华蟾毒配基抑制组蛋白乙酰化诱导肝癌细胞死亡的机制
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摘要
目的探讨华蟾毒配基通过改变表观遗传修饰调控肝癌细胞死亡的机制。方法通过CCK-8法检测不同浓度(0、0.75、1.5、2.5、5、10μmol/L)华蟾毒配基作用于肝癌HepG2细胞24 h细胞增殖能力的变化;5μmol/L华蟾毒配基作用HepG2细胞12、24 h后,流式细胞术检测细胞周期,蛋白印迹检测钙/钙调素依赖蛋白激酶2β(CaMK2B)、甲基化CpG结合蛋白2(MeCP2)、磷酸化MeCP2(p-MeCP2)、乙酰化组蛋白H3.1(Ac-Histone3.1)和甲基化组蛋白(Met-Histone3.1)的表达变化;5μmol/L华蟾毒配基作用HepG2细胞24 h后,细胞免疫荧光结合扫描共聚焦检测CaMK2B和MeCP2相互作用。结果华蟾毒配基能显著抑制肝癌HepG2细胞生长,抑制效果呈现浓度依赖性,差异有统计学意义(P<0.05),药物作用细胞后,细胞周期发生G_2/M期阻滞,差异有统计学意义(P<0.05)。5μmol/L华蟾毒配基可促进CaMK2B和MeCP2在细胞核内结合,Western blot结果显示华蟾毒配基作用HepG2细胞后上调CaMK2B和p-MeCP2表达(P<0.05),抑制Ac-Histone3.1表达,促进Met-Histone3.1(P<0.05)。结论华蟾毒配基通过激活CaMK2B-MeCP2信号通路抑制组蛋白乙酰化、促进甲基化继而诱导肝癌细胞死亡。
Objective To investigate the mechanism of cinobufagin in regulating the death of liver cells by regulating epigenetic modification. Methods The CCK-8 assay was used to detect the changes of proliferation ability after different concentrations(0, 0.75, 1.5, 2.5, 5, 10 μmol/L) of cinobufagin acting on the HepG2 cells for 24 h; after 5 μmol/L of cinobufagin acting on the HepG2 cells for 12, 24 h, the cell cycle was detected by flow cytometry, and the expression levels of Ca~(2+)/Calmodulin-dependent protein kinase 2β(CaMK2B), methylated-cpg binding protein(MeCP2),phosphorylation-MeCP2(p-MeCP2), acetylated histone H3.1(Ac-Histone3.1) and methylated histones(Met-Histone3.1) were detected by Western blot; after 5 μmol/L of cinobufagin acting on the HepG2 cells for 24 h, the interaction between CaMK2B and MeCP2 in HepG2 cells was detected by cell immunofluorescence combined with confocal scanning. Results Cinobufagin could inhibit the growth of HepG2 cells in a dose-independent manner,the difference was statistically significant( P < 0.05), after the drug acting on the cells, the cell cycle comes up G_2/M phase arrest, the difference was statistically significant(P < 0.05). 5 μmol/L of cinobufagin could promote the incorporation of CaMK2B and MeCP2 in cell nucleus, the results of Western blot showed that the cinobufagin could increase the expression of CaMK2B and MeCP2 after acting on HepG2 cells( P < 0.05), inhibit the expression of the Ac-Histone3.1, and promote the Met-Histone3.1(P < 0.05). Conclusion Cinobufagin can induce the death of liver cells by activating the CaMK2B-MeCP2 signal path, inhibiting histone acetylation and promoting histone methy-lation.
Objective To investigate the mechanism of cinobufagin in regulating the death of liver cells by regulating epigenetic modification. Methods The CCK-8 assay was used to detect the changes of proliferation ability after different concentrations(0, 0.75, 1.5, 2.5, 5, 10 μmol/L) of cinobufagin acting on the HepG2 cells for 24 h; after 5 μmol/L of cinobufagin acting on the HepG2 cells for 12, 24 h, the cell cycle was detected by flow cytometry, and the expression levels of Ca~(2+)/Calmodulin-dependent protein kinase 2β(CaMK2B), methylated-cpg binding protein(MeCP2),phosphorylation-MeCP2(p-MeCP2), acetylated histone H3.1(Ac-Histone3.1) and methylated histones(Met-Histone3.1) were detected by Western blot; after 5 μmol/L of cinobufagin acting on the HepG2 cells for 24 h, the interaction between CaMK2B and MeCP2 in HepG2 cells was detected by cell immunofluorescence combined with confocal scanning. Results Cinobufagin could inhibit the growth of HepG2 cells in a dose-independent manner,the difference was statistically significant( P < 0.05), after the drug acting on the cells, the cell cycle comes up G_2/M phase arrest, the difference was statistically significant(P < 0.05). 5 μmol/L of cinobufagin could promote the incorporation of CaMK2B and MeCP2 in cell nucleus, the results of Western blot showed that the cinobufagin could increase the expression of CaMK2B and MeCP2 after acting on HepG2 cells( P < 0.05), inhibit the expression of the Ac-Histone3.1, and promote the Met-Histone3.1(P < 0.05). Conclusion Cinobufagin can induce the death of liver cells by activating the CaMK2B-MeCP2 signal path, inhibiting histone acetylation and promoting histone methy-lation.
引文
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[3]徐忠伟,王凤梅,王聪聪,等.钠钾ATP酶抑制剂通过调节DNA损伤感应复合体Mre11/Rad50/Nbs1的表达诱导肝癌HepG2细胞周期阻滞[J].中国药理学通报,2016,32(3):323-327.
[4]王志美,徐忠伟,王佳宝,等.蟾酥活性成分协调索拉非尼通过下调Akt/NF-κB信号途径抑制肝癌HepG2细胞生长[J].中国药理学通报,2017,33(11):1510-1516.
[5]Kim N,Yim HY,He N,et al.Cardiac glycosides display selective efficacy for STK11 mutant lung cancer[J].Sci Rep,2016,6:29721.
[6]Karasneh RA,Murray LJ,Cardwell CR.Cardiac glycosides and breast cancer risk:A systematic review and metaanalysis of observational studies[J].Int J Cancer,2017,140(5):1035-1041.
[7]Prassas I,Karagiannis GS,Batruch I,et al.Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks[J].Mol Cancer Ther,2011,10(11):2083-2093.
[8]Xu Z,Wang F,Fan F,et al.Quantitative Proteomics Reveals That the Inhibition of Na(+)/K(+)-ATPase Activity Affects S-Phase Progression Leading to a Chromosome Segregation Disorder by Attenuating the Aurora A Function in Hepatocellular Carcinoma Cells[J].J Proteome Res,2015,14(11):4594-4602.
[9]Clark T,Maximin S,Meier J,et al.Hepatocellular Carcinoma:Review of Epidemiology,Screening,Imaging Diagnosis,Response Assessment,and Treatment[J].Curr Probl Diagn Radiol,2015,44(6):479-486.
[10]Xu ZW,Wang FM,Gao MJ,et al.Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting[J].Biol Pharm Bull,2010,33(5):743-751.
[11]Xu ZW,Wang FM,Gao MJ,et al.Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth[J].J Steroid Biochem Mol Biol,2011,125(3-5):181-191.
[12]Nakanishi A,Hatano N,Fujiwara Y,et al.AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin(Ca M)dependence of Ca2+/Ca M-dependent protein kinase kinaseβ[J].J Biol Chem,2017,292(48):19804-19813.
[13]York B,Li F,Lin F,et al.Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD[J].Sci Rep,2017,7(1):11793.
[14]Chen X,Wu Q,You L,et al.Propofol attenuates pancreatic cancer malignant potential via inhibition of NMDA receptor[J].Eur J Pharmacol,2017,795:150-159.
[15]Marcelo KL,Means AR,York B.The Ca(2+)/Calmodulin/CaMKK2 Axis:Nature′s Metabolic Ca Mshaft[J].Trends Endocrinol Metab,2016,27(10):706-718.
[16]Raynal NJ,Lee JT,Wang Y,et al.Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer[J].Cancer Res,2016,76(6):1494-1505.
[17]Mushtaq AU,Lee Y,Hwang E,et al.Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA[J].Biochem Biophys Res Commun,2018,495(1):145-150.
[18]Good KV,Martínez de Paz A,Tyagi M,et al.Trichostatin A decreases the levels of MeCP2 expression and phosphorylation and increases its chromatin binding affinity[J].Epigenetics,2017,12(11):934-944.
[19]Zhang J,Zhao J,Gao N,et al.MECP2 expression in gastric cancer and its correlation with clinical pathological parameters[J].Medicine(Baltimore),2017,96(31):e7691.
[20]Ohashi M,Korsakova E,Allen D,et al.Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence[J].Stem Cell Reports,2018,10(5):1453-1463.