EZH2抑制剂GSK126对肺腺癌细胞增殖和凋亡的影响

doi:10.13481/j.1671-587x.20200419

摘要/Abstract

摘要： 目的：探讨zeste基因增强子同源物2（EZH2）特异性抑制剂GSK2816126（GSK126）对肺腺癌A549细胞增殖和凋亡的影响，阐明其可能机制。方法：常规体外培养A549细胞，将细胞分为对照组和不同浓度（1.0、2.5、5.0、10.0和15.0μmol·L^-1） GSK126组。MTT比色法检测各组细胞存活率，克隆形成实验检测各组细胞集落形成数并计算克隆形成率，EdU细胞增殖成像法检测各组细胞增殖率，Hoechst-33342荧光染色法观察各组细胞凋亡形态表现，流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中蛋白激酶B（AKT）、磷酸化AKT（p-AKT）、B细胞淋巴瘤-2原癌基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、天冬氨酸特异性半胱氨酸蛋白酶3（Caspase-3）和活化型天冬氨酸特异性半胱氨酸蛋白酶3（Cleaved-Caspase-3）蛋白表达水平。结果：MTT比色法，与对照组比较，随着GSK126浓度升高，不同浓度GSK126组细胞存活率明显降低（P<0.05）；克隆形成实验，与对照组比较，随着GSK126浓度升高，不同浓度GSK126组克隆形成率逐渐降低（P<0.05）；EdU成像，与对照组比较，随着GSK126浓度升高，不同浓度GSK126组增殖率明显降低（P<0.01）；Hoechst-33342染色，与对照组比较，随着GSK126浓度的升高，5、10和15μmol·L^-1GSK126组凋亡细胞数明显增加；流式细胞术检测，与对照组比较，随着GSK126浓度升高，不同浓度GSK126组细胞凋亡率明显升高（P<0.01）。Western blotting法，与对照组比较，随着GSK126浓度升高，不同浓度GSK126组细胞中p-AKT和Bcl-2蛋白表达水平逐渐降低（P<0.05），Bax和Cleaved-Caspase-3蛋白表达水平逐渐升高（P<0.05）。结论：GSK126可能通过降低AKT磷酸化水平抑制细胞增殖，并且通过诱导Bax/Bcl-2/Caspase-3信号通路活化，激活凋亡途径，促进肺癌细胞凋亡。

关键词: zeste基因增强子同源物2, 肺肿瘤, GSK2816126, 细胞增殖, 细胞凋亡

Abstract: Objective: To investigate the effects of GSK2816126(GSK126), a specific inhibitor of zeste gene enhancer homolog 2 (EZH2), on the proliferation and apoptosis of lung adenocarcinoma A549 cells, and to elucidate their possible mechanisms. Methods: The A549 cells were cultured in vitro and divided into control group and different concentrations(1.0,2.5,5.0,10.0,15.0 μmol·L^-1)of GSK126 groups. MTT colorimetry was used to detect the survival rates of cells in various groups.Clone formation experiment was used to detect the number of colony formation and the colony formation rate was calculated. EdU imaging was used to detect the proliferation rates of cells in various groups. Hoechst-33342 fluorescence staining was used to observe the apoptotic morphology of cells in various groups; flow cytometry was used to detect the apoptotic rates of A549 cells in various groups. Western blotting method was used to detect the expression levels of protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), B-cell lymphoma-2 proto oncogene (Bcl-2), Bcl-2 related X protein (Bax), aspartate specific cysteine proteinase-3 (caspase-3) and activated aspartate specific cysteine proteinase-3 (Cleaved-Caspase-3) proteins in the cells in various groups. Results: The MTT results showed that compared with control group, the survival rates of A549 cells in different concentrations of GSK126 groups were decreased with the increase of GSK126 concentration (P<0.05).The results of clone formation experiment showed that compared with control group, the number of colonies in different concentrations of GSK126 groups were decreased gradually with the increase of GSK126 concentration (P<0.05).The EdU imaging results showed that the proliferation rates of the cells in different concentrations of GSK126 groups were decreased significantly with the increase of GSK126 concentration compared with control group (P<0.01).The Hoechst-33342 staining and flow cytometry results showed that compared with control group, the apoptotic rates of the cells in different concentrations of GSK126 groups were increased significantly with the increase of GSK126 concentration (P<0.01).The Western blotting results showed that compared with control group, the expression levels of p-AKT and Bcl-2 proteins were decreased gradually with the increase of GSK126 concentration (P<0.05), and the expression levels of Bax and Cleaved-Caspase-3 proteins were increased gradually (P<0.05). Conclusion: GSK126 may inhibit the cell proliferation by reducing the phosphorylation level of AKT, and promote the apoptosis of lung cancer cells by inducing Bax/Bcl-2/Caspase-3 signal pathway activation and activating apoptosis pathway.

Key words: zeste gene enhancer homolog 2, lung neoplasms, GSK2816126, cell proliferation, apoptosis

中图分类号:

R734.2

张爽爽, 贺武斌, 苏荣健, 杜晓媛. EZH2抑制剂GSK126对肺腺癌细胞增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2020, 46(04): 786-791.

ZHANG Shuangshuang, HE Wubin, SU Rongjian, DU Xiaoyuan. Effects of EZH2 inhibitor GSK126 on proliferation and apoptosis of lung adenocarcinoma cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 786-791.

参考文献

[1] FENG R M, ZONG Y N, CAO S M, et al. Current cancer situation in China:good or bad news from the 2018 Global Cancer Statistics?[J].Cancer Commun (Lond), 2019, 39(1):22.
[2] SHAHABIPOUR F, CARAGLIA M, MAJEED M, et al. Naturally occurring anti-cancer agents targeting EZH2[J]. Cancer Lett, 2017, 400:325-335.
[3] 李杰宝, 喻晓程, 田野. 果蝇EZH2与乳腺癌分子分型及临床病理特征的关系[J]. 中国现代医学杂志, 2017, 27(6):50-53.
[4] OKI S, SONE K, ODA K, et al. Oncogenic histone methyltransferase EZH2:a novel prognostic marker with therapeutic potential in endometrial cancer[J]. Oncotarget, 2017, 8(25):40402-40411.
[5] KUMARI K, DAS B, ADHYA A, et al. Nicotine associated breast cancer in smokers is mediated through high level of EZH2 expression which can be reversed by methyltransferase inhibitor DZNepA[J]. Cell Death Dis, 2018, 9(2):152.
[6] ZHOU J F, NIE D N, LI J, et al. PTEN is fundamental for elimination of leukemia stem cells mediated by GSK126 targeting EZH2 in chronic myelogenous leukemia[J]. Clin Cancer Res, 2018, 24(1):145-157.
[7] ZHANG H K, QI J, REYES J M, et al. Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer[J]. Cancer Discov, 2016, 6(9):1006-1021.
[8] WEI S, LIU J H, LI X, et al. Repression of lncRNA-SVUGP2 mediated by EZH2 contributes to the development of non-small cell lung cancer via brisking Wnt/β-catenin signal[J]. Artif Cells Nanomed Biotechnol, 2019, 47(1):3400-3409.
[9] TAKESHIMA H, WAKABAYASHI M, HATTORI N, et al. Identification of coexistence of DNA methylation and H3K27me3 specifically in cancer cells as a promising target for epigenetic therapy[J]. Carcinogenesis, 2015, 36(2):192-201.
[10] 林伟仁,朱锋,应荣彪,等.EZH2抑制剂GSK126对Neuro-2a细胞增殖分化的影响及其可能机制[J].中国药理学通报,2017,33(2):289-290
[11] YU P, GUO Y W, YUSUFU M, et al. Decreased expression of EZH2 reactivates RASSF2A by reversal of promoter methylation in breast cancer cells[J]. Cell Biol Int, 2016, 40(10):1062-1070.
[12] QIU Z G,ZHU W L,MENG H,et al.CDYL promotes the chemoresistance of small cell lung cancer by regulating H3K27 trimethylation at the CDKN1C promoter[J].Theranostics, 2019,9(16):4717-4729.
[13] SERRESI M, SITEUR B, HULSMAN D, et al. Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and associated vulnerabilities[J]. J Exp Med, 2018, 215(12):3115-3135.
[14] LIU X Y, LU X Y, ZHEN F X, et al. LINC00665 induces acquired resistance to gefitinib through recruiting EZH2 and activating PI3K/AKT pathway in NSCLC[J]. Mol Ther Nucleic Acids, 2019, 16:155-161.
[15] LI J, ZHOU Z, XU F C, et al. MicroRNA-374b accelerates the development of lung cancer through downregulating PTEN expression via activating PI3K/Akt pathway[J]. Eur Rev Med Pharmacol Sci, 2019, 23(3):1116-1124.
[16] RIQUELME E, BEHRENS C, LIN H Y, et al. Modulation of EZH2 expression by MEK-ERK or PI3K-AKT signaling in lung cancer is dictated by different KRAS oncogene mutations[J]. Cancer Res, 2016, 76(3):675-685.
[17] KOSALAI S T, MORSY M H A, PAPAKONSTANTINOU N, et al. EZH2 upregulates the PI3K/AKT pathway through IGF1R and MYC in clinically aggressive chronic lymphocytic leukaemia[J]. Epigenetics, 2019, 14(11):1125-1140.
[18] WANG Z Q,CAI Q,HU L,et al.Long noncoding RNA UCA1 induced by SP1 promotes cell proliferation via recruiting EZH2 and activating AKT pathway in gastric cancer[J].Cell Death Dis,2017,8(6):e2839.
[19] SI X Q, XU F X, XU F H, et al. CADM1 inhibits ovarian cancer cell proliferation and migration by potentially regulating the PI3K/Akt/mTOR pathway[J]. Biomed Pharmacother, 2020, 123:109717.
[20] WARREN C F A, WONG-BROWN M W, BOWDEN N A. BCL-2 family isoforms in apoptosis and cancer[J]. Cell Death Dis, 2019, 10(3):177.
[21] JIN J F, XIONG Y, CEN B. Bcl-2 and Bcl-xL mediate resistance to receptor tyrosine kinase-targeted therapy in lung and gastric cancer[J]. Anticancer Drugs, 2017, 28(10):1141-1149.
[22] PAL M K,JAISWAR S P,SRIVASTAV A K,et al.Synergistic effect of piperine and paclitaxel on cell fate via cyt-c, Bax/Bcl-2-caspase-3 pathway in ovarian adenocarcinomas SKOV-3 cells[J]. Eur J Pharmacol, 2016,791:751-762.