药学学报, 2021, 56(11): 3074-3081
引用本文:
郭婉霜, 宋琬晨, 田静, 陈露, 詹明杰, 郑伟娟, 华子春. FADD敲除增强A549细胞对依托泊苷的敏感性[J]. 药学学报, 2021, 56(11): 3074-3081.
GUO Wan-shuang, SONG Wan-chen, TIAN Jing, CHEN Lu, ZHAN Ming-jie, ZHENG Wei-juan, HUA Zi-chun. FADD knockout enhances the sensitivity of A549 cells to etoposide[J]. Acta Pharmaceutica Sinica, 2021, 56(11): 3074-3081.

FADD敲除增强A549细胞对依托泊苷的敏感性
郭婉霜1, 宋琬晨1, 田静2, 陈露2, 詹明杰3, 郑伟娟1*, 华子春1,2,3*
1. 南京大学生命科学学院, 江苏 南京 210023;
2. 南京中医药大学药学院, 江苏 南京 210023;
3. 中国药科大学生物药物学院, 江苏 南京 211198
摘要:
本文主要探究Fas相关死亡域蛋白 (Fas-associated death domain protein,FADD) 敲除后,化疗抗癌药物依托泊苷 (etoposide,VP16) 对非小细胞肺癌 (non-small cell lung cancer,NSCLC) A549细胞在增殖、迁移和凋亡方面的影响。通过CRISPR/Cas9技术构建了FADD敲除A549 (FADD KO A549) 及其对照 (control A549) 细胞。采用CCK-8法检测不同浓度的依托泊苷对control A549细胞和FADD KO A549细胞活力的抑制作用;采用划痕实验检测两株细胞的迁移情况,比较依托泊苷对两株细胞迁移的抑制作用;采用流式细胞术检测两株细胞的凋亡情况,比较依托泊苷对二者凋亡的促进作用。Western blot检测增殖蛋白[c-Raf (raf proto-oncogene serine/threonine-protein kinase) 和p-ERK (extracellular signal-regulated kinase of phosphorylation)]、凋亡蛋白[BCL2 (B-cell lymphoma 2)、cleaved-caspase-3 (cleaved cysteinyl aspartate specific proteinase 3) 以及cleaved-caspase-9] 和迁移蛋白[MMP2 (matrix metalloproteinase 2)] 的表达变化。结果显示,与control A549细胞相比,FADD KO A549细胞迁移和增殖能力减弱,凋亡增加,且对依托泊苷的敏感性增加;c-Raf、p-ERK、MMP2和BCL2蛋白减弱趋势显著;cleaved-caspase-3和cleaved-caspase-9蛋白增加趋势显著。结合GEPIA数据库得到的Kaplan-Meier (KM) 生存曲线分析,初步判断在肺腺癌中FADD基因水平高的患者预后不良。本文提示,FADD可作为肺腺癌治疗潜在的生物标志物,为肺腺癌治疗提供个性化治疗方案。
关键词:    肺腺癌      A549      依托泊苷      Fas相关死亡域蛋白      敏感性      预后     
FADD knockout enhances the sensitivity of A549 cells to etoposide
GUO Wan-shuang1, SONG Wan-chen1, TIAN Jing2, CHEN Lu2, ZHAN Ming-jie3, ZHENG Wei-juan1*, HUA Zi-chun1,2,3*
1. School of Life Sciences, Nanjing University, Nanjing 210023, China;
2. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China;
3. School of Biopharmacy, China Pharmaceutical University, Nanjing 211198, China
Abstract:
We aimed to explore the involvement of Fas-associated death domain protein (FADD) in the inhibitory effects of etoposide (VP16) on the proliferation, migration, and apoptosis of A549 non-small cell lung cancer (NSCLC) cells. FADD knockout (KO) and control A549 cells were constructed using the CRISPR/Cas9 system. The cell counting kit-8 (CCK-8) assay, the scratch wounding assay, and the Annexin V/PI staining-based flow cytometry were used to assess the effect of FADD KO on viability, migration, and apoptosis of A549 cells with or without the presence of etoposide, respectively. The expression pattern of several proteins involved in proliferation[raf proto-oncogene serine/threonine-protein kinase (c-Raf) and extracellular signal-regulated kinase of phosphorylation (p-ERK)], apoptosis[B-cell lymphoma 2 (BCL2), cleaved cysteinyl aspartate specific proteinase 3 (cleaved-caspase-3), and cleaved-caspase-9] and migration[matrix metalloproteinase 2 (MMP2)] was detected by Western blot. We found that FADD KO attenuated proliferation and migration of A549 cells. Consistently, we demonstrated that FADD KO enhanced etoposide-mediated inhibition of proliferation and migration in A549 cells. We further demonstrated that FADD KO obviously enhanced etoposide-mediated apoptosis in A549 cells. For mechanism exploration, we found that etoposide sensitivity enhanced by FADD KO may be partly explained by reduced expression of c-Raf, p-ERK, MMP2, and increased cleavage of caspase-3 and -9. Combined with the Kaplan-Meier (KM) survival curve analysis obtained from the GEPIA database, it is preliminarily judged that patients with high FADD gene levels in lung adenocarcinoma have a poor prognosis. Our study suggests that FADD can be used as a potential biomarker for the treatment of lung adenocarcinoma, providing a personalized treatment plan for the treatment of lung adenocarcinoma.
Key words:    lung adenocarcinoma    A549    etoposide    Fas-associated death domain protein    sensitivity    prognosis   
收稿日期: 2021-04-07
DOI: 10.16438/j.0513-4870.2021-0509
基金项目: 国家自然科学基金资助项目(81630092).
通讯作者: 郑伟娟,Tel:13814039758,E-mail:zchua@nju.edu.cn;华子春,Tel:13705153568,E-mail:wjzheng@nju.edu.cn
Email: zchua@nju.edu.cn;wjzheng@nju.edu.cn
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