研究目的：肺癌是常见的恶性肿瘤之一，在我国的肺癌患者中85%为非小细胞肺癌（non-small cell lung cancer, NSCLC）。目前临床上针对晚期非小细胞肺癌多采用靶向药物联合放射治疗，研究表明表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）和血管内皮生长因子受体（Vascular Endothelial Growth Factor Receptor, VEGFR）的异常激活通常会促进癌细胞的生长增殖，它们作为经典靶点在NSCLC的治疗中发挥重要作用。凡德他尼（Vandetanib）是已上市的多靶点酪氨酸激酶抑制剂，课题组在前期合成了以其结构为基础的一系列衍生化合物，筛选出活性最强的小分子化合物IRM-39。本研究旨在研究IRM-39对非小细胞肺癌的抑制效果，确证对细胞中p-EGFR和p-VEGFR2的靶向作用，探索IRM-39联合放射抑制非小细胞肺癌的作用机制。
研究方法：（1）通过体外激酶实验验证小分子化合物IRM-39对p-EGFR/p-VEGFR2的抑制效果。免疫印迹实验检测筛选出p-EGFR/p-VEGFR2表达较高的肿瘤细胞。采用CCK-8法检测不同浓度IRM-39对NSCLC细胞活力的影响，平板克隆形成实验检测IRM-39联合放射对NSCLC细胞的抑制效果。单细胞凝胶电泳实验和免疫荧光实验联合检测 IRM-39联合照射对NSCLC细胞DNA损伤的影响。通过流式细胞术检测IRM-39联合放射对细胞内活性氧水平的影响，观察其对细胞周期分布及细胞凋亡的影响，通过蛋白免疫印迹实验检测细胞凋亡信号通路变化。（2）通过免疫印迹实验研究IRM-39对促凋亡蛋白的降解途径的影响，透射电镜实验检测IRM-39联合放射对NSCLC细胞内自噬体的影响，免疫印迹实验检测相关信号通路变化。转染mRFP-GFP-LC3质粒，免疫荧光实验检测IRM-39联合放射对自噬标志物LC3的影响。免疫印迹实验研究IRM-39联合放射通过调控细胞自噬对促凋亡蛋白以及氧化应激蛋白的影响，流式细胞术检测调控自噬-溶酶体途径对细胞凋亡的影响。免疫印迹实验检测IRM-39联合照射调控EGFR/VEGFR下游通路以及对自噬信号的影响。（3）建立裸鼠皮下移植肿瘤模型，观察IRM-39联合放射对体内肿瘤生长以及裸鼠体重的影响。免疫印迹实验检测IRM-39联合照射对体内肿瘤中p-EGFR及p-VEGFR2蛋白的抑制作用。利用HE染色检测IRM-39对裸鼠脏器的影响。
研究结果：（1）IRM-39对p-EGFR/p-VEGFR2的抑制能力优于阳性药凡德他尼。A549和H1975细胞p-EGFR/p-VEGFR2蛋白的表达更高。IRM-39对A549和H1975细胞的平均IC50分别为3.45 μM和2.89 μM，且联合放射对两种细胞有较强的增殖抑制效果。IRM-39联合放射显著增加NSCLC细胞内的活性氧水平，且加重了放射诱导的DNA损伤。IRM-39联合照射降低G2/M期细胞检查点的阻滞从而发生凋亡，相关促凋亡蛋白表达水平增加。（2）实验发现促凋亡蛋白的降解途径受到了抑制。IRM-39联合放射诱导NSCLC细胞自噬发生，同时增强了自噬标志物LC3绿色荧光水平，说明抑制了细胞自噬途径。接着发现IRM-39联合放射通过调控自噬-溶酶体途径抑制促凋亡蛋白的降解导致细胞凋亡从而发挥抗肿瘤作用。（3）IRM-39联合照射显著抑制了裸鼠体内肿瘤的生长，但不影响裸鼠体重，并且药物对裸鼠脏器无明显影响。IRM-39联合照射能够降低体内肿瘤中p-EGFR及p-VEGFR2蛋白的水平。
研究结论：本研究验证了小分子化合物IRM-39对EGFR/VEGFR靶点在体内外的抑制作用，并联合放射对非小细胞肺癌的增殖抑制效果优于阳性药凡德他尼。实验证明，IRM-39联合放射对NSCLC具有较强的抑制效果，其可能机制为通过调控细胞自噬途径抑制促凋亡蛋白的降解，从而导致NSCLC细胞的凋亡。本研究为临床治疗NSCLC提供了参考。
 

Objective: Lung cancer is one of the most common malignancies, and 85% of lung cancer patients in China are non-small cell lung cancer (NSCLC). Currently, most advanced NSCLC is treated clinically with targeted drugs in combination with radiation therapy, and studies have shown that abnormal activation of the Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor (VEGFR) is often associated with abnormal activation of the EGFR and VEGFR receptors. The aberrant activation of EGFR and VEGFR usually promotes the growth and proliferation of cancer cells and therefore plays an important role as a classical target in the treatment of NSCLC. Vandetanib is a marketed multi-target tyrosine kinase inhibitor, and we has synthesized a series of derivatives based on its structure and selected the most active small molecule, IRM-39. The study was to investigate the inhibitory effect of IRM-39 on NSCLC, to confirm the targeting of p-EGFR and p-VEGFR2 in cells, and to explore the mechanism of IRM-39 in combination with radiation inhibition of NSCLC.
Methods: (1) The inhibition of p-EGFR/p-VEGFR2 by the small molecule IRM-39 was verified by in vitro kinase assay. Tumour cells with high p-EGFR/p-VEGFR2 expression were screened by immunoblotting assay. The CCK-8 assay was used to detect the effect of different concentrations of IRM-39 on the viability of NSCLC cells, and the clone formation assay was used to detect the inhibition of IRM-39 combined with radiation on NSCLC cells. Single cell gel electrophoresis assay and immunofluorescence assay were used to detect the effect of IRM-39 combined with radiation on DNA damage in NSCLC cells. The effect of IRM-39 combined radiation on intracellular reactive oxygen levels was detected by flow cytometry, and its effect on cell cycle distribution and apoptosis was observed, and changes in apoptotic signaling pathways were detected by protein immunoblotting assay. (2) The effect of IRM-39 on the degradation pathway of pro-apoptotic proteins was investigated by immunoblotting assay, the effect of IRM-39 combined with radiation on autophagosomes in NSCLC cells was detected by transmission electron microscopy, and the changes of related signaling pathways were detected by immunoblotting assay. The mRFP-GFP-LC3 plasmid was transfected and the effect of IRM-39 co-radiation on autophagy marker LC3 was detected by immunofluorescence assay. Immunoblotting assay was performed to investigate the effect of IRM-39 co-radiation on pro-apoptotic proteins and oxidative stress proteins through regulation of autophagy, and flow cytometry was performed to detect the effect of regulation of autophagy-lysosome pathway on apoptosis. Immunoblotting assays were performed to examine the effect of IRM-39 combined irradiation on the regulation of the EGFR/VEGFR downstream pathway and on autophagic signalling. (3) A subcutaneous transplantation tumor model was established in nude mice to observe the effects of IRM-39 combined irradiation on tumor growth and body weight in vivo. Immunoblotting assay was performed to detect the inhibition of p-EGFR and p-VEGFR2 expression in tumors in vivo by IRM-39 combined irradiation. The effect of IRM-39 on the organs of nude mice was detected using HE staining.
Results: (1) IRM-39 inhibited p-EGFR/p-VEGFR2 better than the positive drug Vandetanib. Higher expression of p-EGFR/p-VEGFR2 protein are in A549 and H1975 cells. The IC50 of IRM-39 was 3.45 μM and 2.89 μM in A549 and H1975 cells, respectively, and the combined radiation had a stronger effect on both cell proliferation inhibition. The combined radiation of IRM-39 reduced blockage of G2/M phase cellular checkpoints which induced apoptosis in NSCLC cells and increased the expression of related pro-apoptotic proteins.  (2) IRM-39 combined with radiation induced autophagy in NSCLC cells and enhanced the green fluorescence level of LC3, a marker of autophagy, indicating that the autophagy pathway was inhibited. It was then found that IRM-39 combined with radiation inhibited the degradation of pro-apoptotic proteins through modulation of the autophagy-lysosome pathway, leading to apoptosis and thus exerting anti-tumor effects. (3) IRM-39 combined radiation significantly inhibited tumor growth in nude mice, but did not affect their body weight, and the drug had no significant effect on nude mice organs. IRM-39 combined radiation reduced the levels of p-EGFR and p-VEGFR2 proteins in tumours in vivo.
Conclusion: In the study, the inhibition of small molecule IRM-39 on EGFR/VEGFR targets in vitro and in vivo was verified, and the proliferation inhibition effect of IRM-39 in combination with radiation was superior to that of the positive drug Vandetanib in NSCLC. The study demonstrated that IRM-39 combined with radiation has a strong inhibition on NSCLC. The study provides a reference for the clinical treatment of NSCLC.