1A5下调对细胞增殖的影响。此外，通过集落形成试验确定药物对集落形成能力的影响。利用免疫荧光和蛋白质印迹检测凋亡和自噬相关蛋白表达。利用DAPI染色检测药物对细胞核的影响。使用透射电子显微镜观察细胞自噬体和细胞核等亚微观结构的变化。使用mCherry-GFP-LC3B串联荧光蛋白检测自噬通量水平。利用荷瘤裸鼠模型检测V9302对阿美替尼抗肿瘤作用的影响。结果，阿美替尼抑制H1975和A549细胞增殖，这取决于其剂量和治疗持续时间，并且它还诱导凋亡。具有野生型EGFR的A549细胞对阿美替尼敏感性较低。在NSCLC细胞中，低浓度阿美替尼刺激可上调SLC1A5的表达。SLC1A5在具有野生型EGFR的A549细胞中高表达。谷氨酰胺缺失或SLC1A5抑制/沉默可抑制NSCLC细胞增殖，并降低细胞谷氨酰胺摄取。SLC1A5抑制剂V9302与阿美替尼联合对NSCLC细胞增殖具有协同抑制作用。V9302增强了阿美替尼诱导NSCLC细胞凋亡的作用。V9302与阿美替尼联合可能通过抑制自噬诱导凋亡。
