药学学报, 2021, 56(5): 1384-1390
引用本文:
张凤霞, 张建东, 单保恩, 储金秀. Zebularine通过SFRP2/Dkk3去甲基化调控Wnt/β-catenin信号通路诱导食管癌细胞凋亡[J]. 药学学报, 2021, 56(5): 1384-1390.
ZHANG Feng-xia, ZHANG Jian-dong, SHAN Bao-en, CHU Jin-xiu. Zebularine induces apoptosis of esophageal cancer cells via demethylation SFRP2/Dkk3 to regulate Wnt/β-catenin signaling pathway[J]. Acta Pharmaceutica Sinica, 2021, 56(5): 1384-1390.

Zebularine通过SFRP2/Dkk3去甲基化调控Wnt/β-catenin信号通路诱导食管癌细胞凋亡
张凤霞1, 张建东2, 单保恩3, 储金秀1,4*
1. 华北理工大学基础医学院, 河北省慢性疾病基础医学重点实验室, 河北 唐山 063200;
2. 河北医科大学第三医院检验科, 河北 石家庄 050000;
3. 河北医科大学第四医院科研中心, 河北 石家庄 050000;
4. 华北理工大学, 河北 唐山 063200
摘要:
研究去甲基化药物zebularine对食管癌细胞凋亡的影响及其作用机制。采用不同浓度(25、50、100、200和400 μmol·L-1)zebularine处理食管癌ECA109和KYSE170细胞,CCK-8法检测细胞活力。选用浓度为100 μmol·L-1 zebularine处理细胞,流式细胞术检测凋亡率,Western blot检测凋亡蛋白(Bcl-2蛋白、Bax蛋白、cleaved-caspase-3蛋白和cleaved-PARP蛋白)和Wnt(wingless-related integration site)信号通路分子(β-catenin蛋白、cyclin D1蛋白和c-Myc蛋白)的表达量,实时荧光定量PCR检测Wnt信号通路上游负调控基因表达水平,甲基化特异性PCR(methylation specific PCR,MSP)法检测人分泌型卷曲相关蛋白2(secreted frizzled related protein 2,SFRP2)和Dkk3(dickkopf 3)基因的甲基化状态。敲低SFRP2Dkk3观察对zebularine诱导凋亡的影响。结果显示,zebularine能抑制ECA109和KYSE170细胞的活力,呈剂量和时间依赖性;zebularine可诱导细胞凋亡,下调Bcl-2蛋白,上调Bax、cleaved-caspase-3和cleaved-PARP蛋白的表达,并抑制β-catenin、cyclin D1和c-Myc蛋白表达,与阴性对照组比较,差异均具有统计学意义(P<0.05);zebularine作用后,Dkk3SFRP2的mRNA表达水平明显升高,SFRP2Dkk3启动子甲基化水平降低;敲低SFRP2Dkk3可降低zebularine诱导的细胞凋亡。综上所述,zebularine降低SFRP2Dkk3基因启动子甲基化水平,促进SFRP2Dkk3基因表达,进而通过抑制Wnt/β-catenin信号通路诱导食管癌细胞凋亡。
关键词:    食管癌      zebularine      细胞凋亡      人分泌型卷曲相关蛋白2      dickkopf 3      Wnt/β-catenin信号通路     
Zebularine induces apoptosis of esophageal cancer cells via demethylation SFRP2/Dkk3 to regulate Wnt/β-catenin signaling pathway
ZHANG Feng-xia1, ZHANG Jian-dong2, SHAN Bao-en3, CHU Jin-xiu1,4*
1. Hebei Key Laboratory for Chronic Diseases of Basic Medical College, North China University of Science and Technology, Tangshan 063200, China;
2. Department of Laboratory Medicine, the Third Hospital of Hebei Medical University, Shijiazhuang 050000, China;
3. Research Center, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050000, China;
4. North China University of Science and Technology, Tangshan 063200, China
Abstract:
To explore the effect and mechanisms of demethylation drug zebularine on esophageal cancer cells apoptosis, ECA109 cells and KYSE170 cells were treated with zebularine at different concentrations (25, 50, 100, 200, and 400 μmol·L-1). The cell viability was measured by CCK-8. Flow cytometry was used to detect the cell apoptosis rate, Western blot was performed to determine the expression of apoptosis protein (Bcl-2, Bax, cleaved-caspase-3, and cleaved-PARP) and Wnt signal pathway molecules (β-catenin, cyclin D1, and c-Myc), real-time quantitative PCR was used to detect the expression level of negative regulatory genes of Wnt signaling pathway, methylation specific PCR (MSP) was used to detect the methylation status of secreted frizzled related protein 2 (SFRP2) and dickkopf 3 (Dkk3) genes. After knockdown of SFRP2 and Dkk3, the effect of zebularine on apoptosis was detected. The studies showed that zebularine could inhibit the activity of ECA109 and KYSE170 cells in a dose-dependent and time-dependent manner; zebularine could induce cell apoptosis, down-regulate the expression of Bcl-2 protein, up-regulate the expression of Bax, cleaved-caspase-3, and cleaved-PARP protein, and inhibit the expression of β-catenin, cyclin D1, and c-Myc protein (P<0.05); the mRNA expression levels of Dkk3 and SFRP2 were significantly up-regulated by zebularine, while the methylation levels of SFRP2 and Dkk3 promoters were decreased; knockdown of SFRP2 and Dkk3 could reduce the apoptosis induced by zebularine. In summary, zebularine could reduce the methylation level of SFRP2 and Dkk3 gene promoter, promote the expression of SFRP2 and Dkk3 gene, and then induce the apoptosis of esophageal cancer cells by inhibiting Wnt/β-catenin signaling pathway.
Key words:    esophageal cancer    zebularine    apoptosis    SFRP2    Dkk3    Wnt/β-catenin signaling pathway   
收稿日期: 2020-11-26
DOI: 10.16438/j.0513-4870.2020-1828
基金项目: 国家自然科学基金资助项目(81673642);河北省医学科学研究重点课题计划项目(20170640).
通讯作者: 储金秀,Tel:86-311-88603328,E-mail:chujx19@126.com
Email: chujx19@126.com
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参考文献:
[1] Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012[J]. CA Cancer J Clin, 2015, 65:87-108.
[2] Lin Y, Totsuka Y, He Y, et al. Epidemiology of esophageal cancer in Japan and China[J]. J Epidemiol, 2013, 23:233-242.
[3] Huang D, Wang Y, He Y, et al. Paraoxonase 3 is involved in the multi-drug resistance of esophageal cancer[J]. Cancer Cell Int, 2018, 18:168-172.
[4] Andersen JB, Factor VM, Marquardt JU, et al. An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer[J]. Sci Transl Med, 2010, 2:54ra77.
[5] Cheng JC, Matsen CB, Gonzales FA, et al. Inhibition of DNA methylation and reactivation of silenced genes by zebularine[J]. J Natl Cancer Inst, 2003, 95:399-409.
[6] Cheng JC, Weisenberger DJ, Gonzales FA, et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells[J]. Mol Cell Biol, 2004, 24:1270-1278.
[7] Villanueva L, Álvarez-Errico D, Esteller M. The contribution of epigenetics to cancer immunotherapy[J]. Trends Immuno, 2020, 41:676-691.
[8] Cao J, Yan Q. Cancer epigenetics, tumor immunity, and immunotherapy[J]. Trends Cancer, 2020, 6:580-592.
[9] Skvortsova K, Stirzaker C, Taberlay P. The DNA methylation landscape in cancer[J]. Essays Biochem, 2019, 63:797-811.
[10] Krzeminski P, García-Sanz R, Gutiérrez NC. Zebularine-induced myeloma cell death is accompanied by decreased c-Myc expression[J]. Cell Oncol (Dordr), 2020, 43:743-750.
[11] Ye K, Wang S, Wang J, et al. Zebularine enhances apoptosis of human osteosarcoma cells by suppressing methylation of ARHI[J]. Cancer Sci, 2016, 107:1851-1857.
[12] You BR, Park WH. Zebularine inhibits the growth of A549 lung cancer cells via cell cycle arrest and apoptosis[J]. Mol Carcinog, 2014, 53:847-857.
[13] You BR, Park WH. Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis[J]. Mol Biol Rep, 2012, 39:9723-9731.
[14] Takemura Y, Satoh M, Hatanaka K, et al. Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells[J]. Biosci Biotechnol Biochem, 2018, 82:1159-1164.
[15] Tan W, Zhou W, Yu HG, et al. The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo[J]. Biochem Biophys Res Commun, 2013, 430:250-255.
[16] Nakamura K, Nakabayashi K, Htet AK, et al. DNA methyltransferase inhibitor zebularine induces human cholangiocarcinoma cell death through alteration of DNA methylation status[J]. PLoS One, 2015, 10:e0120545.
[17] MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling:components, mechanisms, and diseases[J]. Dev Cell, 2009, 17:9-26.
[18] Zhang J, Xing B, Song J, et al. Associated analysis of DNA methylation for cancer detection using CCP-based FRET technique[J]. Anal Chem, 2014, 86:346-350.
[19] Wang B, Yu L, Luo X, et al. Detection of OPCML methylation, a possible epigenetic marker, from free serum circulating DNA to improve the diagnosis of early-stage ovarian epithelial cancer[J]. Oncol Lett, 2017, 14:217-223.