多西他赛诱导的多倍体肿瘤细胞在肿瘤复发中的作用

doi:10.3760/cma.j.cn371439-20200103-00032

摘要/Abstract

摘要：

目的研究多西他赛诱导的多倍体肿瘤细胞迁移、上皮间质转化特性及免疫细胞对其杀伤作用,以探讨多倍体肿瘤细胞在肿瘤复发中的潜在作用。方法使用1 μmol/L多西他赛处理人非小细胞肺癌A549细胞24 h后收集的细胞为Doc 1 d组,撤除药物,细胞继续在新鲜的完全培养基中培养至第3天或第5天后收集的细胞分别为Doc 3 d组和Doc 5 d组,以DMSO处理A549细胞24 h作为对照组。采用免疫荧光染色法检测细胞形态,流式细胞术检测细胞倍性,划痕实验检测细胞迁移能力,Western blotting检测细胞上皮间质转化相关蛋白的表达,乳酸脱氢酶释放法评估细胞因子诱导杀伤(CIK)细胞的杀伤活性。结果与对照组相比,Doc 1 d组、Doc 3 d组、Doc 5 d组细胞多数发生凋亡,少数存活的细胞体积明显增大,且细胞核呈多核状态。对照组、Doc 1 d组、Doc 3 d组、Doc 5 d组多倍体细胞亚群(DNA含量>4N)比例分别为(1.93±0.55)%、(22.97±2.37)%、(51.30±12.51)%、(67.87±8.31)%,4组间差异具有统计学意义(F=26.521,P<0.001),Doc 1 d组、Doc 3 d组、Doc 5 d组多倍体细胞亚群比例均明显高于对照组,差异均具有统计学意义(均P<0.001),且随着撤药时间的延长,Doc 3 d组、Doc 5 d组多倍体细胞亚群比例明显高于Doc 1 d组,差异均具有统计学意义(P=0.009;P=0.004)。对照组细胞在培养24 h、48 h后,伤口愈合率均达100%;Doc 3 d组在培养24 h、48 h后,伤口愈合率分别为(39.10±2.12)%、(46.13±5.32)%,差异无统计学意义(t=2.126,P=0.051);与对照组24 h、48 h相比,Doc 3 d组细胞迁移能力明显降低,差异均具有统计学意义(t=49.756,P<0.001;t=30.825,P<0.001)。与对照组相比,Doc 1 d组、Doc 3 d组和Doc 5 d组细胞中E-cadherin蛋白表达逐渐下调,Vimentin蛋白表达逐渐上调,Snail蛋白、N-cadherin蛋白表达均未发生明显改变。CIK细胞对对照组、Doc 3 d组、Doc 5 d组细胞的杀伤效率分别为(27.27±1.91)%、(17.87±2.35)%、(9.47±0.51)%,3组间差异具有统计学意义(F=11.294,P<0.001),Doc 3 d组、Doc 5 d组细胞的杀伤效率明显低于对照组,差异均具有统计学意义(P=0.004;P<0.001),Doc 5 d组细胞的杀伤效率明显低于Doc 3 d组,差异具有统计学意义(P=0.003)。结论多西他赛诱导的多倍体肿瘤细胞迁移能力减弱,但易发生上皮间质转化,且免疫细胞对其杀伤作用降低。

关键词: 癌,非小细胞肺, 多倍体肿瘤细胞, 多西他赛, 上皮间质转化

Abstract:

Objective To study the migration of polyploid tumor cells induced by docetaxel, the characteristics of epithelial-mesenchymal transition, and the killing effect of immune cells on them, in order to explore the potential role of polyploid tumor cells in tumor recurrence. Methods The human non-small cell lung cancer A549 cells were treated with 1 μmol/L docetaxel for 24 h, and the cells were collected as Doc 1 d group. After drug removal, the cells were cultured in fresh and complete medium for 3 or 5 days, then the cells were collected as Doc 3 d group or Doc 5 d group respectively. The A549 cells were treated with DMSO for 24 h as control group. Immunofluorescence staining was used to detect cell morphology, flow cytometry was used to analyze cell ploidy, scratch test was used to detect cell migration, Western blotting was used to detect the expression of epithelial-mesenchymal transition related proteins, and lactate dehydrogenase release method was used to evaluate the killing activity of cytokine-induced killer (CIK) cells. Results Compared with the control group, most of the cells in the Doc 1 d group, Doc 3 d group and Doc 5 d group were apoptotic, a few of the surviving cells were significantly larger, and the nucleus was polynuclear. The proportions of polyploid cell subset (DNA content > 4N) in the control group, Doc 1 d group, Doc 3 d group and Doc 5 d group were (1.93±0.55)%, (22.97±2.37)%, (51.30±12.51)% and (67.87±8.31)% respectively, and the difference among the four groups was statistically significant (F=26.521, P<0.001). The proportion of polyploid cell subset in Doc 1 d group, Doc 3 d group and Doc 5 d group was significantly higher than that in the control group (all P<0.001). With the prolongation of withdrawal time, the proportion of polyploid cell subset in Doc 3 d group and Doc 5 d group was significantly higher than that in Doc 1 d group (P=0.009; P=0.004). After 24 h and 48 h culture, the wound healing rates of the control group were both 100%, and the wound healing rates of the Doc 3 d group were (39.10±2.12)% and (46.13±5.32)% respectively, with no significant difference (t=2.126, P=0.051). Compared with the control group at 24 h and 48 h, the cell migration abilities of Doc 3 d group were significantly lower (t=49.756, P<0.001; t=30.825, P<0.001). Compared with the control group, the expression of E-cadherin protein decreased gradually in the Doc 1 d group, Doc 3 d group and Doc 5 d group, the expression of Vimentin protein increased gradually, and the expressions of Snail protein and N-cadherin protein did not change significantly. The killing efficiencies of CIK cells against the cells of the control group, Doc 3 d group and Doc 5 d group were (27.27±1.91)%, (17.87±2.35)%, (9.47±0.51)% respectively, and the difference was statistically significant (F=11.294, P<0.001). The killing efficiency of Doc 3 d group and Doc 5 d group was significantly lower than that of the control group (P=0.004; P<0.001). The killing efficiency of Doc 5 d group was significantly lower than that of Doc 3 d group (P=0.003). Conclusion The migration ability of polyploid tumor cells induced by docetaxel is weakened, but epithelial-mesenchymal transition is likely to occur, and the killing effect of immune cells on them is reduced.

Key words: Carcinoma,non-small-cell lung, Polyploid tumor cells, Docetaxel, Epithelial-mesenchymal transition

王丽丽, 赵松, 欧阳明玥, 谢晓冬, 邢思宁, 刘硕, 于卉影. 多西他赛诱导的多倍体肿瘤细胞在肿瘤复发中的作用[J]. 国际肿瘤学杂志, 2020, 47(6): 340-345.

Wang Lili, Zhao Song, Ouyang Mingyue, Xie Xiaodong, Xing Sining, Liu Shuo, Yu Huiying. Role of docetaxel induced polyploid tumor cells in tumor recurrence[J]. Journal of International Oncology, 2020, 47(6): 340-345.

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参考文献 20

[1]	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. DOI: 10.1038/onc.2013.96. doi: 10.1038/onc.2013.96
[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. DOI: 10.3389/fonc.2014.00123. doi: 10.3389/fonc.2014.00123 pmid: 24904834
[3]	Amend SR, Torga G, Lin KC, et al. Polyploid giant cancer cells: unrecognized actuators of tumorigenesis, metastasis, and resistance[J]. Prostate, 2019,79(13):1489-1497. DOI: 10.1002/pros.23877. doi: 10.1002/pros.23877 pmid: 31376205
[4]	杜均祥, 彭东旭, 潘莹, 等. Tau低表达对肺腺癌患者多西他赛化疗敏感性及疾病进展时间关系的研究[J]. 临床肺科杂志, 2019,24(6):1111-1115. DOI: 10.3969/j.issn.1009-6663.2019.06.037.
[5]	鲁凯, 刘燕文, 李惠, 等. BRCA1在乳腺癌中的表达及与多西他赛疗效的关系[J]. 中华内分泌外科杂志, 2016,10(5):382-385. DOI: 10.3760/cma.j.issn.1674-6090.2016.05.008.
[6]	Ishida S, Masuguchi K, Kawashiri T, et al. Effects of diluent volume and administration time on the incidence of anaphylaxis following docetaxel therapy in breast cancer[J]. Biol Pharm Bull, 2020,43(4):663-668. DOI: 10.1248/bpb.b19-00876. doi: 10.1248/bpb.b19-00876 pmid: 32238707
[7]	于卉影, 孙英慧, 蔺迪, 等. 肿瘤患者自体CIK细胞输注增强再次制备时CD3⁺CD56⁺细胞的体外扩增能力 [J]. 细胞与分子免疫学杂志, 2014,30(7):748-753, 758.
[8]	李淑青, 陈亚萍. 卵巢癌对顺铂和紫杉醇耐药的分子机制[J]. 国际妇产科学杂志, 2016,43(2):145-150.
[9]	张赛, 邱明宁, 汤焕城, 等. 去势抵抗性前列腺癌发生多西他赛耐药的相关机制及其治疗进展[J]. 肿瘤, 2016,36(10):1165-1170. DOI: 10.3781/j.issn.1000-7431.2016.55.436.
[10]	Ogden A, Rida PC, Knudsen BS, et al. Docetaxel-induced polyploidization may underlie chemoresistance and disease relapse[J]. Cancer Lett, 2015,367(2):89-92. DOI: 10.1016/j.canlet.2015.06.025. doi: 10.1016/j.canlet.2015.06.025 pmid: 26185000
[11]	Tsubaki M, Takeda T, Ogawa N, et al. Overexpression of survivin via activation of ERK1/2, Akt, and NF-κB plays a central role in vincristine resistance in multiple myeloma cells[J]. Leuk Res, 2015,39(4):445-452. DOI: 10.1016/j.leukres.2015.01.016. doi: 10.1016/j.leukres.2015.01.016 pmid: 25726084
[12]	Mittal K, Donthamsetty S, Kaur R, et al. Multinucleated polyploidy drives resistance to docetaxel chemotherapy in prostate cancer[J]. Br J Cancer, 2017,116(9):1186-1194. DOI: 10.1038/bjc.2017.78. doi: 10.1038/bjc.2017.78 pmid: 28334734
[13]	Yeung KT, Yang J. Epithelial-mesenchymal transition in tumor metastasis[J]. Mol Oncol, 2017,11(1):28-39. DOI: 10.1002/1878-0261.12017. doi: 10.1002/1878-0261.12017 pmid: 28085222
[14]	Mittal V. Epithelial mesenchymal transition in tumor metastasis[J]. Annu Rev Pathol, 2018,13:395-412. DOI: 10.1146/annurev-pathol-020117-043854. doi: 10.1146/annurev-pathol-020117-043854 pmid: 29414248
[15]	Papadaki MA, Stoupis G, Theodoropoulos PA, et al. Circulating tumor cells with stemness and epithelial-to-mesenchymal transition features are chemoresistant and predictive of poor outcome in metasta-tic breast cancer[J]. Mol Cancer Ther, 2019,18(2):437-447. DOI: 10.1158/1535-7163.MCT-18-0584. doi: 10.1158/1535-7163.MCT-18-0584 pmid: 30401696
[16]	van Staalduinen J, Baker D, Ten Dijke P, et al. Epithelial-mesenchymal-transition-inducing transcription factors: new targets for tackling chemoresistance in cancer?[J]. Oncogene, 2018,37(48):6195-6211. DOI: 10.1038/s41388-018-0378-x. doi: 10.1038/s41388-018-0378-x pmid: 30002444
[17]	Schmeel LC, Schmeel FC, Coch C, et al. Cytokine-induced killer (CIK) cells in cancer immunotherapy: report of the international re-gistry on CIK cells (IRCC)[J]. J Cancer Res Clin Oncol, 2015,141(5):839-849. DOI: 10.1007/s00432-014-1864-3. doi: 10.1007/s00432-014-1864-3 pmid: 25381063
[18]	Introna M. CIK as therapeutic agents against tumors[J]. J Autoi-mmun, 2017,85:32-44. DOI: 10.1016/j.jaut.2017.06.008.
[19]	梁雪峰, 郑振东, 朴瑛, 等. 自体CIK细胞治疗联合 DP 方案一线化疗治疗晚期肺腺癌临床研究[J]. 肿瘤预防与治疗, 2013,26(2):68-71. DOI: 10.3969/j.issn.1674-0904.2013.02.003.
[20]	李宁, 田永巍, 高岭, 等. 自体CIK细胞联合放化疗治疗中晚期子宫颈癌的疗效[J]. 中国肿瘤生物治疗杂志, 2016,23(6):830-834. DOI: 10.3872/j.issn.1007-385X.2016.06.016.