熊果酸对人胰腺癌细胞株SW1990的作用及其机制研究
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摘要
目的:研究熊果酸(ursolic acid,UA)对人胰腺癌细胞SW1990的生长抑制作用,并探讨其作用机制。
方法:用不同浓度的UA处理人胰腺癌细胞SW1990,采用MTT法测定UA对SW1990细胞的生长抑制作用、流式细胞术分析细胞周期改变和细胞凋亡;Hoechst 33258荧光染色、透射电镜等方法观察给药前后细胞的形态变化;RT-PCR检测UA处理SW1990细胞前后的基因表达水平变化;同时引入p53的抑制剂PFT-α(pifithrin alpha,PFT-α)做对照研究。应用免疫组织化学染色方法检测胰腺癌中P53和Bcl-2的表达情况。
结果:UA对SW1990细胞生长具有显著的抑制作用,且此作用呈明显的时间、剂量依赖趋势。流式细胞术分析显示,UA可以诱导人胰腺癌SW1990细胞凋亡,引起细胞发生S期阻滞;Hoechst 33258荧光染色、透射电镜观察证实UA可以诱导SW1990细胞发生凋亡。RT-PCR分析结果显示UA(40μmol/L)作用于胰腺癌SW1990细胞,可以调节细胞凋亡相关基因的表达,上调p53、cytochrome C、caspase-3的表达,下调Bcl-2表达。用p53的抑制剂PFT-α预处理SW1990细胞后再给予UA处理,细胞凋亡现象受到抑制,RT-PCR结果显示下调p53、cytochrome C、caspase-3的表达,上调Bcl-2表达。免疫组织化学染色检测结果显示胰腺癌中存在着高频率的p53突变。
结论:UA能明显抑制人胰腺癌SW1990细胞的生长,并诱导其凋亡,这种作用可能与p53、cytochrome C、caspase-3、Bcl-2基因的表达变化相关。
Objective: To study the effects and mechanisms of ursolic acid on human pancr- eastic carcinoma cell line SW1990.
Metheods: After UA treatment, the growth inhibition of SW1990 cells were assessed by MTT assay. Cells were evaluated with flow cytometric analysis, Hoechst 33258 staining and transmission electron microscope after it was induced by UA;It’s mechanisms was determined by RT-PCR analysis. The inhibitor of p53-pifithrin alpha (PFT-a) was applied to investigate the effect of p53 in the study. The p53 and Bcl-2 expression in SW1990 was examined by immunocytochemistry method.
Results: The proliferation of SW1990 cells was significantly inhibited in a dose-and time-dependent manner after UA treatment. Cell apoptosis ratio and cell cycle arrest was examined by FCM; UA-induced apoptosis was confirmed by Hoechst 33258 staining and transmission electron microscope. After cells were treated by UA(40μmol/L), the expressions of p53, cytochrome C, caspase-3 increased whereas bcl-2 decreased. After the pretreatment of PFT-a on the SW1990 cell , the ratio of cell apoptosis became weakly.The immunocytochemistrical examination showed the expression of p53 mutated.
Conclusions: It is significant that the growth of human pancreatic carcinoma cell line SW1990 could be inhibitated when treated by UA. UA can induce cell apoptosis. These effects might be related with the expressions of p53, Bcl-2, cytochrome C, caspase-3.
方法:用不同浓度的UA处理人胰腺癌细胞SW1990,采用MTT法测定UA对SW1990细胞的生长抑制作用、流式细胞术分析细胞周期改变和细胞凋亡;Hoechst 33258荧光染色、透射电镜等方法观察给药前后细胞的形态变化;RT-PCR检测UA处理SW1990细胞前后的基因表达水平变化;同时引入p53的抑制剂PFT-α(pifithrin alpha,PFT-α)做对照研究。应用免疫组织化学染色方法检测胰腺癌中P53和Bcl-2的表达情况。
结果:UA对SW1990细胞生长具有显著的抑制作用,且此作用呈明显的时间、剂量依赖趋势。流式细胞术分析显示,UA可以诱导人胰腺癌SW1990细胞凋亡,引起细胞发生S期阻滞;Hoechst 33258荧光染色、透射电镜观察证实UA可以诱导SW1990细胞发生凋亡。RT-PCR分析结果显示UA(40μmol/L)作用于胰腺癌SW1990细胞,可以调节细胞凋亡相关基因的表达,上调p53、cytochrome C、caspase-3的表达,下调Bcl-2表达。用p53的抑制剂PFT-α预处理SW1990细胞后再给予UA处理,细胞凋亡现象受到抑制,RT-PCR结果显示下调p53、cytochrome C、caspase-3的表达,上调Bcl-2表达。免疫组织化学染色检测结果显示胰腺癌中存在着高频率的p53突变。
结论:UA能明显抑制人胰腺癌SW1990细胞的生长,并诱导其凋亡,这种作用可能与p53、cytochrome C、caspase-3、Bcl-2基因的表达变化相关。
Objective: To study the effects and mechanisms of ursolic acid on human pancr- eastic carcinoma cell line SW1990.
Metheods: After UA treatment, the growth inhibition of SW1990 cells were assessed by MTT assay. Cells were evaluated with flow cytometric analysis, Hoechst 33258 staining and transmission electron microscope after it was induced by UA;It’s mechanisms was determined by RT-PCR analysis. The inhibitor of p53-pifithrin alpha (PFT-a) was applied to investigate the effect of p53 in the study. The p53 and Bcl-2 expression in SW1990 was examined by immunocytochemistry method.
Results: The proliferation of SW1990 cells was significantly inhibited in a dose-and time-dependent manner after UA treatment. Cell apoptosis ratio and cell cycle arrest was examined by FCM; UA-induced apoptosis was confirmed by Hoechst 33258 staining and transmission electron microscope. After cells were treated by UA(40μmol/L), the expressions of p53, cytochrome C, caspase-3 increased whereas bcl-2 decreased. After the pretreatment of PFT-a on the SW1990 cell , the ratio of cell apoptosis became weakly.The immunocytochemistrical examination showed the expression of p53 mutated.
Conclusions: It is significant that the growth of human pancreatic carcinoma cell line SW1990 could be inhibitated when treated by UA. UA can induce cell apoptosis. These effects might be related with the expressions of p53, Bcl-2, cytochrome C, caspase-3.
引文
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4 Jie Liu .Pharmacology of oleanolic acid and ursolic acid Journal of Ethnophar- macology 1995,49:57-68
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7 Ollosy F,Idei M,Csorba G,et al. Activation of caspase-3 protease during the process of ursolic acid and its derivative-induced apoptosis. Anticancer Res. 2001,21(5) :3485.
8 Harmand PO,Duval R,Liagre B,et al. Ursolic acid induces apoptosis through caspase-3 activation and cell cycle arrest in HaCat cells. Int J Oncol,2003, 23 (1):105.
9 Lanthier F , Taillet L , Trouillas P , et al. Ursolic acid triggers calcium-dependent apoptosis in human Daudi cells. Anticancer Drugs. 2000,11(9):737.
10 Shishodia S,Majumdar S,Banerjee S,et al. Ursolic acid inhibits nuclear factor- kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation :correlation with down-regulation of cyclooxygenase 2,matrix metalloproteinase 9,and cyclin D1. Cancer Res 2003 ,63(15):4375
11 Andersson D,Liu JJ,Nilsson A,et al. Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase. Anticancer Res. 2003,23(4):3317.
12 Hollosy F,Meszaros G,Bokonyi G,et al. Cytostatic,cytotoxic and protein tyrosine kinase inhibitory activity of ursolic acid in A431 human tumor cells. Anticancer Res. 2000,20(6B):4563.
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. Life Sci. 2004, 75(19):2303-2316.
14 Cardenas C,Quesada AR,Medina MA. Effects of ursolic acid on different steps of the angiogenic process. Biochem Biophys Res Commun. 2004,320(2):402-408.
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2 Ho, Kuen-Yao MD; Kuo, Wen-Rei MD; Chai, Chee-Yin MD, PhD et al, A Prospective Study of p53 Expression and Its Correlation With Clinical Response of Radiotherapy in Nasopharyngeal Carcinoma. Laryngoscope. January 2001,111(1): 131-136.
3 Ovesna Z,Vachalkova A,Horvathova K,et al Pentacyclic triterpenoic acids: new chemoprotective compounds,Neoplasma (Bratislava). 2004,51(5):327-333
4 Jie Liu .Pharmacology of oleanolic acid and ursolic acid Journal of Ethnophar- macology 1995,49:57-68
5 Baglin I,. Mitaine-Offer AC, Nour M, et al A Review of Natural and Modified Betulinic, Ursolic and Echinocystic Acid Derivatives as Potential Antitumor and Anti-HIV Agents. Med Chem, 2003, 3, 525-539
6李开泉,陈武,熊筱娟等.乌索酸的化学、药理及临床应用进展.中成药. 2002,24(9):709-711
7 Ollosy F,Idei M,Csorba G,et al. Activation of caspase-3 protease during the process of ursolic acid and its derivative-induced apoptosis. Anticancer Res. 2001,21(5) :3485.
8 Harmand PO,Duval R,Liagre B,et al. Ursolic acid induces apoptosis through caspase-3 activation and cell cycle arrest in HaCat cells. Int J Oncol,2003, 23 (1):105.
9 Lanthier F , Taillet L , Trouillas P , et al. Ursolic acid triggers calcium-dependent apoptosis in human Daudi cells. Anticancer Drugs. 2000,11(9):737.
10 Shishodia S,Majumdar S,Banerjee S,et al. Ursolic acid inhibits nuclear factor- kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation :correlation with down-regulation of cyclooxygenase 2,matrix metalloproteinase 9,and cyclin D1. Cancer Res 2003 ,63(15):4375
11 Andersson D,Liu JJ,Nilsson A,et al. Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase. Anticancer Res. 2003,23(4):3317.
12 Hollosy F,Meszaros G,Bokonyi G,et al. Cytostatic,cytotoxic and protein tyrosine kinase inhibitory activity of ursolic acid in A431 human tumor cells. Anticancer Res. 2000,20(6B):4563.
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. Life Sci. 2004, 75(19):2303-2316.
14 Cardenas C,Quesada AR,Medina MA. Effects of ursolic acid on different steps of the angiogenic process. Biochem Biophys Res Commun. 2004,320(2):402-408.
15. Aparecida Resende F, de Andrade Barcala CA, da Silva Faria MC.Antimuta- genicity of ursolic acid and oleanolic acid against doxorubicin-induced clastogenesis in Balb/c mice. Life Sci. 2006,79(13):1268-1273.
16.Cha HJ,Bae SK,Lee HY, et al. Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells. Cancer Res. 1996,56(10):2281-2284
17.向敏,顾振纶,梁中琴等.熊果酸对小鼠B16黑色素瘤细胞的分化诱导作用,中国药理通讯2003,20(1);67-69
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