Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme

Mingpu LIU; Hongmei WANG; Yuanli WU; Duanfang ZHOU; Weiying ZHOU

doi:10.16016/j.2097-0927.202402065

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Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme

Basic Medicine | 更新时间：2026-01-09

- Construction and mechanism of a small cell lung cancer cell line resistant to EP chemotherapy scheme
- Journal of Army Medical University Vol. 46, Issue 18, Pages: 2092-2100(2024)
- 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：
- 基金信息：
  
  the National Natural Science Foundation of China(82373901);Chongqing Natural Science Foundation Innovation and Development Joint Fund(2022NSCQ-LZX0068);the Innovation Research Group in Colleges and Universities Program of Chongqing Municipal Education Commission(CXQT20012)
- DOI：10.16016/j.2097-0927.202402065
  CLC： R734.2;R915;R979.1
- Received：22 February 2024，
  
  Revised：2024-04-16，
  
  Published：30 September 2024
- 稿件说明：
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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：

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

中华医学会肿瘤学分会, 中华医学会杂志社. 中华医学会肺癌临床诊疗指南(2023版)[J]. 中华医学杂志, 2023, 103(27): 2037-2074. DOI: 10.3760/cma.j.cn112137-20230510-00767.

Oncology Branch of Chinese Medical Association, Editorial office of Journal of Chinese Medical Association . Chinese Medical Association guideline for clinical diagnosis a nd treatment of lung cancer (2023 edition)[J ] . Natl Med J China, 2023, 103(27): 2037-2074. DOI: 10.3760/cma.j.cn112137-20230510-00767.

SUNG H, FERLAY J, SIEGEL R L, et al . Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J ] . CA Cancer J Clin, 2021, 71(3): 209-249. DOI: 10.3322/caac.21660.

PENG A M, LI G S, XIONG M T, et al . Role of surgery in patients with early stage small-cell lung cancer[J ] . Cancer Manag Res, 2019, 11: 7089-7101. DOI: 10.2147/CMAR.S202283.

PAZ-ARES L, DVORKIN M, CHEN Y B, et al . Durvalumab plus platinum-etoposide versus platinum-etoposide in first-line treatment of extensive-stage small-cell lung cancer (CASPIAN): a randomised, controlled, open-label, phase 3 trial[J ] . Lancet, 2019, 394(10212): 1929-1939. DOI: 10.1016/S0140-6736(19)32222-6.

THAI A A, SOLOMON B J, SEQUIST L V, et al . Lung cancer[J ] . Lancet, 2021, 398(10299): 535-554.

HERZOG B H, DEVARAKONDA S, GOVINDAN R. Overcoming chemotherapy resistance in SCLC[J]. J Thorac Oncol, 2021, 16(12): 2002-2015. DOI: 10.1016/j.jtho.2021.07.018.

WANG Y L, YAN Y L, ZHOU N J, et al . Mechanism of multidrug resistance of human small cell lung cancer cell line H446/VP[J ] . Chin Med J, 2010, 123(22): 3299-3303.

CHIKAMORI M, TAKIGAWA N, KIURA K, et al . Establishment of a 7-ethyl-10-hydroxy-camptothecin-resistant small cell lung cancer cell line[J ] . Anticancer Res, 2004, 24(6): 3911-3916.

AMARAL M V S, DE SOUSA PORTILHO A J, DA SILVA E L, et al . Establishment of drug-resistant cell lines as a model in experimental oncology: a review[J ] . Anticancer Res, 2019, 39(12): 6443-6455. DOI: 10.21873/anticanres.13858.

俞毅麟, 肖艺, 赵娟娟, 等. 三阴性乳腺癌耐药细胞模型的构建及其生物学特性考察[J]. 中国药学杂志, 2022, 57(22): 1901-1910. DOI: 10.11669/cpj.2022.22.006.

YU Y L, XIAO Y, ZHAO J J, et al . Establishment of doxorubicin hydrochloride resistant triple negative breast cancer cell model and inves-tigation of its biological characteristics[J ] . Chin Pharm J, 2022, 57(22): 1901-1910. DOI: 10.11669/cpj.2022.22.006.

荣小丽. LIMK1基因稳定转染骨肉瘤耐药细胞系的建立及其增殖侵袭能力的研究[D]. 长春: 吉林大学, 2016.

RONG X L. Establishment of drug-resistant osteosarcoma cell line stably transfected with LIMK1 gene and study on its proliferation and invasion ability[D]. Changchun: Jilin University, 2016.

延冰, 刘国勤, 姜永胜, 等. 高剂量法诱导胃癌铂类耐药细胞株的建立及其生物学特性[J]. 山东大学学报(医学版), 2014, 52(4): 49-52, 57. DOI: 10.6040/j.issn.1671-7554.0.2013.556.

YAN B, LIU G Q, JIANG Y S, et al . Establishment of cisplatin-resistant gastric cancer cell line and the biological characteristics[J ] . J Shandong Univ Health Sci, 2014, 52(4): 49-52, 57. DOI: 10.6040/j.issn.1671-7554.0.2013.556.

CHANG H C, ZOU Z Z. Targeting autophagy to overcome drug resistance: further developments[J]. J Hematol Oncol, 2020, 13(1): 159. DOI: 10.1186/s13045-020-01000-2.

GARCIA-MAYEA Y, MIR C, MASSON F, et al . Insights into new mechanisms and models of cancer stem cell multidrug resistance[J ] . Semin Cancer Biol, 2020, 60: 166-180. DOI: 10.1016/j.semcancer.2019.07.022.

LU J F, POKHAREL D, BEBAWY M. MRP1 and its role in anticancer drug resistance[J]. Drug Metab Rev, 2015, 47(4): 406-419. DOI: 10.3109/03602532.2015.1105253.

ZATTONI I F, DELABIO L C, DUTRA J P, et al . Targeting breast cancer resistance protein (BCRP/ABCG2): Functional inhibitors and expression modulators[J ] . Eur J Med Chem, 2022, 237: 114346. DOI: 10.1016/j.ejmech.2022.114346.

HANSSEN K M, HABER M, FLETCHER J I. Targeting multidrug resistance-associated protein 1 (MRP1)-expressing cancers: Beyond pharmacological inhibition[J]. Drug Resist Updat, 2021, 59: 100795. DOI: 10.1016/j.drup.2021.100795.

OMORI M, NORO R, SEIKE M, et al . Inhibitors of ABCB1 and ABCG2 overcame resistance to topoisomerase inhibitors in small cell lung cancer[J ] . Thorac Cancer, 2022, 13(15): 2142-2151. DOI: 10.1111/1759-7714.14527.

KUO T H, LIU F Y, CHUANG C Y, et al . To predict response chemotherapy using technetium-99m tetrofosmin chest images in patients with untreated small cell lung cancer and compare with p-glycoprotein, multidrug resistance related protein-1, and lung resistance-related protein expression[J ] . Nucl Med Biol, 2003, 30(6): 627-632. DOI: 10.1016/S0969-8051(03)00058-1.

SOUZA I D, MONTEIRO L K S, GUEDES C B, et al . High levels of NRF2 sensitize temozolomide-resistant glioblastoma cells to ferr optosis via ABCC1/MRP1 upregulation[J ] . Cell Death Dis, 2022, 13(7): 591. DOI: 10.1038/s41419-022-05044-9.

ZHANG J, HU J, LI W W, et al . Rapamycin antagonizes BCRP-Mediated drug resistance through the PI3K/Akt/mTOR signaling pathway inmPRα-Positive breast cancer[J ] . Front Oncol, 2021, 11: 608570. DOI: 10.3389/fonc.2021.608570.

GROELLY F J, FAWKES M, DAGG R A, et al . Targeting DNA damage response pathways in cancer[J ] . Nat Rev Cancer, 2023, 23(2): 78-94. DOI: 10.1038/s41568-022-00535-5.

MCMULLEN M, KARAKASIS K, MADARIAGA A, et al . Overcoming platinum and PARP-inhibitor resistance in ovarian cancer[J ] . Cancers, 2020, 12(6): 1607. DOI: 10.3390/cancers12061607.

YUAN J S, ADAMSKI R, CHEN J J. Focus on histone variant H2AX: to be or not to be[J]. FEBS Lett, 2010, 584(17): 3717-3724. DOI: 10.1016/j.febslet.2010.05.021.

JIN M H, OH D Y. ATM in DNA repair in cancer[J]. Pharmacol Ther, 2019, 203: 107391. DOI: 10.1016/j.pharmthera.2019.07.002.

STAGNI V, CIROTTI C, BARILÀ D. Ataxia -telangiectasia mutated kinase in the control of oxidative stress, mitochondria, and autophagy in cancer: a maestro with a large orchestra[J ] . Front Oncol, 2018, 8: 73. DOI: 10.3389/fonc.2018.00073.

FOO T K, XIA B. BRCA1-dependent and independent recruitment of PALB2-BRCA2-RAD51 in the DNA damage response and cancer[J]. Cancer Res, 2022, 82(18): 3191-3197. DOI: 10.1158/0008-5472.CAN-22-1535.

LORD C J, ASHWORTH A. BRCAness revisited[J]. Nat Rev Cancer, 2016, 16(2): 110-120. DOI: 10.1038/nrc.2015.21.

CRUZ C, CASTROVIEJO-BERMEJO M, GUTIÉRREZ-ENRÍQUEZ S, et al . RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer[J ] . Ann Oncol, 2018, 29(5): 1203-1210. DOI: 10.1093/annonc/mdy099.

POILLET-PEREZ L, WHITE E. Role of tumor and host autophagy in cancer metabolism[J]. Genes Dev, 2019, 33(11/12): 610-619. DOI: 10.1101/gad.325514.119.

LI X H, HE S K, MA B Y. Autophagy and autophagy-related proteins in cancer[J]. Mol Cancer, 2020, 19(1): 12. DOI: 10.1186/s12943-020-1138-4.

KLIONSKY D J, ABDEL-AZIZ A K, ABDELFATAH S, et al . Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)[J ] . Autophagy, 2021, 17(1): 1-382. DOI: 10.1080/15548627.2020.1797280.

LAMARK T, SVENNING S, JOHANSEN T. Regulation of selective autophagy: the p62/SQSTM1 paradigm[J]. Essays Biochem, 2017, 61(6): 609-624. DOI: 10.1042/EBC20170035.

ZHANG Y B, WANG J Q, HUI B N, et al . Pristimerin enhances the effect of cisplatin by inhibiting the miR-23a/Akt/GSK3β signaling pathway and suppressing autophagy in lung cancer cells[J ] . Int J Mol Med, 2019, 43(3): 1382-1394. DOI: 10.3892/ijmm.2019.4057.

MA K Y, LI S X, HUO X Y, et al . Exploring the mechanism of cisplatin resistance by transcriptome sequencing and reversing the chemoresistance by autophagy inhibition in small cell lung cancer[J ] . Biochem Biophys Res Commun, 2020, 533(3): 474-480. DOI: 10.1016/j.bbrc.2020.09.023.

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