马齿苋醇提取物对结肠癌细胞及其干细胞体外增殖作用的机理研究
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摘要
目的探讨马齿苋醇提取物对结肠癌细胞及其干细胞体外增殖能力的影响及其作用机制。方法对结肠癌干细胞进行无血清培养,CCK-8法测定不同浓度马齿苋醇提取物对结肠癌HT-29细胞及HT-29干细胞的生长抑制情况,流式细胞仪检测其对HT-29细胞及其干细胞的凋亡情况及对细胞周期的影响。RT-PCR法以及Western blot分析马齿苋醇提取物对HT-29细胞及其干细胞Notch、Wnt双信号通路中Notch1、Notch2及β-catenin基因及蛋白表达的影响。结果马齿苋醇提取物对HT-29细胞及HT-29干细胞增殖有抑制作用,其作用时间、剂量与细胞增殖抑制率呈正相关,且对HT-29细胞增殖的抑制作用及凋亡率的影响较HT-29干细胞显著(P<0.05)。马齿苋醇提取物可下调HT-29细胞及其干细胞Notch1、β-catenin基因表达,并下调Notch1、Notch2及β-catenin蛋白表达(P<0.05)。结论马齿苋醇提取物可以通过下调Notch-1、Notch-2、β-catenin蛋白的表达发挥体外抑制结肠癌细胞及其干细胞增殖的作用。
Objective To discuss the influence of ethanol extract from Machixian(Herba Portulacae,Portulaca) on colon cancer cells and their stem cell proliferation in vitro and effective mechanism.Methods The stem cells of colon cancer were cultivated with serum free medium.The inhibitory effects of ethanol extract from Machixian in difference doses on growth of HT-29 cells and HT-29 stem cells were detected by using CCK-8 assay.The influence of ethanol extract from Machixian on apoptosis and cell cycle of HT-29 cells and HT-29 stem cells was detected by using flow cytometer.The influence of ethanol extract from Machixian on genetic and protein expressions of Notch1,Notch2 and β-catenin in Notch signal pathway and Wnt signal pathway of HT-29 cells and HT-29 stem cells was analyzed by using reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting method.Results The ethanol extract from Machixian had inhibitory effect on proliferation of HT-29 cells and HT-29 stem cells,its effective time and dose were positively correlated to cellular proliferation inhibition rate,and its inhibitory effect on proliferation and improve effect on apoptosis rate were more significant on HT-29 cells than those on HT-29 stem cells(P < 0.05).The ethanol extract from Machixian could down-regulate genetic expressions of Notch1 and β-catenin and protein expressions of Notch1,Notch2 and β-catenin of HT-29 cells and HT-29 stem cells(P < 0.05).Conclusion The ethanol extract from Machixian can inhibit in vitro the proliferation of colon cancer cells and their stem cells through down-regulating protein expressions of Notch1,Notch2 and β-catenin.
Objective To discuss the influence of ethanol extract from Machixian(Herba Portulacae,Portulaca) on colon cancer cells and their stem cell proliferation in vitro and effective mechanism.Methods The stem cells of colon cancer were cultivated with serum free medium.The inhibitory effects of ethanol extract from Machixian in difference doses on growth of HT-29 cells and HT-29 stem cells were detected by using CCK-8 assay.The influence of ethanol extract from Machixian on apoptosis and cell cycle of HT-29 cells and HT-29 stem cells was detected by using flow cytometer.The influence of ethanol extract from Machixian on genetic and protein expressions of Notch1,Notch2 and β-catenin in Notch signal pathway and Wnt signal pathway of HT-29 cells and HT-29 stem cells was analyzed by using reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting method.Results The ethanol extract from Machixian had inhibitory effect on proliferation of HT-29 cells and HT-29 stem cells,its effective time and dose were positively correlated to cellular proliferation inhibition rate,and its inhibitory effect on proliferation and improve effect on apoptosis rate were more significant on HT-29 cells than those on HT-29 stem cells(P < 0.05).The ethanol extract from Machixian could down-regulate genetic expressions of Notch1 and β-catenin and protein expressions of Notch1,Notch2 and β-catenin of HT-29 cells and HT-29 stem cells(P < 0.05).Conclusion The ethanol extract from Machixian can inhibit in vitro the proliferation of colon cancer cells and their stem cells through down-regulating protein expressions of Notch1,Notch2 and β-catenin.
引文
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[10]Mariangela M,Brfigida C,Francesco M,et al.Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cell[J].Food and Chemical 2015,86(12):16-24.
[11]Ghaleb AM,Aggarwal G,Bialkowska AB,et al.Notch inhibits expression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium[J].Mol Cancer Res,2008,6(12):1920-1927.
[12]Chu D,Zhang Z,Zhou Y,et al.Notchl and Notch2 have opposite prognostic effects on patients with colorectal cancer[J].Ann Oncol,2011,22(11):2440-2447.
[13]Chu D,Zheng J,Wang W,et al.Notch2 expression is decreased in colorecta I cancer and related to tumor differentiation status[J].Ann Surg Oncol,2009,16(12):3259-3266.
[14]Gregorieff A,Clevers H.Wnt signaling in the intestinal epithelium:From endoderm to cancer[J].Genes Dev,2005,19(8):877-890.
[15]Boman BM,Huang E.Human colon cancer stem cells:a new paradigm in gastrointestinal oncology[J].Clinical Oncology,2008,26(17):2828-2838.
[16]Felipe DSEM,Vermeulen L.Wnt Signaling in Cancer Stem Cell Biology[J].Cancers,2016,8(7):60-62.
[2]陈竺.全国第三次死因回顾抽样调查报告[M].北京:中国协和医科大学出版社,2008.Chen Z.Report on National Third Retrospective Sampling Survey of Causes of Death[M].Beijing:Peking Union Medical College Press,2008.
[3]Zeuner A,Todaro M,Stassi G,et al.Colorectal cancer stem cells:from the crypt to the clinic[J].Cell Stem Cell,2014,15(6):692-705.
[4]Zhou YX,Xin HL,RAHMAN K,et al.Portulaca oleracea L:a review of phytochemistry and pharmacological effects[J].Biomed Res Int,2015,2015:925631.
[5]邓超,陈豪,金黑鹰,等.结肠癌干细胞的肿瘤形成特征及其临床价值[J].世界华人消化杂志,2015,23(3):363-372.Deng C,Chen H,Jin HY,et al.Tumorigenic features of colon cancer stem cells[J].World Chinese Journal of Digestology,2015,23(3):363-372.
[6]Wilson AA,Kotton DN.Another notch in stem cell biology:Drosophila intestinal stem cells and the specification of cell fates[J].Bioessays,2008,30(2):107-109.
[7]Vaiopoulos AG,Kostakis BID,Koutsukueris BM,et al.Concise review:Colorectal cancer stem cells[J].Stem Cells,2012,30(3):363-371.
[8]周阿成,金黑鹰,谈瑄忠,等.洋葱提取物对结肠癌细胞增殖的抑制作用[J].世界华人消化杂志,2011,19(19):2011-2015.Zhou AC,Jin HY,Tan XZ,et al.Allium cepa extracts inhibit the proliferation of colorectal cancer in vitro[J].World Chinese Journal of Digestology,2011,19(19):2011-2015.
[9]戴功建,金黑鹰,丁义江,等.茶多酚对微卫星不稳定结直肠癌裸小鼠的抗肿瘤作用及其作用机制[J].中西医结合学报,2008,12(12):1263-1266.Dai GJ,Jin HY,Ding YJ.et al.Anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability in nude mice[J].Journal of Chinese Integrative Medicine,2008,12(12):1263-1266.
[10]Mariangela M,Brfigida C,Francesco M,et al.Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cell[J].Food and Chemical 2015,86(12):16-24.
[11]Ghaleb AM,Aggarwal G,Bialkowska AB,et al.Notch inhibits expression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium[J].Mol Cancer Res,2008,6(12):1920-1927.
[12]Chu D,Zhang Z,Zhou Y,et al.Notchl and Notch2 have opposite prognostic effects on patients with colorectal cancer[J].Ann Oncol,2011,22(11):2440-2447.
[13]Chu D,Zheng J,Wang W,et al.Notch2 expression is decreased in colorecta I cancer and related to tumor differentiation status[J].Ann Surg Oncol,2009,16(12):3259-3266.
[14]Gregorieff A,Clevers H.Wnt signaling in the intestinal epithelium:From endoderm to cancer[J].Genes Dev,2005,19(8):877-890.
[15]Boman BM,Huang E.Human colon cancer stem cells:a new paradigm in gastrointestinal oncology[J].Clinical Oncology,2008,26(17):2828-2838.
[16]Felipe DSEM,Vermeulen L.Wnt Signaling in Cancer Stem Cell Biology[J].Cancers,2016,8(7):60-62.