EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨

刘明菩; 王红梅; 伍元丽; 周端方; 周维英

doi:10.16016/j.2097-0927.202402065

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EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨

基础医学 | 更新时间：2026-01-09

- EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨
- Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme
- 陆军军医大学学报 2024年46卷第18期页码：2092-2100
- 作者机构：
  
  1.400016 重庆，重庆医科大学：药学院药理学系
  2.400016 重庆，重庆医科大学：药物代谢研究重庆市重点实验室
  3.400016 重庆，重庆医科大学：附属第一医院药学部
  4.401147 重庆，重庆市妇幼保健院药学部
- 作者简介：
  
  [周维英, E-mail: wyzhou0118@cqmu.edu.cnZHOU Weiying, E-mail: wyzhou0118@cqmu.edu.cn
- 基金信息：
  
  国家自然科学基金(82373901);重庆市自然科学基金创新发展联合基金(2022NSCQ-LZX0068);重庆市高校创新研究群体项目(CXQT20012)
- DOI：10.16016/j.2097-0927.202402065
  中图分类号： R734.2;R915;R979.1
- 收稿：2024-02-22，
  
  修回：2024-04-16，
  
  纸质出版：2024-09-30
- 稿件说明：
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刘明菩, 王红梅, 伍元丽, 等. EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨[J]. 陆军军医大学学报, 2024,46(18):2092-2100.

Mingpu LIU, Hongmei WANG, Yuanli WU, et al. Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme[J]. Journal of Army Medical University, 2024, 46(18): 2092-2100.
刘明菩, 王红梅, 伍元丽, 等. EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨[J]. 陆军军医大学学报, 2024,46(18):2092-2100. DOI： 10.16016/j.2097-0927.202402065.

Mingpu LIU, Hongmei WANG, Yuanli WU, et al. Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme[J]. Journal of Army Medical University, 2024, 46(18): 2092-2100. DOI： 10.16016/j.2097-0927.202402065.

摘要

目的

构建依托泊苷(etoposide，VP-16)联合顺铂(cisplatin，DDP)化疗方案(EP联合方案)耐药的小细胞肺癌(small cell lung cancer，SCLC)细胞株H446/EP，并进行耐药特性鉴定及机制探讨。

方法

以药物浓度递增法，使用VP-16联合DDP处理NCI-H446细胞构建H446/EP细胞株。以H446/EP与NCI-H446细胞为研究对象，MTT法检测细胞活力，计算H446/EP细胞耐药指数(resistance index，RI)；细胞克隆实验、Incucyte细胞增殖(无标记)法检测细胞增殖能力；转录组学测序后，对2种细胞差异表达基因进行富集分析；Western blot检测细胞耐药、DNA损伤修复(repair of DNA damage，DDR)、自噬标志分子的蛋白表达量。

结果

MTT结果显示，H446/EP细胞对VP-16、DDP与DOX的RI分别为6.14、3.43与1.96；细胞克隆实验与Incucyte细胞增殖(无标记)法结果显示，H446/EP细胞增殖能力显著高于NCI-H446细胞(

0.01)；转录组学测序、通路富集分析显示差异表达基因在肿瘤化疗耐药、DDR、自噬相关通路富集；Western blot结果显示，相比NCI-H446细胞，H446/EP细胞MRP1、BCRP、RAD51、γ-H2AX及LC3-II/LC3-I蛋白表达量显著升高，p62蛋白表达量显著降低(

0.05)。

结论

EP联合方案耐药的SCLC细胞株H446/EP构建成功，增殖能力增强；细胞外排转运蛋白表达量增加、DDR及自噬水平升高可能是SCLC对EP联合方案产生耐药的机制。

Abstract

Objective

To construct the etoposide (VP-16) combined with cisplatin (DDP) chemotherapy scheme (EP chemotherapy scheme) resistant small cell lung cancer (SCLC) cell line H446/EP and to identify the drug resistance characteristics and explore the mechanism.

Methods

NCI-H446 cells were treated with increasing concentrations of VP-16 and DDP to construct an H446/EP cell line. H446/EP and NCI-H446 cells were used as the research objects. The cell viability was detected by MTT assay

and the resistance index (RI) of H446/EP cells was calculated. Cell cloning assay and Incucyte cell proliferation (label-free) assay were used to detect cell proliferation ability. Transcriptome sequencing was performed to analyze the enrichment of differentially expressed genes (DEGs) in the 2 cell lines. Western blotting was applied to detect the protein expression levels of drug resistance

DNA damage repair (DDR)

and autophagy markers.

Results

MTT assay showed that the resistance index (RI) of H446/EP cells to VP-16

DDP

and DOX were 6.14

3.43

and 1.96

respectively. The results of cell cloning assay and Incucyte cell proliferation assay indicated that the proliferation ability was significantly higher in the H446/EP cells than the NCI-H446 cells (

0.01). Transcriptome sequencing and pathway enrichment analysis displayed that the DEGs between H446/EP and NCI-H446 cells were enriched in tumor chemoresistance

DDR

and autophagy pathways. Western blot results showed the expression levels of MRP1

BCRP

RAD51

γ-H2AX

and LC3-II/LC3-I were significantly increased

and that of p62 was obviously decreased in the H446/EP cells when compared with the NCI-H446 cells (

0.05).

Conclusion

An EP chemotherapy-resistant H446/EP cell line is successfully constructed. Stronger proliferation ability

increased expression of efflux transporters

and enhanced DDR and autophagy may be the mechanisms of the resistance of SCLC to EP chemotherapy scheme.

关键词

Keywords

references

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