姜黄素通过Wnt3a/β-catenin/EMT信号通路抑制胃癌细胞的增殖、迁移及侵袭
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摘要
探讨姜黄素对人胃癌细胞增殖、迁移、侵袭及凋亡的作用及其机制。采用CCK-8法检测不同浓度(2. 5,5,10,20,40,80,160μmol·L~(-1))和不同干预时间(12,24,48,72 h)姜黄素对人胃癌细胞SGC7901,MKN45,NCI N87增殖的影响;划痕试验检测细胞迁移能力,Transwell小室侵袭试验检测细胞侵袭能力,流式细胞术检测细胞凋亡率,Western blot法检测N-cadherin,E-cadherin,snail1,Wnt3a,p-β-catenin,p-LRP6,Bcl-2,Bax的表达,试剂盒检测caspase-3,caspase-8,caspase-9酶活性的变化。结果表明,姜黄素对MKN45细胞的增殖具有较强抑制作用(IC50=21. 93μmol·L~(-1)),呈浓度、时间依赖性,显著抑制MKN45细胞的迁移和侵袭,下调N-cadherin,snail1,Wnt3a,p-β-catenin,p-LRP6,Bcl-2的表达,上调E-cadherin和Bax的表达,增加caspase-3,caspase-8,caspase-9活性,并诱导其凋亡,其机制可能与抑制Wnt3a/β-catenin/EMT通路,调控Bcl-2家族及caspase途径有关。
The aim of this paper was to investigate the effects of curcumin on the proliferation,migration,invasion and apoptosis of human gastric cancer cells and to explore the potential mechanisms. SGC7901,MKN45 and NCI N87 cells lines were cultured under different concentrations of curcumin( 2. 5,5,10,20,40,80 and 160 μmol·L~(-1)) at different time points( 12,24,48 and 72 h),and the effect of curcumin on cell proliferation was detected by CCK-8 assay. The migration and invasiveness of cells were determined by wound healing and Transwell assays,the apoptosis rate was assessed by flow cytometry,the expression of N-cadherin,E-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6,Bcl-2 and Bax were detected by Western blot,and the enzymatic activity of caspase-3,caspase-8 and caspase-9 was evaluated via caspase kit. Results indicated that the proliferation of MKN45 cells was significantly inhibited by curcumin in a dose-and time-dependent manner( IC50= 21. 93 μmol·L~(-1)). Moreover,curcumin could inhibit the migration and invasion of MKN45 cells,downregulate the expression of N-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6 and Bcl-2,and upregulate the expression of E-cadherin and Bax,it could increase the activity of caspase-3,caspase-8,caspase-9 and induce apoptosis as well. The potential mechanism is through inhibiting the Wnt3 a/β-catenin/EMT pathway,regulating Bcl-2 signaling and caspase pathway,which might provide new potential strategies for gastric cancer treatment.
The aim of this paper was to investigate the effects of curcumin on the proliferation,migration,invasion and apoptosis of human gastric cancer cells and to explore the potential mechanisms. SGC7901,MKN45 and NCI N87 cells lines were cultured under different concentrations of curcumin( 2. 5,5,10,20,40,80 and 160 μmol·L~(-1)) at different time points( 12,24,48 and 72 h),and the effect of curcumin on cell proliferation was detected by CCK-8 assay. The migration and invasiveness of cells were determined by wound healing and Transwell assays,the apoptosis rate was assessed by flow cytometry,the expression of N-cadherin,E-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6,Bcl-2 and Bax were detected by Western blot,and the enzymatic activity of caspase-3,caspase-8 and caspase-9 was evaluated via caspase kit. Results indicated that the proliferation of MKN45 cells was significantly inhibited by curcumin in a dose-and time-dependent manner( IC50= 21. 93 μmol·L~(-1)). Moreover,curcumin could inhibit the migration and invasion of MKN45 cells,downregulate the expression of N-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6 and Bcl-2,and upregulate the expression of E-cadherin and Bax,it could increase the activity of caspase-3,caspase-8,caspase-9 and induce apoptosis as well. The potential mechanism is through inhibiting the Wnt3 a/β-catenin/EMT pathway,regulating Bcl-2 signaling and caspase pathway,which might provide new potential strategies for gastric cancer treatment.
引文
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[29] Lee H P,Li T M,Tsao J Y,et al. Curcumin induces cell apoptosis in human chondrosarcoma through extrinsic death receptor pathway[J]. Int Immunopharmacol,2012,13(2):163.
[30] Cao A,Li Q,Yin P,et al. Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29cells through mitochondrial dysfunction and endoplasmic reticulum stress[J]. Apoptosis,2013,18(11):1391.
[2] Wang C Y,Bai X Y,Wang C H. Traditional Chinese medicine:a treasured natural resource of anticancer drug research and development[J]. Am J Chin Med,2014,42(3):543.
[3] Liu J,Wang S,Zhang Y,et al. Traditional Chinese medicine and cancer:history,present situation,and development[J].Thorac Cancer,2015,6(5):561.
[4] Panahi Y,Ahmadi Y,Teymouri M,et al. Curcumin as a potential candidate for treating hyperlipidemia:a review of cellular and metabolic mechanisms[J]. J Cell Physiol,2018,233(1):141.
[5]袁旭,李政,王晓天,等.中药及其有效成分在抗癫痫中的作用与机制[J].中国中药杂志,2019,44(1):9.
[6]马致洁,张祎,董捷鸣,等.基于代谢组学方法的姜黄素抗抑郁作用生物标志物初步筛查[J].中国中药杂志,2017,42(18):3596.
[7]张育光,吴声振,刘远亮,等.姜黄素对S180肉瘤小鼠的抗肿瘤作用及对血清IL-2和IL-12水平影响的研究[J].今日药学,2011,21(12):736.
[8] Seo J A,Kim B,Dhanasekaran D N,et al. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ATPase activity in ovarian cancer cells[J]. Cancer Lett,2016,371(1):30.
[9] Wang D,Kong X,Li Y,et al. Curcumin inhibits bladder cancer stem cells by suppressing Sonic Hedgehog pathway[J]. Biochem Biophys Res Commun,2017,493(1):521.
[10] Buhrmann C,Kraehe P,Lueders C,et al. Curcumin suppresses crosstalk between colon cancer stem cells and stromal fibroblasts in the tumor microenvironment:potential role of EMT[J]. PLoS ONE,2014,9(9):e107514.
[11]李宁,王雪婷,蒋少鸿,等.姜黄素-羟丙基-β-环糊精包合物的制备及其体外抗肿瘤活性评价[J].中国实验方剂学杂志,2018,24(19):31.
[12] Ruiz de Porras V,Bystrup S,Martinez-Cardus A,et al. Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC chemokine/NF-κB signaling pathway[J]. Sci Rep,2016,6:24675.
[13]任海玉,孙剑经,李多,等.姜黄素通过Wnt2/β-catenin通路逆转食管癌Eca-109/VCR细胞多药耐药性研究[J].中国药理学通报,2018,34(10):1455.
[14]刘文虎,袁江北,杨兰,等.姜黄素逆转胃癌细胞赫赛汀耐药机制研究[J].药学学报,2018,53(11):1817.
[15] Liu W H,Yuan J B,Liu Z Z,et al. Label-free quantitative proteomics combined with biological validation reveals activation of Wnt/β-catenin pathway contributing to trastuzumab resistance in gastric cancer[J]. Int J Mol Sci,2018,19(7):1981.
[16] Qi L,Song W,Liu Z,et al. Wnt3a promotes the vasculogenic mimicry formation of colon cancer via Wnt/β-catenin signaling[J]. Int J Mol Sci,2015,16(8):18564.
[17] Qi L,Sun B,Liu Z,et al. Wnt3a expression is associated with epithelial-mesenchymal transition and promotes colon cancer progression[J]. J Exp Clin Cancer Res,2014,33:107.
[18] Kotiyal S,Bhattacharya S. Breast cancer stem cells,EMT and therapeutic targets[J]. Biochem Biophys Res Commun,2014,453(1):112.
[19] Ciurea M E,Georgescu A M,Purcaru S O,et al. Cancer stem cells:biological functions and therapeutically targeting[J]. Int J Mol Sci,2014,15(5):8169.
[20] Tiwari N,Gheldof A,Tatari M,et al. EMT as the ultimate survival mechanism of cancer cells[J]. Semin Cancer Biol,2012,22(3):194.
[21] Brabletz T,Kalluri R,Nieto M A,et al. EMT in cancer[J]. Nat Rev Cancer,2018,18(2):128.
[22] Lamouille S,Xu J,Derynck R. Molecular mechanisms of epithelial-mesenchymal transition[J]. Nat Rev Mol Cell Biol,2014,15(3):178.
[23] Kim W,Kim M,Jho E H. Wnt/β-catenin signaling:from plasma membrane to nucleus[J]. Biochem J,2013,450(1):9.
[24] Zheng R Z,Deng Q H,Liu Y H,et al. Curcumin inhibits gastric carcinoma cell growth and induces apoptosis by suppressing the Wnt/β-catenin signaling pathway[J]. Med Sci Monit,2017,23:163.
[25] Pfeffer C M,Singh A T K. Apoptosis:a target for anticancer therapy[J]. Int J Mol Sci,2018,19(2):E448.
[26]辛文秀,郑小卫,李清林,等.长链非编码RNA NEAT1在肿瘤中的功能和作用机制研究进展[J].中国现代应用药学,2018,35(10):1580.
[27] Wu H,Che X,Zheng Q,et al. Caspases:a molecular switch node in the crosstalk between autophagy and apoptosis[J]. Int J Biol Sci,2014,10(9):1072.
[28]倪欣,黄坤,马俊杰. Procaspase-3小分子激活剂的研究进展[J].中国现代应用药学,2019,36(3):368.
[29] Lee H P,Li T M,Tsao J Y,et al. Curcumin induces cell apoptosis in human chondrosarcoma through extrinsic death receptor pathway[J]. Int Immunopharmacol,2012,13(2):163.
[30] Cao A,Li Q,Yin P,et al. Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29cells through mitochondrial dysfunction and endoplasmic reticulum stress[J]. Apoptosis,2013,18(11):1391.