南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (9): 1493-1499.doi: 10.12122/j.issn.1673-4254.2023.09.06

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阿美替尼具有较好的抗神经母细胞瘤作用：基于下调MMP2和MMP9的表达

刘雪柔，杨玉梅，蔡 慧，张耀帅，范方田，李 娴，李姗姗   

  1. 蚌埠医学院药学院，安徽省生化药物工程技术研究中心，安徽 蚌埠 233030
  • 出版日期:2023-09-20 发布日期:2023-09-28

Aumolertinib inhibits proliferation, invasion and migration and promotes apoptosis of neuroblastoma cells by downregulating MMP2 and MMP9 expression

LIU Xuerou, YANG Yumei, CAI Hui, ZHANG Yaoshuai, FAN Fangtian, LI Xian, LI Shanshan   

  1. School of Pharmacy, Bengbu Medical College, Anhui Provincial Engineering Technology Research Center of Biochemical Pharmaceuticals, Bengbu 233030, China
  • Online:2023-09-20 Published:2023-09-28

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摘要： 目的 探究表皮生长因子受体酪氨酸激酶抑制剂阿美替尼对神经母细胞瘤SH-SY5Y的作用。方法 CCK-8法、集落克隆法和流式细胞术检测不同浓度阿美替尼（0、2、4、8 μmol/L）对SH-SY5Y细胞增殖、细胞存活和细胞凋亡的影响；透射电镜观察阿美替尼处理后SH-SY5Y细胞亚显微结构；Transwell小室实验检测阿美替尼对SH-SY5Y细胞侵袭及迁移的作用；Western blot实验检测阿美替尼对SH-SY5Y细胞凋亡和侵袭迁移相关蛋白Bax、Bcl-2、E-Cadherin、Vimentin、MMP2、MMP9表达的影响；SH-SY5Y细胞注射到5周龄BALB/C裸鼠皮下，建立裸鼠皮下瘤动物模型，每组6只，设置空白对照组、阿美替尼处理组（15 mg/kg）、阳性药环磷酰胺处理组（20 mg/kg），测量裸鼠肿瘤体积及体质量变化，HE染色观察阿美替尼对荷瘤裸鼠心、肝、脾、肺、肾的毒副作用。结果 CCK-8结果表明，相比于对照组，阿美替尼能够抑制SH-SY5Y细胞增殖（P<0.05），其作用24、48、72 h的IC50分别为5.004、3.728、3.228 μmol/L；集落克隆结果表明阿美替尼能够抑制SH-SY5Y细胞存活；流式细胞术结果表明阿美替尼诱导 SH-SY5Y 细胞发生凋亡（P<0.05），透射电镜下观察到阿美替尼处理后具有典型的细胞凋亡形态学特征；Transwell小室实验结果表明阿美替尼能够抑制SH-SY5Y细胞的侵袭迁移能力（P<0.01）；Western blot实验结果表明阿美替尼处理后SH-SY5Y细胞中E-Cadherin、Bax蛋白表达水平上调，Bcl-2、Vimentin、MMP2、MMP9蛋白表达水平下调（P<0.05）；动物实验结果表明阿美替尼能够抑制神经母细胞瘤的生长，HE染色结果表明阿美替尼没有对小鼠脏器产生明显毒性。结论 阿美替尼能够抑制神经母细胞瘤SH-SY5Y细胞增殖并诱导细胞发生凋亡，通过下调 MMP2、MMP9 蛋白表达，从而抑制神经母细胞瘤侵袭和迁移。

关键词: 神经母细胞瘤；阿美替尼；增殖；凋亡；侵袭迁移；上皮间质转化

Abstract: Objective To investigate the effects of aumolertinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), on biological behaviors of neuroblastoma SH-SY5Y cells. Methods CCK-8 assay, colony-forming assay, Transwell assay and flow cytometry were used to assess the effects of 2, 4 and 8 μmol/L aumolertinib on proliferation, survival, migration, invasion and apoptosis of SH-SY5Y cells, and the changes in ultrastructure of the cells were observed using transmission electron microscopy. The protein expressions of Bax, Bcl-2, E-cadherin, vimentin, MMP2, and MMP9 in the treated cells were detected using Western blotting. A nude mouse model bearing subcutaneous SH-SY5Y cell xenograft were treated with aumolertinib (15 mg/kg) or cyclophosphamide (20 mg/kg), and the tumor volume and body mass changes was measured. HE staining was used to observe adverse effects of the treatment on the heart, liver, spleen, lungs and kidneys. Results Aumolertinib significantly inhibited the proliferation and viability of SH-SY5Y cells (P<0.05) with IC50 of 5.004, 3.728 and 3.228 μmol/L at 24, 48 and 72 h, respectively. Aumolertinib treatment induced obvious apoptosis of the cells, which showed characteristic morphological changes of apoptosis under transmission electron microscope. The treatment also inhibited the invasion and migration abilities of SH-SY5Y cells (P<0.01), up-regulated the expression levels of E-cadherin and Bax and lowered the expression levels of Bcl-2, vimentin, MMP2 and MMP9 (P<0.05). In the nude mouse models, treatment with aumolertinib effectively inhibited the growth of neuroblastoma without causing significant toxicity to the vital organs. Conclusion Aumolertinib inhibits proliferation, survival, invasion and migration and induces apoptosis in SH-SY5Y cells by downregulating MMP2 and MMP9 expression.

Key words: neuroblastoma; aumolertinib; proliferation; apoptosis; invasion; migration; epithelial mesenchymal transition