顺铂诱导多倍体肿瘤巨细胞模型的构建

doi:10.3969/j.issn.1004-616x.2024.06.010

摘要/Abstract

摘要： 目的： 探讨顺铂在体外诱导肿瘤细胞构建多倍体肿瘤巨细胞(PGCC)模型的方法，并对构建的细胞模型特性进行考察。方法： 分别采用1.5、3、6、12、24 μg/mL的顺铂处理A549细胞24 h，以及3 μg/mL的顺铂处理3、4、5 d，流式细胞术检测其DNA含量变化，选择DNA含量最高的组合作为顺铂诱导A549、HepG2、SK-OV-3细胞形成PGCC的浓度和时间；Giemsa染色观察3种已处理细胞的表面积和DNA核面积变化；逆转录定量PCR检测3种已处理细胞的干性基因Nanog、Sox-2、OCT-4、c-Myc的mRNA表达情况，流式细胞术检测3种已处理细胞的干性表面标志物CD44、CD133的表达情况；β-半乳糖苷酶染色检测3种已处理细胞的衰老情况；MTT法检测0.75、1.5、3、6、12、24、48、96 μg/mL顺铂作用于3种已处理细胞72 h的细胞活力。结果： 3 μg/mL顺铂处理3 d可有效诱导3种细胞系形成多倍体(>4N)细胞。诱导后的细胞表现出以下特征：细胞和核面积显著增大；干性标志物CD44、CD133表达不同程度上调，部分干性基因表达增加；部分A549细胞呈现衰老表型；与对照组相比，源于A549和SK-OV-3细胞的PGCC对顺铂的耐受性增强(P<0.01)。结论： 成功建立了顺铂诱导的源于A549细胞的PGCC模型，该模型诱导的A549 PGCC具有增大的细胞表面积和核面积，表达干性基因Nanog、Sox-2、OCT-4，对顺铂的耐受度增加，为进一步研究肿瘤耐药机制提供了有效的实验平台。

关键词: 顺铂, 多倍体肿瘤巨细胞, A549细胞, HepG2细胞, SK-OV-3细胞

Abstract: OBJECTIVE： To investigate the characteristics and mechanisms of cisplatin-induced polyploid giant cancer cells (PGCCs) in vitro. METHODS： Cultured cells were treated with cisplatin at concentrations of 1.5，3，6，12，and 24 μg/mL for 3，4，and 5 days. DNA content was analyzed by flow cytometry to determine the optimal combination of concentration and duration for PGCC induction in A549，HepG2，and SK-OV-3 cells. Morphological changes and DNA nuclear area were examined through morphological observation and Giemsa staining. Expression of stemness genes (Nanog，Sox-2，OCT-4，c-Myc) was evaluated by RT-qPCR，while stemness surface markers (CD44，CD133) were analyzed by flow cytometry. Cell senescence was assessed by β-galactosidase staining. After 72-hour treatment with cisplatin at concentrations ranging from 0.75 to 96 μg/mL，cell viability was measured by the MTT assay. RESULTS： Treatment with 3 μg/mL cisplatin for 3 d effectively induced polyploidy (>4N) in all three cell lines. The induced cells exhibited significantly increased cell and nuclear areas；varying degrees of upregulation in stemness markers CD44 and CD133，along with partial increase in stemness gene expression；partial senescence phenotype in A549 cells；and enhanced cisplatin tolerance in A549 and SK-OV-3 PGCCs compared to controls. CONCLUSION： The cisplatin-induced PGCC model in A549 cells demonstrated enlarged cell and nuclear areas，expressed stemness genes (Nanog，Sox-2，OCT-4)，and showed increased cisplatin tolerance. The model may be useful for investigating，drug resistance mechanisms.

Key words: cisplatin, polyploid giant cancer cell, A549 cells, HepG2 cells, SK-OV-3 cells

中图分类号:

R730.2

李子烜, 黄吉, 孙震晓. 顺铂诱导多倍体肿瘤巨细胞模型的构建[J]. 癌变·畸变·突变, 2024, 36(6): 483-490.

LI Zixuan, HUANG Ji, SUN Zhenxiao. Construction of a cisplatin-induced polyploid giant cancer cell model[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(6): 483-490.

参考文献

[1] GJELSVIK K J, BESEN-MCNALLY R, LOSICK V P. Solving the polyploid mystery in health and disease[J]. Trends Genet, 2019, 35(1): 6-14.
[2] COWARD J, HARDING A. Size does matter: why polyploid tumor cells are critical drug targets in the war on cancer[J]. Front Oncol, 2014, 4: 123.
[3] RABIK C A, DOLAN M E. Molecular mechanisms of resistance and toxicity associated with platinating agents[J]. Cancer Treat Rev, 2007, 33(1): 9-23.
[4] DAVIS E, TENG H, BILICAN B, et al. Ectopic Tbx2 expression results in polyploidy and cisplatin resistance[J]. Oncogene, 2008, 27(7): 976-984.
[5] ZHANG S, MERCADO-URIBE I, XING Z, et al. Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[J]. Oncogene, 2014, 33(1): 116-128.
[6] WHITE-GILBERTSON S, VOELKEL-JOHNSON C. Giants and monsters: Unexpected characters in the story of cancer recurrence[J]. Adv Cancer Res, 2020, 148: 201-232.
[7] JIAO Y Q, YU Y J, ZHENG M Y, et al. Dormant cancer cells and polyploid giant cancer cells: the roots of cancer recurrence and metastasis[J]. Clin Transl Med, 2024, 14(2): e1567.
[8] ZHANG D, YANG X Y, YANG Z D, et al. Daughter cells and erythroid cells budding from PGCCs and their clinicopathological significances in colorectal cancer[J]. J Cancer, 2017, 8(3): 469-478.
[9] LIU K, ZHENG M Y, ZHAO Q, et al. Different p53 genotypes regulating different phosphorylation sites and subcellular location of CDC25C associated with the formation of polyploid giant cancer cells[J]. J Exp Clin Cancer Res, 2020, 39(1): 83.
[10] 夏天, 纪妍, 陆颖娜, 等. 自噬通过诱导休眠多倍体巨大肿瘤细胞形成促进鼻咽癌复发的研究[J].-中华耳鼻咽喉头颈外科杂志, 2022, 57(09): 1102-1109.
[11] PUSTOVALOVA M, BLOKHINA T, ALHADDAD L, et al. CD44⁺ and CD133⁺ non-small cell lung cancer cells exhibit DNA damage response pathways and dormant polyploid giant cancer cell enrichment relating to their p53 status[J]. Int J Mol Sci, 2022, 23(9): 4922.
[12] NIU N, MERCADO-URIBE I, LIU J. Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells[J]. Oncogene, 2017, 36(34): 4887-4900.
[13] MAHERALI N, HOCHEDLINGER K. Guidelines and techniques for the generation of induced pluripotent stem cells[J]. Cell Stem Cell, 2008, 3(6): 595-605.
[14] 张忆雪, 周明, 谯英固, 等. 脱水淫羊藿素维持人尿源性干细胞干性的作用研究[J]. 癌变·畸变·突变, 2022, 34(6): 449-454, 458.
[15] DASARI S, TCHOUNWOU P B. Cisplatin in cancer therapy: molecular mechanisms of action[J]. Eur J Pharmacol, 2014, 740: 364-378.
[16] MIRZAYANS R, ANDRAIS B, MURRAY D. Do multiwell plate high throughput assays measure loss of cell viability following exposure to genotoxic agents?[J]. Int J Mol Sci, 2017, 18(8): 1679.
[17] NAJAFI M, MORTEZAEE K, MAJIDPOOR J. Cancer stem cell (CSC) resistance drivers[J]. Life Sci, 2019, 234: 116781.
[18] AMEND S R, TORGA G, LIN K C, et al. Polyploid giant cancer cells: Unrecognized actuators of tumorigenesis, metastasis, and resistance[J]. Prostate, 2019, 79(13): 1489-1497.
[19] FEI F, ZHANG D, YANG Z D, et al. The number of polyploid giant cancer cells and epithelial-mesenchymal transition-related proteins are associated with invasion and metastasis in human breast cancer[J]. J Exp Clin Cancer Res, 2015, 34: 158.
[20] SWAIN N, THAKUR M, PATHAK J, et al. SOX2, OCT4 and NANOG: The core embryonic stem cell pluripotency regulators in oral carcinogenesis[J]. J Oral Maxillofac Pathol, 2020, 24(2): 368-373.