华蟾素注射液对人肝癌HepG-2细胞增殖、凋亡及周期的影响
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摘要
目的：研究华蟾素注射液对人肝癌HepG-2细胞增殖、凋亡及细胞周期的影响,初步探讨可能的机制。
方法：以不同浓度的华蟾素注射液作用于人肝癌HepG-2细胞,采用噻唑蓝还原法(MTT)观察华蟾素注射液对HepG-2细胞增殖的影响；采用倒置显微镜观察细胞形态学的变化；采用原位缺口末端标记法(TUNEL)及AnnexinV/PI双染色法检测细胞凋亡情况：采用流式细胞术(FCM)检测HepG-2细胞周期分布；采用免疫组化法检测凋亡相关基因Bcl-2/Bax表达的变化；采用逆转录-多聚酶链反应技术(RT-PCR)检测凋亡周期蛋白CyclinA.CDK2 mRNA及TOPOⅠ.ⅡmRNA水平表达的影响；采用比色定量法检测HepG-2细胞CyclinA和CDK2活性的变化。
结果：
1、≥0.006μg/ml的华蟾素注射液对HepG-2细胞增殖具有抑制作用,其抑制效应呈时间和浓度依赖性。
2、华蟾素注射液作用HepG-2细胞后可见凋亡形态学变化,部分细胞体积变小,染色质浓缩边集,呈时间和浓度依赖性(P<0.05)。
3、华蟾素注射液作用HepG-2细胞后,细胞被阻滞在S期,呈时间依赖性(P<0.05)。
4、华蟾素注射液抑制HepG-2细胞凋亡相关基因Bcl-2表达,促进Bax表达,呈浓度依赖性。
5、华蟾素注射液抑制HepG-2细胞CDK2、CyclinA、TOPOⅠ、TOPOⅡmRNA水平表达,呈浓度依赖性。
6.华蟾素注射液可使HepG-2细胞CyclinA、CDK2活性降低,呈浓度依赖性(P<0.05)。
结论：华蟾素注射液能够抑制人肝癌HepG-2细胞增殖,诱导其凋亡,将细胞阻滞于S期,下调CDK2、CyclinA、TOPOⅠ、TOPOⅡmRNA,抑制CDK2/CyclinA活性,影响Bcl-2、Bax表达,可能是华蟾素注射液实现药效机制之一。
Objective:To investigate the effect of cinobufacin injection on the proliferation,apoptosis and cell cycle of human hepatoma HepG-2 cells, and discuss the possible mechanism.
Method:After the intervention of Cinobufacini injection different concentrations of, cell proliferation of HepG-2 cells was assessed by MTT assay,cell morphologic was observed by the inverted microscopy, Anne-xinV/PI stain was used to detect the apoptosis and necrosis of the tumor cells,Flow cytometry (FCM) was emplayed to detect tumor cell cycle distribution,Immunohistochemistry assay was used to analyse expression of apoptosis-related genes Bcl-2/Bax.RT-PCR was used to analyse expression of CyclinA,CDK2 mRNA levels in HepG-2 cells,and the expression of TOPOⅠmRNA and TOPOⅡmRNA were also examined by RT-PCR. Quantitative colorimetric assay was used to analyse cyclinA/CDK2 activity in HepG-2 cells.
Result:
1.Cinobufacini injection with the concentration of 0.006μg/ml or more can significantly inhibited HepG-2 cells proliferation in doses and time dependent ways.
2.After Cinobufacini injection intervention,HepG-2 cells showed typical apoptotic morphological changes:the partial cell become smaller volume than before,and the chromatin looseness in doses and time-dependent ways (P<0.05)
3. FCM analysis showed cinobufacin injection induced cell cycle arrest at S phase in time-dependent ways (P< 0.05)
4.Cinobufacin injection can inhibited the expression of apoptosis-related genes Bcl-2 and promote the expression of genes Bax in doses-dependent way.
5.Cinobufac ininjection down-regulated CDK2,CyclinA,TOPOⅠand TOPOⅡexpression at mRNA levels in doses-dependent ways.
6.Cinobufacin injection deceased cyclinA/CDK2 activity in HepG-2 cells in doses-dependent way (P<0.05)
Conclusion:Cinobufacini injection can inhibit human hepotocarcinoma HepG-2 cell growth, induce tumor cell apoptosis, and induce cell cycle arrest at S phase, the mechanism might be partly related to the down-regulation of CDK2, CyclinA, TOPOⅠand TOPOⅡexpression at mRNA level, inhibition of cyclinA/CDK2 activity and influence the expression of genes Bcl-2 and Bax.
方法：以不同浓度的华蟾素注射液作用于人肝癌HepG-2细胞,采用噻唑蓝还原法(MTT)观察华蟾素注射液对HepG-2细胞增殖的影响；采用倒置显微镜观察细胞形态学的变化；采用原位缺口末端标记法(TUNEL)及AnnexinV/PI双染色法检测细胞凋亡情况：采用流式细胞术(FCM)检测HepG-2细胞周期分布；采用免疫组化法检测凋亡相关基因Bcl-2/Bax表达的变化；采用逆转录-多聚酶链反应技术(RT-PCR)检测凋亡周期蛋白CyclinA.CDK2 mRNA及TOPOⅠ.ⅡmRNA水平表达的影响；采用比色定量法检测HepG-2细胞CyclinA和CDK2活性的变化。
结果：
1、≥0.006μg/ml的华蟾素注射液对HepG-2细胞增殖具有抑制作用,其抑制效应呈时间和浓度依赖性。
2、华蟾素注射液作用HepG-2细胞后可见凋亡形态学变化,部分细胞体积变小,染色质浓缩边集,呈时间和浓度依赖性(P<0.05)。
3、华蟾素注射液作用HepG-2细胞后,细胞被阻滞在S期,呈时间依赖性(P<0.05)。
4、华蟾素注射液抑制HepG-2细胞凋亡相关基因Bcl-2表达,促进Bax表达,呈浓度依赖性。
5、华蟾素注射液抑制HepG-2细胞CDK2、CyclinA、TOPOⅠ、TOPOⅡmRNA水平表达,呈浓度依赖性。
6.华蟾素注射液可使HepG-2细胞CyclinA、CDK2活性降低,呈浓度依赖性(P<0.05)。
结论：华蟾素注射液能够抑制人肝癌HepG-2细胞增殖,诱导其凋亡,将细胞阻滞于S期,下调CDK2、CyclinA、TOPOⅠ、TOPOⅡmRNA,抑制CDK2/CyclinA活性,影响Bcl-2、Bax表达,可能是华蟾素注射液实现药效机制之一。
Objective:To investigate the effect of cinobufacin injection on the proliferation,apoptosis and cell cycle of human hepatoma HepG-2 cells, and discuss the possible mechanism.
Method:After the intervention of Cinobufacini injection different concentrations of, cell proliferation of HepG-2 cells was assessed by MTT assay,cell morphologic was observed by the inverted microscopy, Anne-xinV/PI stain was used to detect the apoptosis and necrosis of the tumor cells,Flow cytometry (FCM) was emplayed to detect tumor cell cycle distribution,Immunohistochemistry assay was used to analyse expression of apoptosis-related genes Bcl-2/Bax.RT-PCR was used to analyse expression of CyclinA,CDK2 mRNA levels in HepG-2 cells,and the expression of TOPOⅠmRNA and TOPOⅡmRNA were also examined by RT-PCR. Quantitative colorimetric assay was used to analyse cyclinA/CDK2 activity in HepG-2 cells.
Result:
1.Cinobufacini injection with the concentration of 0.006μg/ml or more can significantly inhibited HepG-2 cells proliferation in doses and time dependent ways.
2.After Cinobufacini injection intervention,HepG-2 cells showed typical apoptotic morphological changes:the partial cell become smaller volume than before,and the chromatin looseness in doses and time-dependent ways (P<0.05)
3. FCM analysis showed cinobufacin injection induced cell cycle arrest at S phase in time-dependent ways (P< 0.05)
4.Cinobufacin injection can inhibited the expression of apoptosis-related genes Bcl-2 and promote the expression of genes Bax in doses-dependent way.
5.Cinobufac ininjection down-regulated CDK2,CyclinA,TOPOⅠand TOPOⅡexpression at mRNA levels in doses-dependent ways.
6.Cinobufacin injection deceased cyclinA/CDK2 activity in HepG-2 cells in doses-dependent way (P<0.05)
Conclusion:Cinobufacini injection can inhibit human hepotocarcinoma HepG-2 cell growth, induce tumor cell apoptosis, and induce cell cycle arrest at S phase, the mechanism might be partly related to the down-regulation of CDK2, CyclinA, TOPOⅠand TOPOⅡexpression at mRNA level, inhibition of cyclinA/CDK2 activity and influence the expression of genes Bcl-2 and Bax.
引文
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3 Masahiro Kurosawa,Satoshi Nishimur, Yoshihiro Tani,el al.ERK signaling mediates hhe induction of inflammatory cytokines by bufalin in human mono-cytic cellsAm[J].J. Physiol.Cell Physiol 2000,278:500-508.
4 Numazawa S.Inous N,Nakura H,et al.A cardiotonic steroid bufalin-iuduced differentiation of THP-1 cells.Involvement of Na+/K+-ATPase inhibition in the early changes in proto-oncogene expression[J]Biochem Pharmacol,1996,52(2): 321-329.
5 Kawazoe N,Aiuchi T,Masuda Y,et al.Induction of apoptosis by bufalin in hnman tumor cells is associated with a change of intracellular concentration of Na+inons[J].JBiochem(Tokyo),1999,126(2):278-286.
6 顾伟,韩克起,苏永华,等,蟾毒灵对肝癌BEL-7402细胞增殖的影响[J].中西医结合肝病杂志,2006,16(1)16-20.
7 吴万垠,柴小姝,刘伟胜,中药联合长春瑞滨对小鼠Lewis肺癌细胞周期及细胞增殖抗原的影响[J]中国癌症杂志,2004,14(4)：363-365.
8 陈娜,陈兴平。华蟾素对小鼠脾淋巴细胞分泌白细胞介素-2水平的影响[J]医药导报,2007,26(1)14-16。
9 赵兴梅,陈晓明,杨祖怡,等,华蟾素注射液对人IL-2水平及LAK细胞活性的影响[J]中药药理与临床,1999,15(6)：33-35.
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28(2):160-168
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3 Masahiro Kurosawa,Satoshi Nishimur, Yoshihiro Tani,el al.ERK signaling mediates hhe induction of inflammatory cytokines by bufalin in human mono-cytic cellsAm[J].J. Physiol.Cell Physiol 2000,278:500-508.
4 Numazawa S.Inous N,Nakura H,et al.A cardiotonic steroid bufalin-iuduced differentiation of THP-1 cells.Involvement of Na+/K+-ATPase inhibition in the early changes in proto-oncogene expression[J]Biochem Pharmacol,1996,52(2): 321-329.
5 Kawazoe N,Aiuchi T,Masuda Y,et al.Induction of apoptosis by bufalin in hnman tumor cells is associated with a change of intracellular concentration of Na+inons[J].JBiochem(Tokyo),1999,126(2):278-286.
6 顾伟,韩克起,苏永华,等,蟾毒灵对肝癌BEL-7402细胞增殖的影响[J].中西医结合肝病杂志,2006,16(1)16-20.
7 吴万垠,柴小姝,刘伟胜,中药联合长春瑞滨对小鼠Lewis肺癌细胞周期及细胞增殖抗原的影响[J]中国癌症杂志,2004,14(4)：363-365.
8 陈娜,陈兴平。华蟾素对小鼠脾淋巴细胞分泌白细胞介素-2水平的影响[J]医药导报,2007,26(1)14-16。
9 赵兴梅,陈晓明,杨祖怡,等,华蟾素注射液对人IL-2水平及LAK细胞活性的影响[J]中药药理与临床,1999,15(6)：33-35.
10 Enomoto A,Rbo MC,Komiyama K,et,al.Inhibitoey effects of bufadieno-lides on interleukin-6 in MH-60 cells[J].J Nat Prod,2004,67 (12):2070-2072.
11王四旺等,安替可胶囊抗肿瘤作用的机理.第四军医大学学报,1997,18(4)：368-372
12 Eeffrth T, DaveyM, Olbrieh A, et al. Activity of drugs from traditional Chinese medicine toward sensitive and MDR I-or MRPI-overexp ressing multidurg-resistant human CCRF-CEM leukemia cells[J]. B lood CellsMol D is,2002,
28(2):160-168
13王玲,刘世坤,周于禄,等.华蟾素对人乳腺癌细胞阿霉素多药耐药性的逆转作用[J].中国药理学通报,2007,23(5)：677-680.
14刘浩,林洪生,花宝金,等。华蟾素调控VEGF/VEGFR-2信号传导抑制肿瘤血管生成的研究。中华中医药学刊,2008,11(26)2489-2491.
15解景田.脂蟾毒配基对人和犬心肌细胞膜反应性与后电位的影响.中国药理学报,1985,6(4)：254-258。
16李新民,金光,张壁钰,等,蟾250治疗恶性肿瘤114例临床观察.陕西中医,1985,(4)：152-153.
17 Pastor N,Corter F.bufalin influenoes the repair of X-rayinduced DNA breaks in Chinese hamster cells[J].DNA Repair,2003,2(12):1353-1360.
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