薯蓣皂苷通过调控PI3K/AKT信号通路抑制人肺腺癌细胞H1299上皮间质转化
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摘要
目的研究薯蓣皂苷(Dioscin,Dio)对人肺腺癌细胞H1299上皮间质转化的影响,并初步探讨其作用机制。方法采用不同浓度Dio(1、2、4、8μmol·L~(-1))处理H1299细胞24、48 h,CCK8法检测细胞的增殖能力。选择低于IC_(50)浓度的Dio(1、2μmol·L~(-1))作用于H1299细胞,采用细胞划痕实验和Transwell穿膜实验观察不同浓度Dio对H1299细胞迁移能力的影响;采用铺有Matrigel胶的Transwell实验检测Dio对H1299细胞侵袭能力的影响;Western Blot法检测Dio对H1299细胞中E-cadherin、Zonula occluden 1(ZO-1)、ZEB1、Cludin-1、N-cadherin、Vimentin、β-catenin、PI3K、AKT、P-AKT蛋白表达的影响;免疫荧光法检测间质标志物Vimentin在细胞中的定位及表达情况。结果 Dio干预24、48 h后的IC_(50)值分别为3.50、3.21μmol·L~(-1),且呈现浓度依赖性。Dio干预后与空白对照组比较,Dio 1、2μmol·L~(-1)浓度组的细胞愈合率显著降低(P<0.05,P<0.001);Dio 1、2μmol·L~(-1)浓度组均能显著抑制H1299细胞的迁移能力(P <0.001),并显著抑制H1299细胞体外侵袭能力(P <0.001);Dio 1、2μmol·L~(-1)浓度组能明显上调E-cadherin、ZO-1和Cludin-1蛋白的表达(P <0.05,P <0.01,P <0.001),下调N-cadherin、Vimentin、ZEB1、β-catenin蛋白及PI3K/AKT信号通路蛋白PI3K、AKT、P-AKT的表达(P <0.05,P <0.01,P <0.001);Dio 1、2μmol·L~(-1)浓度组免疫荧光定位表达结果显示Vimentin表达明显降低。结论 Dio能有效抑制人肺腺癌H1299细胞增殖,低于IC_(50)浓度的Dio(1、2μmol·L~(-1))能明显抑制H1299细胞的迁移与侵袭能力,可能与通过调控PI3K/AKT信号通路有关。
Objective To investigate roles of dioscin(Dio) on epithelial-mesenchymal transition of lungadenocarcinoma H1299 cells,and the mechanism of its role on migration and invasion. Methods H1299 cells weretreated with Dio at a range of 1 to 8 μmol·L~(-1)for 24 h and 48 h respectively. Cell proliferation was assessed byCCK8 assay. Cell migration at different concentrations of Dio(1,2 μmol·L~(-1)) on H1299 cells was assayed by thescratch wound healing and Transwell chamber assay. And Matrigel cell invasion assay was used to detect the effect ofDio on the invasion of H1299 cells. Western Blot was used to detect the protein levels of E-cadherin, Zonulaoccluden 1(ZO-1),ZEB1,Cludin-1,N-cadherin,Vimentin,β-catenin,PI3 K,AKT,P-AKT in the cells.Immunofluorescence was used to detect the localization and expression of interstitial marker Vimentin in cells.Results IC_(50) values were 3.50 μmol·L~(-1)and 3.21 μmol·L~(-1)after cells were treated with Dio for 24 h and 48 h,which showed a dose dependent manner. Compared with the control,the scratch wound healing of the cells were significantly decreased after treating with Dio(1,2 μmol·L~(-1))(P<0.05,P<0.001);and we observed that Dio(1,2 μmol·L~(-1)) significantly suppressed the migration(P < 0.001) and invasion(P < 0.001) activities of H1299 cells. What's more,the levels of E-cadherin,ZO-1 and Cludin-1 proteins' expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001), and the levels of N-cadherin, Vimentin, ZEB1, β-catenin, PI3 K, AKT, P-AKT protein expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001). Conclusion Our datademonstrated that Dio can effectively inhibit the proliferation of human lung adenocarcinoma H1299 cells. Dio doses(1,2 μmol·L~(-1)) lower than IC_(50) can significantly inhibit the migration and invasion of H1299 cells,which may berelated to the regulation of PI3K/AKT signaling pathway.
Objective To investigate roles of dioscin(Dio) on epithelial-mesenchymal transition of lungadenocarcinoma H1299 cells,and the mechanism of its role on migration and invasion. Methods H1299 cells weretreated with Dio at a range of 1 to 8 μmol·L~(-1)for 24 h and 48 h respectively. Cell proliferation was assessed byCCK8 assay. Cell migration at different concentrations of Dio(1,2 μmol·L~(-1)) on H1299 cells was assayed by thescratch wound healing and Transwell chamber assay. And Matrigel cell invasion assay was used to detect the effect ofDio on the invasion of H1299 cells. Western Blot was used to detect the protein levels of E-cadherin, Zonulaoccluden 1(ZO-1),ZEB1,Cludin-1,N-cadherin,Vimentin,β-catenin,PI3 K,AKT,P-AKT in the cells.Immunofluorescence was used to detect the localization and expression of interstitial marker Vimentin in cells.Results IC_(50) values were 3.50 μmol·L~(-1)and 3.21 μmol·L~(-1)after cells were treated with Dio for 24 h and 48 h,which showed a dose dependent manner. Compared with the control,the scratch wound healing of the cells were significantly decreased after treating with Dio(1,2 μmol·L~(-1))(P<0.05,P<0.001);and we observed that Dio(1,2 μmol·L~(-1)) significantly suppressed the migration(P < 0.001) and invasion(P < 0.001) activities of H1299 cells. What's more,the levels of E-cadherin,ZO-1 and Cludin-1 proteins' expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001), and the levels of N-cadherin, Vimentin, ZEB1, β-catenin, PI3 K, AKT, P-AKT protein expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001). Conclusion Our datademonstrated that Dio can effectively inhibit the proliferation of human lung adenocarcinoma H1299 cells. Dio doses(1,2 μmol·L~(-1)) lower than IC_(50) can significantly inhibit the migration and invasion of H1299 cells,which may berelated to the regulation of PI3K/AKT signaling pathway.
引文
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[31]姜爽,任艳平,魏琳,等.人参皂苷CK对MCF-7细胞增殖、凋亡、上皮间质转化、PI3K/Akt信号通路的影响[J].中成药,2018,40(9):1925-1929.
[32]王雷,杜媛鲲,米源,等.Hedgehog/Gli和PI3K/AKT信号通路的串话促进胃癌AZ521细胞上皮-间质转化[J].中国医科大学学报,2018,47(2):151-156.
[33]李洁玭,秦垠,邹玺,等.桂皮醛通过PI3K/Akt信号通路抑制TGF-β_1诱导的结肠癌细胞LoVo上皮间质转化[J].中国实验方剂学杂志,2017,23(18):105-111.
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[36]YAO K,YE P P,TAN J,et al.Involvement of PI3K/Akt pathway in TGF-β2-mediated epithelial mesenchymal transition in human lens epithelial cells[J].Ophthalmic Research,2008,40(2):69-76.
[37]BAKIN A V,TOMLINSON A K,BHOWMICK N A,et al.Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration[J].Journal of Biological Chemistry,2000,275(47):36803-36810.
[38]PORTA C,PAGLINO C,MOSCA A.Targeting PI3K/Akt/mTORSignaling in Cancer[J].Frontiers in Oncology,2014,4(4):64.
[2]KLASTERSKY J,PAESMANS M.Response to chemotherapy,quality of life benefits and survival in advanced non-small cell lung cancer:review of literature results[J].Lung Cancer,2001,34(3):95-101.
[3]MICALIZZI D S,FARABAUGH S M,FORD H L.Epithelialmesenchymal transition in cancer:parallels between normal development and tumor progression[J].Journal of Mammary Gland Biology&Neoplasia,2010,15(2):117-134.
[4]PEREGO M,TORTORETO M,TRAGNI G,et al.Heterogeneous phenotype of human melanoma cells with in vitro and in vivo features of tumor-initiating cells[J].Journal of Investigative Dermatology,2010,130(7):1877-1886.
[5]THOMPSON E W,NEWGREEN D F,TARIN D.Carcinoma invasion and metastasis:a role for epithelial-mesenchymal transition?[J].Cancer Research,2005,65(14):5991-5995.
[6]KRANTZ S B,SHIELDS M A,DANGI-GARIMELLA S,et al.Contribution of epithelial-mesenchymal transition to pancreatic cancer progression[J].Cancers,2010,2(4):2084-2097.
[7]ACLOQUE H,ADAMS M S,FISHWICK K,et al.Epithelialmesenchymal transitions:the importance of changing cell state in development and disease[J].Journal of Clinical Investigation,2009,119(6):1438-1449.
[8]KWON C S,SOHN H Y,KIM S H,et al.Anti-obesity effect of Dioscorea nipponica Makino with lipase-inhibitory activity in rodents[J].Biosci Biotechnol Biochem,2003,67(7):1451-1456.
[9]SAUTOUR M,MITAINEOFFER A C,MIYAMOTO T,et al.A new steroidal saponin from dioscorea cayenensis[J].Chemical&Pharmaceutical Bulletin,2004,52(11):1353-1355.
[10]WANG T,LIU Z,LI J,et al.Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry[J].Journal of Chromatography B,2007,848(2):363-368.
[11]CHO J,CHOI H,LEE J,et al.The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica[J].Biochimica Et Biophysica Acta,2013,1828(3):1153-1158.
[12]ZHAO X,CONG X,ZHENG L,et al.Dioscin,a natural steroid saponin,shows remarkable protective effect against acetaminopheninduced liver damage in vitro and in vivo[J].Toxicology Letters,2012,214(1):69-80.
[13]AQUINO R,CONTI C,DE F S,et al.Antiviral activity of constituents of Tamus communis[J].Journal of Chemotherapy,1991,3(5):305-309.
[14]CHADWICK H.iTRAQ-based proteomic analysis of dioscin on human HCT-116 colon cancer cells[J].Proteomics,2014,14(1):51-73.
[15]LI C,LU Y,DU S,et al.Dioscin exerts protective effects against crystalline silica-induced pulmonary fibrosis in mice[J].Theranostics,2017,7(17):4255-4275.
[16]KOU Y,JI L,WANG H,et al.Connexin 43 upregulation by dioscin inhibits melanoma progression via suppressing malignancy and inducing M1 polarization[J].International Journal of Cancer,2017,141(8):1690-1703.
[17]MJAATVEDT C H,MARKWALD R R.Induction of an epithelialmesenchymal transition by an in vivo adheron-like complex[J].Developmental Biology,1989,136(1):118-128.
[18]THOMPSON E W,TORRI J,SABOL M,et al.Oncogeneinduced basement membrane invasiveness in human mammary epithelial cells[J].Clinical&Experimental Metastasis,1994,12(3):181-194.
[19]SAVAGNER P,BOYER B,VALLES A M,et al.Modulations of the epithelial phenotype during embryogenesis and cancer progression[J].Cancer Treat Res,1994,71:229-249.
[20]XU J,ZHU W,XU W,et al.Up-regulation of MBD1 promotes pancreatic cancer cell epithelial-mesenchymal transition and invasion by epigenetic down-regulation of E-cadherin[J].Current Molecular Medicine,2013,13(3):387-400.
[21]BOREDDY S R,SRIVASTAVA S K.Deguelin suppresses pancreatic tumor growth and metastasis by inhibiting epithelial-to-mesenchymal transition in an orthotopic model[J].Oncogene,2013,32(34):3980-3991.
[22]SáNCHEZTILLóE,LIU Y,BARRIOS O D,et al.EMT-activating transcription factors in cancer:beyond EMT and tumor invasiveness[J].Cellular&Molecular Life Sciences,2012,69(20):3429-3456.
[23]BASTID J.EMT in carcinoma progression and dissemination:facts,unanswered questions,and clinical considerations[J].Cancer Metastasis Rev,2012,31(1-2):277-283.
[24]NAKASHIMA H,HASHIMOTO N,AOYAMA D,et al.Involvement of the transcription factor twist in phenotype alteration through epithelial-mesenchymal transition in lung cancer cells[J].Molecular Carcinogenesis,2012,51(5):400-410.
[25]胡彦建,韩明子,胡彦华.上皮间质转化在原发性肝癌侵袭、转移中的作用[J].胃肠病学和肝病学杂志,2014,23(1):117-120.
[26]WENDT M K,TAYLOR M A,SCHIEMANN B J,et al.Downregulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer[J].Molecular Biology of the Cell,2011,22(14):2423-2435.
[27]RADISKY D C,LEVY D D,LITTLEPAGE L E,et al.Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability[J].Nature,2005,436(7047):123-127.
[28]YOO Y A,KANG M H,LEE H J,et al.Sonic hedgehog pathway promotes metastasis and lymphangiogenesis via activation of Akt,EMT,and MMP-9 pathway in gastric cancer[J].Cancer Research,2011,71(22):7061-7070.
[29]江苏新医学院.中药大辞典(下册)[M].上海:上海人民出版社,1977:1726.
[30]李笑,李洪利,尹崇高.TGF-β_1通过PI3K/AKT/ARK5/Snail信号通路促进非小细胞肺癌SPC-A1细胞的上皮-间质转化[J].中国生物化学与分子生物学报,2015,31(7):710-715.
[31]姜爽,任艳平,魏琳,等.人参皂苷CK对MCF-7细胞增殖、凋亡、上皮间质转化、PI3K/Akt信号通路的影响[J].中成药,2018,40(9):1925-1929.
[32]王雷,杜媛鲲,米源,等.Hedgehog/Gli和PI3K/AKT信号通路的串话促进胃癌AZ521细胞上皮-间质转化[J].中国医科大学学报,2018,47(2):151-156.
[33]李洁玭,秦垠,邹玺,等.桂皮醛通过PI3K/Akt信号通路抑制TGF-β_1诱导的结肠癌细胞LoVo上皮间质转化[J].中国实验方剂学杂志,2017,23(18):105-111.
[34]王月松.PI3K/Akt信号通路在子宫内膜腺癌上皮间质转化中的作用[D].青岛:青岛大学,2017.
[35]李少星.PI3K/Akt信号通路对上皮性卵巢癌细胞顺铂耐药与上皮间质转化关系的影响[D].石家庄:河北医科大学,2013.
[36]YAO K,YE P P,TAN J,et al.Involvement of PI3K/Akt pathway in TGF-β2-mediated epithelial mesenchymal transition in human lens epithelial cells[J].Ophthalmic Research,2008,40(2):69-76.
[37]BAKIN A V,TOMLINSON A K,BHOWMICK N A,et al.Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration[J].Journal of Biological Chemistry,2000,275(47):36803-36810.
[38]PORTA C,PAGLINO C,MOSCA A.Targeting PI3K/Akt/mTORSignaling in Cancer[J].Frontiers in Oncology,2014,4(4):64.