重楼总皂苷对LiCl诱导人胃癌MKN-45细胞迁移及侵袭的影响
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摘要
目的探讨重楼总皂苷(Rhizoma Paridis total saponin,RPTS)在体外对人胃癌MKN-45细胞增殖、迁移和侵袭能力的影响,探讨其作用的可能机制。方法体外培养MKN-45细胞,取对数生长期细胞,加入不同质量浓度RPTS(2.5、5.0、10.0、20.0、40.0μg/m L)处理24h,采用MTT法检测细胞增殖情况;细胞划痕实验、Transwell小室实验检测RPTS对MKN-45细胞迁移和侵袭的影响;酶联免疫吸附实验(ELISA)检测RPTS对LiCl诱导的MKN-45细胞上清液中基质金属蛋白酶-9(MMP-9)的上调表达的影响;免疫印迹技术(Western blotting)和实时荧光定量PCR(qRT-PCR)技术分别检测RPTS(10、20、40μg/m L)对Li Cl诱导的MKN-45细胞中肿瘤侵袭转移相关分子血管内皮生长因子(VEGF)、环加氧酶-2(COX-2)以及Li Cl作用靶点糖原合酶激酶-3β(GSK-3β)蛋白和基因表达水平的影响。结果与对照组比较,RPTS 5.0、10.0、20.0、40.0μg/m L可明显下调MKN-45细胞的增殖活性(P<0.05、0.001);RPTS 2.5、5.0、10.0μg/m L可抑制MKN-45细胞的迁移和侵袭能力(P<0.01、0.001)。与模型组比较,RPTS能够显著下调Li Cl诱导的MKN-45细胞上清液中MMP-9的表达水平(P<0.05、0.01);下调细胞中VEGF、COX-2 mRNA和蛋白表达水平;上调Li Cl作用位点GSK-3βm RNA和蛋白表达水平(P<0.05、0.001)。结论 RPTS具有体外抑制MKN-45细胞迁移和侵袭的能力,其作用机制可能与调控Wnt/β-catenin信号通路有关。
Objective To investigate the effect and the possible mechanism of Rhizoma Paridis total saponin(RPTS) on human gastric cancer cell line MKN-45 proliferation, migration and invasion in vitro. Methods MKN-45 cells were cultured in vitro and treated respectively with indicated concentrations of RPTS(2.5, 5.0, 10.0, 20.0, and 40.0 μg/m L) for 24 h, and cell viability of cell proliferation was detected by MTT assay; The invasive and metastatic ability of MKN-45 treated with indicated concentrations of RPTS(2.5, 5.0, 10.0 μg/mL) was detected by Transwell migration assay and wound healing assay; Elisa assay was employed to detect the concentrations of MMP-9 induced by LiCl after RPTS administration(10, 20, and 40 μg/mL) in the cell supernatant;Western blotting and qRT-PCR were respectively performed to investigate the invasion and migration related protein and mRNA level of VEGF, COX-2, and GSK-3β in RPTS-treated MKN-45 after LiCl stimulation for 24 h. Results Compared with the control group, RPTS(10, 20, and 40 μg/mL) significantly inhibited the proliferation of MKN-45 cells(P < 0.05 and P < 0.001); RPTS(2.5,5.0, 10.0 μg/m L) suppressed the invasion and migration of MKN-45 cells(P < 0.05 and P < 0.001); Compared with the model group,RPTS significantly downregulated the expression of MMP-9 in the cell supernatant of MKN-45 cells induced by LiCl(P < 0.05 and P <0.01), and RPTS also decreased the protein and mRNA expression level of VEGF and COX-2, but it significantly upregulated the expression of GSK-3β at the protein and m RNA level(P < 0.05 and P < 0.001). Conclusion RPTS play a pivotal role in suppressing the invasion and migration of MKN-45 cells in vitro, and its mechanism may be related to the regulating effects of the Wnt/β-catenin pathway in the human gastric adenocarcinoma cell.
Objective To investigate the effect and the possible mechanism of Rhizoma Paridis total saponin(RPTS) on human gastric cancer cell line MKN-45 proliferation, migration and invasion in vitro. Methods MKN-45 cells were cultured in vitro and treated respectively with indicated concentrations of RPTS(2.5, 5.0, 10.0, 20.0, and 40.0 μg/m L) for 24 h, and cell viability of cell proliferation was detected by MTT assay; The invasive and metastatic ability of MKN-45 treated with indicated concentrations of RPTS(2.5, 5.0, 10.0 μg/mL) was detected by Transwell migration assay and wound healing assay; Elisa assay was employed to detect the concentrations of MMP-9 induced by LiCl after RPTS administration(10, 20, and 40 μg/mL) in the cell supernatant;Western blotting and qRT-PCR were respectively performed to investigate the invasion and migration related protein and mRNA level of VEGF, COX-2, and GSK-3β in RPTS-treated MKN-45 after LiCl stimulation for 24 h. Results Compared with the control group, RPTS(10, 20, and 40 μg/mL) significantly inhibited the proliferation of MKN-45 cells(P < 0.05 and P < 0.001); RPTS(2.5,5.0, 10.0 μg/m L) suppressed the invasion and migration of MKN-45 cells(P < 0.05 and P < 0.001); Compared with the model group,RPTS significantly downregulated the expression of MMP-9 in the cell supernatant of MKN-45 cells induced by LiCl(P < 0.05 and P <0.01), and RPTS also decreased the protein and mRNA expression level of VEGF and COX-2, but it significantly upregulated the expression of GSK-3β at the protein and m RNA level(P < 0.05 and P < 0.001). Conclusion RPTS play a pivotal role in suppressing the invasion and migration of MKN-45 cells in vitro, and its mechanism may be related to the regulating effects of the Wnt/β-catenin pathway in the human gastric adenocarcinoma cell.
引文
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[2]Ferlay J,Soerjomataram I,Dikshit R,et al.Cancer incidence andmortality worldwide:Sources,methods and major patterns in GLOBOCAN 2012[J].Int J Cancer,2015,136(5):359-386.
[3]Kim H S,Kim J H,Kim J W,et al.Chemotherapy in elderly patients with gastric cancer[J].J Cancer,2016,7(1):88-94.
[4]Farran B,Müller S,Montenegro R C.Gastric cancer managment:Kinases as a target therapy[J].Clin Exp Pharmacol Physiol,2017,44(6):613-622.
[5]Sun J,Liu B R,Hu W J,et al.In vitro anticancer activity of aqueous extracts and ethanol extracts of fifteen traditional Chinese medicines on human digestive tumor cell lines[J].Phytother Res,2007,21(11):1102-1104.
[6]陈雪梅,杨焕芝,施敏,等.长柱重楼总皂苷的抗肿瘤活性及急性毒性作用研究[J].药物评价研究,2017,40(7):904-910.
[7]金炜东,陈孝平,蔡红娇.重楼提取物对HepG2细胞的毒性作用[J].华中科技大学学报:医学版,2006,35(1):103-106.
[8]吴夏慧,薛娇,胡文静,等.重楼总皂苷提取分离及其对人胃癌细胞BGC823的抑制作用[J].江苏中医药,2011,43(8):84-86.
[9]朱丽丽,李惠芬.重楼皂苷对SGC-7901细胞增殖抑制及诱导凋亡的实验研究[J].时珍国医国药,2009,20(6):1501-1502.
[10]Cheng Z X,Liu B R,Qian X P,et al.Proteomic analysis of anti-tumor effects by Rhizoma Paridistotal saponintreatment in Hep G2 cells[J].J Ethnopharmacol,2008,122(2):139-137.
[11]陈志红,龚先玲,刘义,等.重楼总皂苷对人鼻咽癌细胞CNE-2Z周期及凋亡的影响[J].中成药,2011,33(1):25-29.
[12]保永亮,龚晓燕,方海雁,等.重楼总皂苷对人胃癌MNK-45和MGC80-3细胞增殖的影响[J].安徽中医学院学报,2012,31(6):51-55.
[13]方海雁,龚晓燕,洪星辉,等.重楼总皂苷对人胃癌MKN-45细胞凋亡及Fas/FasL信号通路的影响[J].中国中药杂志,2015,40(7):1388-1391.
[14]Li V S,Ng S S,Boersema P J,et al.Wnt signaling through inhibition ofβ-catenin degradation in an intact Axin1 complex[J].Cell,2012,149(6):1245-1256.
[15]Holland J D,Klaus A,Garratt A N,et al.Wnt signaling in stem and cancer stem cells[J].Curr Opin Cell Biol,2013,25(2):254-264.
[16]Ilyas M.Wnt signalling and the mechanistic basis of tumour development[J].J Pathol,2015,205(2):130-144.
[17]年四辉,张赫名,郑渝艳.重楼水提工艺的初步研究[J].云南中医学院学报,2006,29(1):13-16.
[18]张娟,路金才.皂苷的提取方法及含量测定研究进展[J].中国现代中药,2006,8(3):25-28.
[19]孙治国,张琳,李凌军,等.重楼总皂苷提取工艺研究[J].中药材,2006,30(6):726-729.
[20]孙治国,张琳,田景奎,等.重楼总皂苷的纯化工艺研究[J].药学实践杂志.2009,27(6):455-469.
[21]杨骁,张振秋.重楼中总皂苷的含量测定[J].中华中医药学刊,2007,25(11):2420-2422.
[22]Chen W Q,Zheng R S,Baade P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.