白杨素通过调控MAPKs信号通路促进SMMC-7721细胞凋亡
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摘要
目的探究白杨素诱导肝癌细胞系SMMC-7721细胞凋亡的效应及分子机制。方法将SMMC-7721细胞分为空白对照组,DMSO溶剂对照组和白杨素处理组(5,10,20,40,60,80,100,150,200μg/mL),采用CCK-8法检测细胞的增殖抑制作用;将SMMC-7721细胞分为空白对照组,DMSO溶剂对照组和白杨素处理组(5,10,20μg/mL),在倒置显微镜下观察细胞形态学变化;DAPI染色后,在荧光倒置显微镜下观察细胞核形态变化;Annexin V-FITC/PI双染法检测细胞凋亡率;将SMMC-7721细胞分别用白杨素,P38特异性抑制剂SB203580(0,5,10μmol/L)、ERK特异性抑制剂U0126(0,5,10μmol/L)和JNK特异性抑制剂SP600125(0,5,10μmol/L)处理后,Western blotting技术检测凋亡相关蛋白PARP、caspase-3和凋亡相关信号通路和MAPKs的磷酸化变化情况。结果白杨素显著抑制SMMC-7721细胞增殖,和对照组相比,DMSO组,5,10,20μg/mL白杨素处理组细胞抑制率分别为96%,76%,71.75%和64.29%,具有剂量依赖性。超过60μg/mL细胞抑制率达到饱和。另外,白杨素促进细胞凋亡,20μg/mL白杨素处理组细胞凋亡率为68.7%,比空白对照组增加了59.4%,PARP和caspase-3显著切割。白杨素激活MAPKs信号通路,具有剂量和时间依赖性。分别用SB203580、U0126、SP600125预处理细胞,白杨素诱导的P38、ERK和JNK的磷酸化和PARP切割被抑制。结论白杨素通过调控MAPKs信号分子的活化,抑制SMMC-7721细胞的增殖并且促进细胞凋亡的发生。
Objective To study the effect of chrysin in inducing apoptosis of human hepatic carcinoma cells and explore the possible mechanism. Methods Human hepatic carcinoma SMMC-7721 cells treated with DMSO or chrysin at different concentrations(5-200 μg/mL) were examined for changes in the cell proliferation using CCK-8 assay. The morphological changes of SMMC-7721 cells were observed in response to treatment with 5, 10, or 20 μg/mL chrysin, and the changes in the cell nuclei were observed using DAPI nuclear staining. Annexin Ⅴ-FITC/PI flow cytometry was used to determine the cell apoptosis rate. The changes in the apoptosis-related proteins(PARP and caspase-3) and MAPKs signal pathway were detected with Western blotting. Results Chrysin treatment obviously suppressed the proliferation of SMMC-7721 cells in a dose-dependent manner below the concentration of 60 μg/mL. Chrysin(20 μg/mL) also caused significantly increased cell apoptosis and significant cleavage of PARP and caspase-3. Chrysin significantly activated MAPKs signaling pathway in a timeand dose-dependent manner, with the peak activation level occurring at 15 min. Pretreatment of the cells with specific inhibitors of the MAPKs pathway obviously inhibited the effect of chrysin in inducing cell apoptosis. Conclusion Chrysin inhibits the proliferation and promotes apoptosis of SMMC-7721 cells by regulating the activation of MAPKs signaling.
Objective To study the effect of chrysin in inducing apoptosis of human hepatic carcinoma cells and explore the possible mechanism. Methods Human hepatic carcinoma SMMC-7721 cells treated with DMSO or chrysin at different concentrations(5-200 μg/mL) were examined for changes in the cell proliferation using CCK-8 assay. The morphological changes of SMMC-7721 cells were observed in response to treatment with 5, 10, or 20 μg/mL chrysin, and the changes in the cell nuclei were observed using DAPI nuclear staining. Annexin Ⅴ-FITC/PI flow cytometry was used to determine the cell apoptosis rate. The changes in the apoptosis-related proteins(PARP and caspase-3) and MAPKs signal pathway were detected with Western blotting. Results Chrysin treatment obviously suppressed the proliferation of SMMC-7721 cells in a dose-dependent manner below the concentration of 60 μg/mL. Chrysin(20 μg/mL) also caused significantly increased cell apoptosis and significant cleavage of PARP and caspase-3. Chrysin significantly activated MAPKs signaling pathway in a timeand dose-dependent manner, with the peak activation level occurring at 15 min. Pretreatment of the cells with specific inhibitors of the MAPKs pathway obviously inhibited the effect of chrysin in inducing cell apoptosis. Conclusion Chrysin inhibits the proliferation and promotes apoptosis of SMMC-7721 cells by regulating the activation of MAPKs signaling.
引文
[1]伍石华,罗招阳,张坚松.白杨素的抗肿瘤作用及机理研究[J].湖南师范大学学报:医学版,2006,3(4):78-81.
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[3]景临林,范小飞,马慧萍,等.以白杨素为原料简便合成A环多氧黄酮的方法[J].化学试剂,2013,35(9):782-4,800.
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[13]李欣,王剑宁,熊习昆,等.白杨素提高肿瘤坏死因子-α诱导肝癌细胞HepG2凋亡能力的研究[J].中草药,2010,41(11):1828-34.
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[19]Su L,Chen X,Wu J,et al.Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress[J].Food Chem Toxicol,2013,62:810-6.
[20]Zheng ZP,Yan Y,Xia J,et al.A phenylacetaldehyde-flavonoid adduct,8-C-(E-phenylethenyl)-norartocarpetin,exhibits intrinsic apoptosis and MAPK pathways-related anticancer potential on HepG2,SMMC-7721 and QGY-7703[J].Food Chem,2016,197(Pt B):1085-92.
[21]Wang D,Lu J,Liu Y,et al.Liquiritigenin induces tumor cell death through mitogen-activated protein kinase-(MPAKs-)mediated pathway in hepatocellular carcinoma cells[J].2014:965316.
[22]Boye A,Wu C,Jiang Y,et al.Compound astragalus and salvia miltiorrhiza extracts modulate MAPK-regulated TGF-β/Smad signaling in hepatocellular carcinoma by multi-target mechanism[J].J Ethnopharmacol,2015,169(169):219-28.
[23]Cargnello M,Roux PP.Activation and function of the MAPKs and their substrates,the MAPK-activated protein kinases[J].Microbiol Mol Biol Rev,2011,75(1):50-83.
[24]Wada T,Penninger JM.Mitogen-activated protein kinases in apoptosis regulation[J].Oncogene,2004,23(16):2838-49.
[25]Kitaoka S,Shiota G,Kawasaki H.Serum levels of interleukin-10,interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type C[J].Hepatogastroenterology,2003,50(53):1569-74.
[26]Song le H,Binh VQ,Duy DN,et al.Serum cytokine profiles associated with clinical presentation in Vietnamese infected with hepatitis B virus[J].J Clin Virol,2003,28(1):93-103.
[27]Crawford ED,Wells JA.Caspase substrates and cellular remodeling[J].Annu Rev Biochem,2011,80:1055-87.
[28]Boatright KM,Salvesen GS.Mechanisms of caspase activation[J].Curr Opin Cell Biol,2003,15(6):725-31.
[29]Chang LF,Karin M.Mammalian MAP kinase signalling cascades[J].Nature,2001,410(6824):37-40.
[30]Xia Z,Dickens M,Raingeaud J,et al.Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis[J].Science,1995,270(5240):1326-31.
[2]曹炜,符军放,索志荣,等.蜂胶中白杨素和高良姜素的HPLC分析[J].药物分析杂志,2007,27(4):522-6.
[3]景临林,范小飞,马慧萍,等.以白杨素为原料简便合成A环多氧黄酮的方法[J].化学试剂,2013,35(9):782-4,800.
[4]Chen YH,Yang ZS,Wen CC,et al.Evaluation of the structureactivity relationship of flavonoids as antioxidants and toxicants of zebrafish larvae[J].Food Chem,2012,134(2):717-24.
[5]Calderwood DA,Ginsberg MH.Talin forges the links between integrins and actin[J].Nat Cell Biol,2003,5(8):694-7.
[6]Gresa-Arribas N,Serratosa J,Saura J,et al.Inhibition of CCAAT/enhancer binding proteinδexpression by chrysin in microglial cells results in anti-inflammatory and neuroprotective effects[J].JNeurochem,2010,115(2):526-36.
[7]齐世美,李强,姜琦,等.白杨素通过JAK-STATs信号通路抑制内毒素诱导的巨噬细胞炎症反应[J].南方医科大学学报,2018,38(3):243-50.
[8]康颖,刘红光,谷依学,等.白杨素对人胃癌SGC-7901细胞的体内抑制作用[J].南华大学学报:医学版,2009,37(2):170-2.
[9]张强,赵新淮.黄酮和黄酮醇通过诱导PIG3表达引发人食管癌细胞凋亡[J].生物化学与生物物理进展,2009,36(2):213-9.
[10]徐成坤,阳波,曹建国.白杨素诱导肝癌Hep3B细胞凋亡机制研究[J].医药导报,2014,33(3):295-8.
[11]邱艳君,刘飞,曹建国,等.白杨素敏化rhsTRAIL诱导人肝癌Bel-7402细胞凋亡[J].湖南师范大学学报:医学版,2009,6(3):5-8.
[12]Zhang QY,Ma S,Liu B,et al.Chrysin induces cell apoptosis via activation of the p53/Bcl-2/caspase-9 pathway in hepatocellular carcinoma cells[J].Exp Ther Med,2016,12(1):469-74.
[13]李欣,王剑宁,熊习昆,等.白杨素提高肿瘤坏死因子-α诱导肝癌细胞HepG2凋亡能力的研究[J].中草药,2010,41(11):1828-34.
[14]Hu KH,Li WX,Sun MY,et al.Cadmium induced apoptosis in MG63cells by increasing ROS,activation of p38 MAPK and inhibition of ERK 1/2 pathways[J].Cell Physiol Biochem,2015,36(2):642-54.
[15]Kim EK,Choi EJ.Pathological roles of MAPK signaling pathways in human diseases[J].Biochimi Biophys Acta-Mol Basis Dis,2010,1802(4):396-405.
[16]Blüthgen N,Legewie S.Systems analysis of MAPK signal transduction[J].Essays Biochem,2008,45:95-107.
[17]Junttila MR,Li SP,Westermarck J.Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival[J].FASEB J,2008,22(4):954-65.
[18]Cuevas BD,Abell AN,Johnson GL.Role of mitogen-activated protein kinase kinase kinases in signal integration[J].Oncogene,2007,26(22):3159-71.
[19]Su L,Chen X,Wu J,et al.Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress[J].Food Chem Toxicol,2013,62:810-6.
[20]Zheng ZP,Yan Y,Xia J,et al.A phenylacetaldehyde-flavonoid adduct,8-C-(E-phenylethenyl)-norartocarpetin,exhibits intrinsic apoptosis and MAPK pathways-related anticancer potential on HepG2,SMMC-7721 and QGY-7703[J].Food Chem,2016,197(Pt B):1085-92.
[21]Wang D,Lu J,Liu Y,et al.Liquiritigenin induces tumor cell death through mitogen-activated protein kinase-(MPAKs-)mediated pathway in hepatocellular carcinoma cells[J].2014:965316.
[22]Boye A,Wu C,Jiang Y,et al.Compound astragalus and salvia miltiorrhiza extracts modulate MAPK-regulated TGF-β/Smad signaling in hepatocellular carcinoma by multi-target mechanism[J].J Ethnopharmacol,2015,169(169):219-28.
[23]Cargnello M,Roux PP.Activation and function of the MAPKs and their substrates,the MAPK-activated protein kinases[J].Microbiol Mol Biol Rev,2011,75(1):50-83.
[24]Wada T,Penninger JM.Mitogen-activated protein kinases in apoptosis regulation[J].Oncogene,2004,23(16):2838-49.
[25]Kitaoka S,Shiota G,Kawasaki H.Serum levels of interleukin-10,interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type C[J].Hepatogastroenterology,2003,50(53):1569-74.
[26]Song le H,Binh VQ,Duy DN,et al.Serum cytokine profiles associated with clinical presentation in Vietnamese infected with hepatitis B virus[J].J Clin Virol,2003,28(1):93-103.
[27]Crawford ED,Wells JA.Caspase substrates and cellular remodeling[J].Annu Rev Biochem,2011,80:1055-87.
[28]Boatright KM,Salvesen GS.Mechanisms of caspase activation[J].Curr Opin Cell Biol,2003,15(6):725-31.
[29]Chang LF,Karin M.Mammalian MAP kinase signalling cascades[J].Nature,2001,410(6824):37-40.
[30]Xia Z,Dickens M,Raingeaud J,et al.Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis[J].Science,1995,270(5240):1326-31.