CDK7抑制剂THZ1对人胶质瘤细胞U251放疗的增敏性

doi:10.6040/j.issn.1671-7554.0.2020.1297

摘要/Abstract

摘要： 目的探讨细胞周期蛋白酶抑制剂7(CDK7)THZ1对人胶质瘤细胞U251的体外放射增敏性。方法体外培养人胶质瘤细胞U251。MTT检测THZ1对U251的毒性作用, 绘制不同浓度(0、8、16、32、64、128、256、512 noml/L)THZ1处理下的细胞存活曲线,并计算其IC50及IC20,将IC20作为后续试验的药物作用浓度。克隆形成实验检测射线联合THZ1对U251的作用,计算放射敏感性参数,绘制集落形成数量曲线。流式细胞术检测各组细胞周期分布。Western blotting检测CDK7细胞周期蛋白、Bax凋亡相关蛋白及γH2AX蛋白表达。多组间比较采用单因素方差分析,两组间比较采用独立样本t检验,2因素组间对比采用析因设计方差分析。结果 THZ1对U251具有增殖抑制作用,且呈浓度和时间依赖性;单独应用THZ1可使CDK7细胞周期蛋白抑制,X射线(6 Gy)联合IC₂₀浓度THZ1(10 nmol/L)能够降低U251的克隆能力,放射增敏比为1.478,同时可使G0/G1期细胞比例由67%减少到11%、G2/M期细胞比例由12%增加到70%; 凋亡相关蛋白Bax表达增加,DNA双链断裂标志γH2AX蛋白表达增加。结论 THZ1对U251具有放射增敏作用,其可能的机制是THZ1联合射线降低细胞损伤修复能力, 诱导细胞凋亡,调节凋亡相关蛋白的表达,改变细胞生长周期。

关键词: 人胶质瘤细胞U251, 放射增敏, 增殖, 细胞周期

Abstract: Objective To investigate the effects of cyclin-dependent kinase 7(CDK7)inhibitor, THZ1, on the radiosensitivity of human glioma cell line U251 in vitro. Methods U251 cells were cultured in vitro. The toxic effects of THZ1 on U251 cells was detected with MTT. The cell survival curves were drawn at different concentrations of THZ1(0, 8, 16, 32, 64, 128, 256, 512 noml/L). IC₅₀ and IC₂₀ were calculated and IC₂₀ was used as the concentration for subsequent experiments. The effects of X-ray combined with THZ1 on U251 was detected with clone formation experiment. Radiosensitivity parameters were calculated and the curve of colony formation number was drawn. Cell cycle distribution in each treatment group was detected with flow cytometry. The expressions of CDK7 cyclin, Bax apoptosis-related proteins and γH2AX were detected with Western blotting. One-factor analysis of variance was used for comparison among groups. Independent sample t test was used for comparison between groups. Factorial design analysis of variance was used for two-factor comparison among groups. Results THZ1 inhibited U251 proliferation in a concentration- and time-dependent manner. THZ1 inhibited CDK7 cyclin. X-ray(6 Gy)combined with IC₂₀ concentration of THZ1(10 nmol/L)reduced the cloning ability of U251(the ratio of enhancing sensitivity being 1.478), decreased the percentage of G0/G1 phase from 67% to 11%, increased the percentage of G2/M phase from 12% to 70%, and increased the expressions of Bax apoptosis-related proteins and γH2AX. Conclusion THZ1 can increase the radiosensitivity of U251. The possible mechanism is that THZ1 combined with X-ray inhibits the repair ability of cells, induces cell apoptosis, regulates expressions of apoptosis-related proteins, and intervenes in the cell cycle.

Key words: Human glioma cell U251, Radiosensitivity, Proliferation, Cell cycle

中图分类号:

R739.41

李文清,叶兰,姜玉华. CDK7抑制剂THZ1对人胶质瘤细胞U251放疗的增敏性[J]. 山东大学学报 (医学版), 2021, 59(1): 8-13.

LI Wenqing, YE Lan, JIANG Yuhua. CDK7 inhibitor THZ1 increases the radiosensitivity of human glioma cell line U251[J]. Journal of Shandong University (Health Sciences), 2021, 59(1): 8-13.

参考文献

[1] Giotta Lucifero A, Luzzi S, Brambilla I, et al. Potential roads for reaching the summit: an overview on target therapies for high-grade gliomas[J]. Acta Biomed, 2020, 91(7-S): 61-78.
[2] Trifiletti DM, Malouff TD, McGovern SL, et al. Repeat radiation in the brain: managing patients with locally recurrent glioma[J]. Semin Radiat Oncol, 2020, 30(3): 218-222.
[3] Ostrom QT, Gittleman H, Liao P, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014[J]. Neuro Oncol, 2017, 19(suppl_5): v1-1v88.
[4] Liu Y, Zhang T, Li G, et al. Radiosensitivity enhancement by Co-NMS-mediated mitochondrial impairment in glioblastoma[J]. J Cell Physiol, 2020, 235(12): 9623-9634.
[5] Farooqi A, Li J, de Groot J, et al. Current role of radiation therapy in the management of malignant central nervous system tumors[J]. Hematol Oncol Clin North Am, 2020, 34(1): 13-28.
[6] Kegelman TP, Wu B, Das SK, et al. Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin[J]. Proc Natl Acad Sci U S A, 2017, 114(2): 370-375.
[7] Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy[J]. Int J Radiat Oncol Biol Phys, 2004, 59(4): 928-942.
[8] Kwiatkowski N, Zhang T, Rahl PB, et al. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor[J]. Nature, 2014, 511(7511): 616-620.
[9] Cheng ZJ, Miao DL, Su QY, et al. THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism[J]. Acta Pharmacol Sin, 2019, 40(6): 814-822.
[10] Zhong S, Zhang Y, Yin X, et al. CDK7 inhibitor suppresses tumor progression through blocking the cell cycle at the G2/M phase and inhibiting transcriptional activity in cervical cancer[J]. Onco Targets Ther, 2019, 12: 2137-2147. doi: 10.2147/OTT.S195655.
[11] Meng W, Wang J, Wang B, et al. CDK7 inhibition is a novel therapeutic strategy against GBM both in vitro and in vivo[J]. Cancer Manag Res, 2018, 10: 5747-5758. doi: 10.2147/CMAR.S183696.
[12] Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation[J]. Development, 2013, 140(15): 3079-3093.
[13] Fisher RP. Cdk7: a kinase at the core of transcription and in the crosshairs of cancer drug discovery[J]. Transcription, 2019, 10(2): 47-56.
[14] Bradner JE, Hnisz D, Young RA. Transcriptional addiction in cancer[J]. Cell, 2017, 168(4): 629-643.
[15] Greenall SA, Lim YC, Mitchell CB, et al. Cyclin-dependent kinase 7 is a therapeutic target in high-grade glioma[J]. Oncogenesis, 2017, 6(5): e336. doi: 10.1038/oncsis.2017.33.
[16] Zhou Y, Lu L, Jiang G, et al. Targeting CDK7 increases the stability of Snail to promote the dissemination of colorectal cancer[J]. Cell Death Differ, 2019, 26(8): 1442-1452.
[17] Lewanski CR, Gullick WJ. Radiotherapy and cellular signalling[J]. Lancet Oncol, 2001, 2(6): 366-370.
[18] 戴寒莹, 徐克前. DNA双链断裂检测技术研究进展[J].中国生物工程杂志, 2020, 40(8): 55-62. DAI Hanying, XU Keqian. Research progress on DNA double-strand break assay[J]. China Biotechnology,2020, 40(8): 55-62.
[19] Tian J, Kong E, Wang X, et al. RSF-1 siRNA enhances tumor radiosensitivity in cervical cancer via enhanced DNA damage, cell cycle redistribution, and promotion of apoptosis[J]. Onco Targets Ther, 2020, 13: 3061-3071. doi: 10.2147/OTT.S246632.
[20] Wei T, Cheng S, Fu XN, et al. miR-219a-5p enhances the radiosensitivity of non-small cell lung cancer cells through targeting CD164[J]. Biosci Rep, 2020, 40(7). doi: 10.1042/BSR20192795.
[21] Elsesy ME, Oh-Hohenhorst SJ, Löser A, et al. Second-generation antiandrogen therapy radiosensitizes prostate cancer regardless of castration state through inhibition of DNA double strand break repair[J]. Cancers(Basel), 2020, 12(9):2467. doi: 10.3390/cancers12092467.
[22] Mladenov E, Iliakis G. Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways[J]. Mutat Res, 2011, 711(1-2): 61-72.
[23] Wu G, Chen G, Zhou J, et al. Liriodenine enhances radiosensitivity in esophageal cancer ECA-109 cells by inducing apoptosis and G2/M arrest[J]. Oncol Lett, 2018, 16(4): 5020-5026.
[24] Desvoyes B, Gutierrez C. Roles of plant retinoblastoma protein: cell cycle and beyond[J]. EMBO J, 2020, 39(19): e105802. doi: 10.15252/embj.2020105802.
[25] Li J, Yang CX, Mei ZJ, et al. Involvement of cdc25c in cell cycle alteration of a radioresistant lung cancer cell line established with fractionated ionizing radiation[J]. Asian Pac J Cancer Prev, 2013, 14(10): 5725-5730.
[26] Ning J, Ma X, Long C, et al. Anti-tumor drug THZ1 suppresses TGFβ2-mediated EMT in lens epithelial cells via Notch and TGFβ/Smad signaling pathway[J]. J Cancer, 2019, 10(16): 3778-3788.
[27] 张晓英, 赵云, 刘桂香, 等. 放射线对人舌鳞癌细胞系Tca8113细胞生物学效应的影响[J]. 山东大学学报(医学版), 2019, 57(2): 70-74. ZHANG Xiaoying, ZHAO Yun, LIU Guixiang, et al. Biological response of human tongue squamous carcinoma cell line Tca8113 to radiation in vitro[J]. Journal of Shandong University, 2019, 57(2): 70-74.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed