熊果酸诱导鼻咽癌细胞株CNE-2Z凋亡的实验研究
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摘要
目的:探讨熊果酸诱导人鼻咽癌细胞CNE-2Z凋亡的作用机制。
方法:用不同浓度的熊果酸处理CNE-2Z细胞,采用:①四甲基偶氮唑蓝比色法(MTT)检测熊果酸对CNE-2Z细胞的增殖抑制效应;②流式细胞术(FCM)分析细胞周期和凋亡率;③应用免疫组织化学SP法检测凋亡相关基因bcl-2,bax和cox-2表达的变化;④Western blot检测CNE-2Z细胞内caspase 3的表达。
结果:1、MTT:熊果酸处理24h ,48h和72h时的IC50分别为30.56μmol/L,31.94μmol/L和33.06μmol/L。熊果酸呈浓度依赖性的抑制CNE-2Z细胞增殖,差异有显著性(P<0.05)。40-50μmol/L熊果酸分别作用24h、48h、72h后, CNE-2Z细胞几乎停止增殖。2、FCM:经熊果酸作用24h后,随着药物浓度的增加,细胞凋亡率逐渐升高,S期细胞的比例逐渐下降, G0/G1期细胞的比例逐渐增加,主要呈现G0/G1期阻滞,并存在浓度依赖性。3、免疫组化SP法:bcl-2,bax和cox-2的表达可见于胞膜和胞浆,在24h时随着药物浓度的升高,bax的表达逐渐增强;bcl-2和cox-2与对照组比较表达则明显降低。4、Western blot:用10～50μmol/L UA处理细胞24 h后,随着UA浓度增加,活化的caspase 3蛋白条带逐渐加深。用Quantity One分析软件测定条带灰度值,求出每组caspase 3与β-actin条带的灰度比值,实验组与对照组的灰度比值差异有统计学意义(t=2.76, t0.05=2.132,p<0.05)。
结论:熊果酸在体外对CNE-2Z细胞有诱导凋亡的作用,其作用机制可能是通过促进caspase 3的活化和bax的表达上调、bcl-2、cox-2的表达下调来实现。
Objective: To investigate the mechanism of ursolic acid inducing the apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z.
Methods: CNE-2Z were treated by ursolic acid with different concentration. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analysed by flow cytometry(FCM). The expressions of bax, bcl-2 and cox-2 were assessed by SP method of immunocytochemistry, while the expression of caspase 3 by Western blot.
Result: 1、MTT:The 3 IC50 of CNE-2Z cells that were treated by ursolic acid for 24h、48h and 72h are 30.56μmol/L、31.94μmol/L and 33.06μmol/L.Ursolic acid inhibited the proliferation of CNE-2Z cells in dosage dependent manner(P<0.05). CNE-2Z cells that were treated by ursolic acid in 40-50μmol/L dosage almost ceased proliferating. 2、FCM: After treated by ursolic acid for 24 hours, the apoptosis rate of the CNE-2Z cells hightened with the increasing of the medicine density. It showed that the cell population reduced in S phase and cell cycle was arrested in G0/G1 phase. 3、Immunocytochemistry:The expression of bcl-2、bax and cox-2 can be shown in cell membrane and endochylema. With the increasing of the medicine density, the expression of bax was up-regulated in the apoptosis, while the expression of bcl-2 and cox-2 down-regulated. 4、Western blot:After treated by ursolic acid for 24 hours, With the increasing of the medicine density, the expression of caspase 3 was strengthened.
Conclusion: Ursolic acid can induce apoptosis of CNE-2Z in vitro. Its molecular mechanism probably is through promoting activate-ion of caspase-3, up-regulating the expression of bax, and down-re-gulating the expressions of bcl-2 and cox-2.
方法:用不同浓度的熊果酸处理CNE-2Z细胞,采用:①四甲基偶氮唑蓝比色法(MTT)检测熊果酸对CNE-2Z细胞的增殖抑制效应;②流式细胞术(FCM)分析细胞周期和凋亡率;③应用免疫组织化学SP法检测凋亡相关基因bcl-2,bax和cox-2表达的变化;④Western blot检测CNE-2Z细胞内caspase 3的表达。
结果:1、MTT:熊果酸处理24h ,48h和72h时的IC50分别为30.56μmol/L,31.94μmol/L和33.06μmol/L。熊果酸呈浓度依赖性的抑制CNE-2Z细胞增殖,差异有显著性(P<0.05)。40-50μmol/L熊果酸分别作用24h、48h、72h后, CNE-2Z细胞几乎停止增殖。2、FCM:经熊果酸作用24h后,随着药物浓度的增加,细胞凋亡率逐渐升高,S期细胞的比例逐渐下降, G0/G1期细胞的比例逐渐增加,主要呈现G0/G1期阻滞,并存在浓度依赖性。3、免疫组化SP法:bcl-2,bax和cox-2的表达可见于胞膜和胞浆,在24h时随着药物浓度的升高,bax的表达逐渐增强;bcl-2和cox-2与对照组比较表达则明显降低。4、Western blot:用10～50μmol/L UA处理细胞24 h后,随着UA浓度增加,活化的caspase 3蛋白条带逐渐加深。用Quantity One分析软件测定条带灰度值,求出每组caspase 3与β-actin条带的灰度比值,实验组与对照组的灰度比值差异有统计学意义(t=2.76, t0.05=2.132,p<0.05)。
结论:熊果酸在体外对CNE-2Z细胞有诱导凋亡的作用,其作用机制可能是通过促进caspase 3的活化和bax的表达上调、bcl-2、cox-2的表达下调来实现。
Objective: To investigate the mechanism of ursolic acid inducing the apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z.
Methods: CNE-2Z were treated by ursolic acid with different concentration. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analysed by flow cytometry(FCM). The expressions of bax, bcl-2 and cox-2 were assessed by SP method of immunocytochemistry, while the expression of caspase 3 by Western blot.
Result: 1、MTT:The 3 IC50 of CNE-2Z cells that were treated by ursolic acid for 24h、48h and 72h are 30.56μmol/L、31.94μmol/L and 33.06μmol/L.Ursolic acid inhibited the proliferation of CNE-2Z cells in dosage dependent manner(P<0.05). CNE-2Z cells that were treated by ursolic acid in 40-50μmol/L dosage almost ceased proliferating. 2、FCM: After treated by ursolic acid for 24 hours, the apoptosis rate of the CNE-2Z cells hightened with the increasing of the medicine density. It showed that the cell population reduced in S phase and cell cycle was arrested in G0/G1 phase. 3、Immunocytochemistry:The expression of bcl-2、bax and cox-2 can be shown in cell membrane and endochylema. With the increasing of the medicine density, the expression of bax was up-regulated in the apoptosis, while the expression of bcl-2 and cox-2 down-regulated. 4、Western blot:After treated by ursolic acid for 24 hours, With the increasing of the medicine density, the expression of caspase 3 was strengthened.
Conclusion: Ursolic acid can induce apoptosis of CNE-2Z in vitro. Its molecular mechanism probably is through promoting activate-ion of caspase-3, up-regulating the expression of bax, and down-re-gulating the expressions of bcl-2 and cox-2.
引文
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[5] Liu XH,Yao S,Kirschenbaum,et al. NS398,a selective Cyclooxygenase 2 inhibitor,induces apoptosis and downregulates bcl-2 expression in LNCap cells[J] . Cancer Res, 1998;58(19):4245-4249.
[6] Niikawa M, Hayashi H, Sato T, et al. Isolation of substances from glossy privet (Ligustrum lucidum Ait.) inhibiting the mutagenicity of benzo[a]pyrene in bacteria[J]. Mutat Res., 1993; 319(1):1-9.
[7] Guevara AP, Amor E, Russell Q Antimutagens from Plumeria acuminata Ait. [J]. Mutat Res., 1996, 361 (23):67-72.
[8] Huang MT, Ho CT, Wang ZY, et al. Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. [J]. Cancer Res., 1994;54(3):701-8.
[9] Kim KA, Lee JS, Park HJ.et al. Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes.[J].Life Sci.,2004 ;74(22):2769-79.
[10] Li J, Guo WJ, Yang QY Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15 .[J]. World J Gastroenterol, 2002; 8 (3):493-495.
[11] Cha HJ, Bae SK, Lee HY, et al. Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase. [J].Cancer Res., 1996,56 (10):2281-2284.
[12] Baek JH, Lee YS, Kang CM, et al. Intercellular Ca2+ release mediates ursolic acid-induced apoptosis in human leukemic HL60 cells. [J].Int J Cancer, 1997;73(5):725 -728.
[13] Hsu YL, Kuo PL, Lin CC. Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells [J]. Life Sci., 2004;75(19):2303-16.
[14] Cardenas C, Quesada AR, Medina MA. Effects of ursolic acid on different steps of the angiogenic process. [J].Biochem Biophys Res Commun, 2004; 320(2):402-8.
[15] Femander-Alnermi T,Litwack G,Alnemrt E S,et a1.Cpp32,a novel human apoptotic protein with homology to eaenorhabaitis elegalls cell death protein ced-e and mammalianICE[J].J boil Chem,l994,269(49):30761-30764.
[16] Eamshaw W C,Martins L M,Kanfmann S H.Mammalian Caspase:structure,activation,substrates,and functions during apoptesis[M]s.Amnu BevBiochem. 1999:383-424.
[17] Srinivasula SM,Femandes-AlnemriT,Zangrilli J,et a1.The ced-3/inter leukin lβ converting enzyme—like homolog Mch6 and the lamin-cleaving enzyme Mch2 are substrates for the apoptotic mediatorCPP32[J].J Biol Chem,1996,271(43):27099-27106.
[18] Zou H,Henzel W J,Liu X,et a1.Apaf-l,a human protein homologous to C.elegans CED-4.participates in cytochromec dependent activation of Caspase-3 [J].Cell,1997,90(3):405-4l3.
[19] Hu Y,BenedletMA,wu D,eta1.Bcl-xL interacts with Apaf-l and inhibits[J].Proc Natl Acad Sci,1998,95:4386-4391.
[20] Pan G,Otourke K,Dixit V M.Caspase-9,Bcl-X1,and Apaf-l form aternary complex[J].JBiol Chem,1998,273:5841-5845.
[21] Kluck RM,Bossy W E,GreenDR,et a1.The release of cytochrome C from mitochondna:a primary site for Bcl-2 regulation of apeptosis[J].Science,l997,275:1132-1136.
[22] Yang J,Iju X,Bhala K,et a1.Prevention of apeptosis by Bcl-2:release of cytochrome C from mitochondria blocked[J].Science,l997,275:1129-1132.
[23] Adams J M,Cory S.The Bcl-2 protein family:arbiters of cell survival [J].Science,l998,28l:l322-l326.
[24] Xiang J,Chao D T,Korsmeyer S J.BAX-induced cell death may not require interleukin l B-converting enzyme-like proteases[J].Proc Nail Acad Sci,l996,93:l4559-l4563.
[25] Harmand PO, Duval R, Delage C, et al. Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells.Int J Cancer. 2005 Mar 10; 114(1):1-11.
[26] Achiwa Y, Hasegawa K, Komiya T, et al. Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer SNG-II cells. OncolRep. 2005 Jan;13(1):51-7.
[27] 杨连君.Bcl-2, Bax 与肿瘤细胞凋亡.中国肿瘤生物治疗杂志,2003, 10 (3): 232-234.
[28] Chulada PC,Thotroson MB,Mahler JF,et a1.Genetic disruption of Ptgs-1,as well as Ptgs-2,reduces intestinal tonaorigenesis in Minmice[J].Cancer Res,2000,60(17).4705-4708.
[29] Schrey MP,Patel KV.Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibmblasts . Regulation by inflanrnatory mediators[J].Br J Cancer,1995,72(6):1412-1419.
[30] Dannenberg AJ,Ahorki NK,Boile JO,et a1.Cydooxygenase 2:a pharmacological target for the prevention of cancer[J].Lancet Oncol,2001,2(9):544-550.
[31] Suns JJ,LeungWK,GoMY,eta1.Cyclooxygenase-2 expression in Helieobacter pylori-associated premalignant and malignant gastric lesions.Am J Pathol,2000,157(3):729-735.
[32] Zinmaenmnn KC,Sarbia M,Weber AA,et a1.Cyclooxygenase-2 expression in human esophageal carcinorm[J].Cancer Res,1999,59(1):198-204.
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