柴胡总皂苷通过Akt/NF-κB信号通路抑制胃癌细胞MGC80-3的增殖和迁移
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摘要
该文以中药柴胡为材料,研究其皂苷成分对胃癌细胞MGC80-3的抑制活性,并探讨其潜在的分子机制。采用已报道的方法提取柴胡的总皂苷成分,通过显微镜观察其对MGC80-3细胞形态的影响,利用MTT法和细胞集落形成实验检测其对MGC80-3细胞增殖的影响,利用细胞黏附实验和细胞划痕实验分析其对MGC80-3细胞迁移的影响,进一步通过Western blot实验检测柴胡总皂苷对Akt/NF-κB信号通路的影响。结果显示,柴胡总皂苷显著抑制胃癌细胞MGC80-3的增殖和迁移能力,并呈现明显的量效关系和时效关系。而且,柴胡总皂苷降低了NF-κB p65的蛋白表达水平,虽然不影响Akt和IKKβ的表达,却显著抑制了二者的磷酸化水平。因此,柴胡总皂苷可能通过Akt/NF-κB信号通路发挥对胃癌细胞MGC80-3的抑制作用,具有开发为抗胃癌药物的潜在价值。
The inhibitory effect of saikosaponin from Bulperum Chinese DC on gastric cancer line MGC80-3 was accessed in this study. And then, the potential molecular mechanism was investigated. Saikosaponin was extracted from Bulperum Chinese DC using the reported method. Effect of saikosaponin on MGC80-3 cell morphology was accessed with the light microscope. Effect of saikosaponin on cell proliferation was determined by MTT method and colony formation assay. Migration capability of MGC80-3 was detected by the cell adhesive assay and wound-healing assay. Western blot was used to access the potential molecular mechanism. Saikosaponin from Bulperum Chinese DC repressed the capability of MGC80-3 cell proliferation and migration in a dose-and time-dependent manner. Saikosaponin significantly reduced the expression level of NF-κB p65, but not affect that of Akt and IKK β. Besides, phosphorylation of Akt and IKK β was decreased in cells treated by saikosaponin. Itis probable that saikosaponin from Bulperum Chinese DC repressed MGC80-3 cell via Akt/NF-κB pathway, and showed potential value in the development of anti-gastric cancer drugs.
The inhibitory effect of saikosaponin from Bulperum Chinese DC on gastric cancer line MGC80-3 was accessed in this study. And then, the potential molecular mechanism was investigated. Saikosaponin was extracted from Bulperum Chinese DC using the reported method. Effect of saikosaponin on MGC80-3 cell morphology was accessed with the light microscope. Effect of saikosaponin on cell proliferation was determined by MTT method and colony formation assay. Migration capability of MGC80-3 was detected by the cell adhesive assay and wound-healing assay. Western blot was used to access the potential molecular mechanism. Saikosaponin from Bulperum Chinese DC repressed the capability of MGC80-3 cell proliferation and migration in a dose-and time-dependent manner. Saikosaponin significantly reduced the expression level of NF-κB p65, but not affect that of Akt and IKK β. Besides, phosphorylation of Akt and IKK β was decreased in cells treated by saikosaponin. Itis probable that saikosaponin from Bulperum Chinese DC repressed MGC80-3 cell via Akt/NF-κB pathway, and showed potential value in the development of anti-gastric cancer drugs.
引文
1 Chen WQ,Zheng RS,Baade PD,Zhang SW,Zeng HM,Bray F,et al.Cancer statistics in China,2015.CA-Cancer J Clin 2016;66(2):115-32.
2涂铭,李林.胃癌防治知识对胃癌患者早期诊断的影响.实用癌症杂志(Tu Ming,Li Lin.Effect of knowledge about prevention of gastric cancer on early diagnosis of gastric cancer.The Practical Journal of Cancer)2014;29(8):972-4.
3石佳佳,曹丽丽,韩倩倩,朱艳琴.含皂苷类中药抗肿瘤作用的研究进展.光明中医(Shi Jiajia,Cao Lili,Han Qianqian,Zhu Yanqin.Research progress on anti-tumor effects of traditional Chinese medicine containing saponins.Guang Ming Zhong Yi)2016;31(23):3526-9.
4孙大鹏,鲁明明,王硕,富力.人参皂苷Rg3通过Ca2+/CaM信号系统抑制胃癌BGC-823细胞增殖及其可能的机制.中国肿瘤生物治疗杂志(Sun Dapeng,Lu Mingming,Wang Shuo,Fu Li.Ginsenoside Rg3 inhibits gastric cancer cell proliferation through Ca2+/CaM kinase downregulation and NF-κB inactivation.Chinese Journal of Cancer Biotherapy)2015;22(2):225-9.
5徐明明.柴胡皂甙D诱导人宫颈癌Hela细胞系凋亡的研究.医药论坛杂志(Xu Ming ming.Study of human cervical cancer Hela apoptosis induced by saikosaponin D.Journal of Medical Forum)2011;32(8):89-71.
6章英宏.桔梗皂苷D对人乳腺癌细胞体外杀伤效应及机制研究.浙江中西医结合杂志(Zhang Yinghong.Viability inhibition of human breast cancer cells treated with platycodin D in vitro and theunderlying mechanism.Zhejiang Journal of Integrated Traditional Chinese and Western Medicine)2015;25(6):547-9.
7陈美娟,赵若琳,郭园园,宁利敏,张旭.麦冬皂苷B对A549细胞株体外黏附、侵袭及迁移的抑制作用及机制研究.中国药理学通报(Chen Meijuan,Zhao Ruolin,Guo Yuanyuan,Ning Limin,Zhang Xu.Inhibitory effect of ophiopogonin-B on adhesion,invasion and migration of A549 cells in vitro.Chinese Pharmacological Bulletin)2015;31(5):660-4.
8 Law BY,Mo JF,Wong VK.Autophagic effects of Chaihu dried roots of Bupleurum Chinense DC or Bupleurum scorzoneraefolium WILD.Chin Med 2014;9:21.
9肖功胜,杨云,刘富岗,许小华.柴胡皂苷提取和含量测定方法研究.中成药(Xiao Gongsheng,Yang Yun,Liu Fugang,Xu Xiaohua.Chinese Traditional Patent Medicine)2009;31(4):595-8.
10 Weitz-schmidt G,Chreng S.Cell adhesion assays.Methods Mol Biol 2012;757:15-30.
11 Chigaev A,Sklar LA.Overview:assays for studying integrin dependent cell adhesion.Methods Mol Biol 2012;757:3-14.
12于蓓蓓,王亮,尹利顺,孙蓉.基于HPLC-DAD-MSn的柴胡皂苷A的体外生物转化研究.中草药(Yu Beibei,Wang Liang,Yin Lishun,Sun Rong.Research on biotransformation of saikosaponin A in vitro based on HPLC-DAD-MSn.Chinese Traditional and Herbal Drugs)2017;48(2):333-8.
13刘丹,王佳贺.柴胡皂苷抗肿瘤作用机制的研究进展.现代药物与临床(Liu Dan,Wang Jiahe.Research progress on antitumor mechanisms of saikosaponin.Drugs&Clinic)2018;33(1):203-8.
14 Wang BF,Dai ZJ,Wang XJ,Bai MH,Lin S,Ma HB,et al.Saikosaponin-d increases the radiosensitivity of smmc-7721hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle.BMC Complement Altern Med2013;13:263.
15 Yao M,Yang J,Cao L,Zhang L,Qu S,Gao H.Saikosaponin-d inhibits proliferation of DU145 human prostate cancer cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase.Mol Med Rep 2014;10(1):365-72.
16 Zhang Z,Zhang H,Chen S,Xu Y,Yao A,Liao Q,et al.Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway.Nutr Res 2017;38:27-33.
17 Tsai YJ,Chen IL,Horng LY,Wu RT.Induction of differentiation in rat C6 glioma cells with saikosaponins.Phytother Res 2002;16(2):117-21.
18 Wong VK,Li T,Law BY,Ma ED,Yip NC,Michelangeli F,et al.Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells.Cell Death Dis 2013;4:e720.
19黄幼异,黄伟,孙蓉.基于肝药酶P450动态变化的柴胡总皂苷小鼠肝毒性剂量-时间-毒性关系研究.中国实验方剂学杂志(Huang Youyi,Huang Wei,Sun Rong.Research on mechanism of hepatotoxicity caused by extracts of saikosaponins in mice.Chinese Journal of Experimental Traditional Medical Formulae)2012;18(22):299-303.
20孙蓉,黄伟.柴胡总皂苷醇洗脱精制品对大鼠慢性肝毒“量-毒”关系研究.中国中药杂志(Sun Rong,Huang Wei.“Dose toxicity”relationsh ip study on rats?chronic hepatoxicity of refined products of saikosaponin by alcohol elution.China Journal of Chinese Materia Medica)2010;35(17):2338-41.
21吕天,牟红元,冯江江,朱旭婷,刘玲,谢莲,等.中药柴胡总皂苷急性肝毒性的代谢组学研究.化学研究与应用(LüTian,Mou Hongyuan,Feng Jiangjiang,Zhun Xuting,Liu Ling,Xie Lian,et al.Metabolomics on acute liver toxicity of saikosaponin in Chinese medicine bupleuri radix.Chemical Research and Application)2013;25(6):789-92.
22盖晓东,历春,李倩,刘艳波,薛昊罡.柴胡皂苷在体外对人白血病细胞株K562/ADM多药耐药性的逆转作用.中国病理生理杂志(Gai Xiaodong,Li Chun,Li Qian,Liu Yanbo,Xue Haogang.Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro.Chinese Journal of Pathophysiology)2012;28(1):76-80.
23张洪峰,王乐,张凯,刘燕娟.柴胡总皂苷的循环超声提取及其对HepG2细胞凋亡的影响.中南药学(Zhang Hongfeng,Wang Le,Zhang Kai,Liu Yanjuan.Circulating ultrasonic extraction of total saikosapon ins from Bulperum Chinese DC and its effect on the apoptosis activity of human HepG2 cells.Central South Pharmacy)2016;14(12):1316-9.
24 Xia Y,Shen S,Verma IM.NF-κB,an active player in human cancers.Cancer Immunol Res 2014;2(9):823-30.
25 Yoong J,Michael M,Leong T.Targeted therapies for gastric cancer:current status.Drugs 2011;71(11):1367-84.
26 Greten FR,Eckmann L,Greten TF,Park JM,Li ZW,Egan LJ,et al.IKK beta links inflam-mation and tumorigenesis in a mouse model of colitis-associated cancer.Cell 2004;118(3):285-96.
27 Mattson MP,Meffert MK.Roles for NF-kappaB in nerve cell survival,plasticity,and disease.Cell Death Differ 2006;13(5):852-60.
28 Czabotar PE,Lessene G,Strasser A,Adams JM.Control of apoptosis by the BCL-2 protein family:implications for physiology and therapy.Nat Rev Mol Cell Biol 2014;15(1):49-63.
2涂铭,李林.胃癌防治知识对胃癌患者早期诊断的影响.实用癌症杂志(Tu Ming,Li Lin.Effect of knowledge about prevention of gastric cancer on early diagnosis of gastric cancer.The Practical Journal of Cancer)2014;29(8):972-4.
3石佳佳,曹丽丽,韩倩倩,朱艳琴.含皂苷类中药抗肿瘤作用的研究进展.光明中医(Shi Jiajia,Cao Lili,Han Qianqian,Zhu Yanqin.Research progress on anti-tumor effects of traditional Chinese medicine containing saponins.Guang Ming Zhong Yi)2016;31(23):3526-9.
4孙大鹏,鲁明明,王硕,富力.人参皂苷Rg3通过Ca2+/CaM信号系统抑制胃癌BGC-823细胞增殖及其可能的机制.中国肿瘤生物治疗杂志(Sun Dapeng,Lu Mingming,Wang Shuo,Fu Li.Ginsenoside Rg3 inhibits gastric cancer cell proliferation through Ca2+/CaM kinase downregulation and NF-κB inactivation.Chinese Journal of Cancer Biotherapy)2015;22(2):225-9.
5徐明明.柴胡皂甙D诱导人宫颈癌Hela细胞系凋亡的研究.医药论坛杂志(Xu Ming ming.Study of human cervical cancer Hela apoptosis induced by saikosaponin D.Journal of Medical Forum)2011;32(8):89-71.
6章英宏.桔梗皂苷D对人乳腺癌细胞体外杀伤效应及机制研究.浙江中西医结合杂志(Zhang Yinghong.Viability inhibition of human breast cancer cells treated with platycodin D in vitro and theunderlying mechanism.Zhejiang Journal of Integrated Traditional Chinese and Western Medicine)2015;25(6):547-9.
7陈美娟,赵若琳,郭园园,宁利敏,张旭.麦冬皂苷B对A549细胞株体外黏附、侵袭及迁移的抑制作用及机制研究.中国药理学通报(Chen Meijuan,Zhao Ruolin,Guo Yuanyuan,Ning Limin,Zhang Xu.Inhibitory effect of ophiopogonin-B on adhesion,invasion and migration of A549 cells in vitro.Chinese Pharmacological Bulletin)2015;31(5):660-4.
8 Law BY,Mo JF,Wong VK.Autophagic effects of Chaihu dried roots of Bupleurum Chinense DC or Bupleurum scorzoneraefolium WILD.Chin Med 2014;9:21.
9肖功胜,杨云,刘富岗,许小华.柴胡皂苷提取和含量测定方法研究.中成药(Xiao Gongsheng,Yang Yun,Liu Fugang,Xu Xiaohua.Chinese Traditional Patent Medicine)2009;31(4):595-8.
10 Weitz-schmidt G,Chreng S.Cell adhesion assays.Methods Mol Biol 2012;757:15-30.
11 Chigaev A,Sklar LA.Overview:assays for studying integrin dependent cell adhesion.Methods Mol Biol 2012;757:3-14.
12于蓓蓓,王亮,尹利顺,孙蓉.基于HPLC-DAD-MSn的柴胡皂苷A的体外生物转化研究.中草药(Yu Beibei,Wang Liang,Yin Lishun,Sun Rong.Research on biotransformation of saikosaponin A in vitro based on HPLC-DAD-MSn.Chinese Traditional and Herbal Drugs)2017;48(2):333-8.
13刘丹,王佳贺.柴胡皂苷抗肿瘤作用机制的研究进展.现代药物与临床(Liu Dan,Wang Jiahe.Research progress on antitumor mechanisms of saikosaponin.Drugs&Clinic)2018;33(1):203-8.
14 Wang BF,Dai ZJ,Wang XJ,Bai MH,Lin S,Ma HB,et al.Saikosaponin-d increases the radiosensitivity of smmc-7721hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle.BMC Complement Altern Med2013;13:263.
15 Yao M,Yang J,Cao L,Zhang L,Qu S,Gao H.Saikosaponin-d inhibits proliferation of DU145 human prostate cancer cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase.Mol Med Rep 2014;10(1):365-72.
16 Zhang Z,Zhang H,Chen S,Xu Y,Yao A,Liao Q,et al.Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway.Nutr Res 2017;38:27-33.
17 Tsai YJ,Chen IL,Horng LY,Wu RT.Induction of differentiation in rat C6 glioma cells with saikosaponins.Phytother Res 2002;16(2):117-21.
18 Wong VK,Li T,Law BY,Ma ED,Yip NC,Michelangeli F,et al.Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells.Cell Death Dis 2013;4:e720.
19黄幼异,黄伟,孙蓉.基于肝药酶P450动态变化的柴胡总皂苷小鼠肝毒性剂量-时间-毒性关系研究.中国实验方剂学杂志(Huang Youyi,Huang Wei,Sun Rong.Research on mechanism of hepatotoxicity caused by extracts of saikosaponins in mice.Chinese Journal of Experimental Traditional Medical Formulae)2012;18(22):299-303.
20孙蓉,黄伟.柴胡总皂苷醇洗脱精制品对大鼠慢性肝毒“量-毒”关系研究.中国中药杂志(Sun Rong,Huang Wei.“Dose toxicity”relationsh ip study on rats?chronic hepatoxicity of refined products of saikosaponin by alcohol elution.China Journal of Chinese Materia Medica)2010;35(17):2338-41.
21吕天,牟红元,冯江江,朱旭婷,刘玲,谢莲,等.中药柴胡总皂苷急性肝毒性的代谢组学研究.化学研究与应用(LüTian,Mou Hongyuan,Feng Jiangjiang,Zhun Xuting,Liu Ling,Xie Lian,et al.Metabolomics on acute liver toxicity of saikosaponin in Chinese medicine bupleuri radix.Chemical Research and Application)2013;25(6):789-92.
22盖晓东,历春,李倩,刘艳波,薛昊罡.柴胡皂苷在体外对人白血病细胞株K562/ADM多药耐药性的逆转作用.中国病理生理杂志(Gai Xiaodong,Li Chun,Li Qian,Liu Yanbo,Xue Haogang.Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro.Chinese Journal of Pathophysiology)2012;28(1):76-80.
23张洪峰,王乐,张凯,刘燕娟.柴胡总皂苷的循环超声提取及其对HepG2细胞凋亡的影响.中南药学(Zhang Hongfeng,Wang Le,Zhang Kai,Liu Yanjuan.Circulating ultrasonic extraction of total saikosapon ins from Bulperum Chinese DC and its effect on the apoptosis activity of human HepG2 cells.Central South Pharmacy)2016;14(12):1316-9.
24 Xia Y,Shen S,Verma IM.NF-κB,an active player in human cancers.Cancer Immunol Res 2014;2(9):823-30.
25 Yoong J,Michael M,Leong T.Targeted therapies for gastric cancer:current status.Drugs 2011;71(11):1367-84.
26 Greten FR,Eckmann L,Greten TF,Park JM,Li ZW,Egan LJ,et al.IKK beta links inflam-mation and tumorigenesis in a mouse model of colitis-associated cancer.Cell 2004;118(3):285-96.
27 Mattson MP,Meffert MK.Roles for NF-kappaB in nerve cell survival,plasticity,and disease.Cell Death Differ 2006;13(5):852-60.
28 Czabotar PE,Lessene G,Strasser A,Adams JM.Control of apoptosis by the BCL-2 protein family:implications for physiology and therapy.Nat Rev Mol Cell Biol 2014;15(1):49-63.