【作者】 孟爱国；

【导师】 姜玲玲；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 土槿乙酸是从松科植物金钱松根皮中分离的新型二萜酸化合物,具有抗真菌、抗生育、抗血管生成和抗肿瘤等作用。近年来研究证实土槿乙酸对肝癌、乳腺癌、宫颈癌、前列腺癌等多种肿瘤细胞具有明显的抗肿瘤活性,且多与p53蛋白表达相关,但确切的作用机制目前仍未清楚。本文拟以表达野生型p53和突变型p53的肿瘤细胞为研究对象,探讨土槿乙酸的抗肿瘤作用机制。因消化道肿瘤(胃癌,肝癌和结肠癌)属于易发生p53突变的恶性肿瘤,而黑色素瘤常常表达野生型p53,很少发生p53的突变,故选择这两类具有代表性和普遍性的人肿瘤细胞系作为研究的对象。我们首先检测了土槿乙酸对不同人肿瘤细胞系的抑瘤作用,然后选择敏感性高且具有代表性的人胃癌MGC803(突变型p53)、AGS(野生型p53)和黑色素瘤SK–28(野生型p53)细胞株作为研究对象,系统研究了土槿乙酸抗肿瘤作用的机制,为土槿乙酸可能成为新型抗肿瘤药物提供实验数据和理论依据。第一部分土槿乙酸对不同人肿瘤细胞系的抑瘤作用目的:检测土槿乙酸对不同人肿瘤细胞系的抑瘤作用,筛选出对土槿乙酸敏感且具有代表性的细胞系,作为土槿乙酸抗肿瘤作用机制研究的细胞模型。方法:采用四唑氮蓝还原法(MTT)检测土槿乙酸对人胃癌MGC803、AGS、肝癌SMMC7721、结肠癌LOVO、人黑色素瘤A375、SK–28和624 mel细胞增殖的抑制作用。运用逆转录聚合酶链式反应(RT–PCR)技术检测PPARγ在人胃癌MGC803、肝癌SMMC7721、结肠癌LOVO细胞中的mRNA表达。结果:1.土槿乙酸对人胃癌MGC803、AGS、肝癌SMMC7721细胞抑制作用显著(P<0.05),且呈时间和剂量依赖性,IC50值分别为AGS(0.89μmol/L)<MGC803(0.91μmol/L)< SMMC7721(3.4μmol/L),对LOVO细胞抑制作用不明显;2.土槿乙酸对人黑色素瘤A375,SK–28和624mel细胞抑制作用显著(P<0.05),且呈时间和剂量依赖性,IC50值分别为SK–28(1.12μmol/L)<A375(1.93μmol/L)<624mel(2.86μmol/L);3. PPARγ在人消化道肿瘤细胞株MGC803、SMMC7721、LOVO中均有表达,这对土槿乙酸发挥抗肿瘤作用可能是必要的。小结:土槿乙酸对表达野生型和突变型p53的肿瘤细胞均具有明显的生长抑制作用,提示土槿乙酸具有较强的抗肿瘤作用,有望成为一种新的肿瘤治疗药物。第二部分土槿乙酸对p53野生型和突变型人胃癌细胞凋亡的影响及其可能机制的对比研究目的:对比研究土槿乙酸对人胃癌MGC803和AGS细胞凋亡的影响及其可能机制。方法:应用Hoechst33342/PI核荧光双染色法、DNA片段化分析检测细胞凋亡的改变;应用流式细胞术分析细胞周期的变化;应用蛋白检测和RT–PCR技术检测参与细胞凋亡和细胞周期运行的相关基因的蛋白和mRNA表达。结果:1. Hoechst33342/PI核荧光双染色法、DNA片段化分析证实,土槿乙酸明显诱导人胃癌AGS、MGC803细胞凋亡产生;2.流式细胞术分析显示,土槿乙酸诱导人胃癌AGS、MGC803细胞G2期阻遏;3. 10μmol/L土槿乙酸作用AGS细胞12h后可见p53表达增加,在36h达到峰值,24h后可见p21WAF1/CIP1表达明显增加;作用MGC803细胞12h后明显上调PPARγmRNA表达,24h后可见p21WAF1/CIP1表达明显增加;4.土槿乙酸作用于AGS、MGC803细胞后,增加了Fas/APO–1蛋白表达和促凋亡蛋白Bax mRNA表达,降低了抑凋亡蛋白Bcl–2 mRNA表达,提高了caspase–3活性。小结: 1.土槿乙酸能明显诱导人胃癌细胞凋亡和细胞周期阻滞;2.土槿乙酸通过Bcl–2介导的线粒体途径和Fas/APO–1介导的死亡受体途径诱导胃癌细胞凋亡。3. p53野生型的胃癌细胞,其凋亡途径的激活可能与p53表达有关;p53突变型的胃癌细胞,其凋亡途径的激活可能与PPARγ表达有关。第三部分土槿乙酸对p53野生型人黑色素瘤细胞抗增殖作用机理的研究目的:研究土槿乙酸抗人黑色素瘤细胞增殖的机制。方法:采用Hoechst33342/PI核荧光双染色法、DNA片段化分析技术检测细胞凋亡的改变;应用流式细胞术分析细胞周期的变化;运用Westernblot和RT–PCR技术检测参与细胞凋亡和细胞周期运行的相关基因的蛋白和mRNA的表达。结果:1. Hoechst33342/PI核荧光双染色法、DNA片段化分析证实土槿乙酸能明显诱导人黑色素瘤SK–28细胞凋亡产生;2.流式细胞术分析显示土槿乙酸诱导人黑色素瘤SK–28细胞G2/M期阻遏: (1)土槿乙酸作用SK–28细胞4h,增加了Cdc2的磷酸化水平,24 h下调了其蛋白水平表达。(2)土槿乙酸降低了Cdc25C磷酸酶蛋白表达,提高了Wee1激酶蛋白表达。(3)土槿乙酸能明显诱导p53下游靶基因p21WAF1/CIP1蛋白表达,降低Cdc2的活性。(4)土槿乙酸增加了ATM激酶活性,提高了细胞周期检测点激酶Chk2和p53蛋白表达及p53磷酸化水平,这些作用可被ATM激酶抑制剂咖啡因阻断;3.土槿乙酸作用于SK–28细胞后,增加了Fas/APO–1蛋白表达和促凋亡蛋白Bax mRNA表达,降低了抑凋亡蛋白Bcl–2 mRNA表达;提高了caspase–3活性。小结: 1.土槿乙酸可通过依赖ATM–p53–p21WAF1/CIP1信号途径和ATM–Chk2–Cdc25C信号途径诱导人黑色素瘤SK–28细胞周期G2/M期阻滞; 2.土槿乙酸可通过Bcl–2介导的线粒体途径和Fas/APO–1介导的死亡受体途径诱导人黑色素瘤SK–28细胞凋亡。3.凋亡途径的激活可能与p53表达有关。结论:1.土槿乙酸具有较强的抗肿瘤作用,有望成为一种新的抗肿瘤药物;2.土槿乙酸可通过Bcl–2介导的线粒体途径和Fas/APO–1介导的死亡受体途径诱导人胃癌和黑色素瘤细胞凋亡,提示诱导细胞凋亡可能是土槿乙酸抗癌作用的机制之一; 3.土槿乙酸可通过依赖ATM–p53–p21WAF1/CIP1信号途径和ATM–Chk2–Cdc25C信号途径诱导人黑色素瘤细胞G2/M期阻滞,提示诱导细胞周期阻滞可能是土槿乙酸抗癌作用的又一机制。更多还原

【Abstract】 Pseudolarix acid B is a natural diterpenoid compound isolated from the root bark of P. kaempferi Gordon, and displays antifungal, antifertil, antivascular and antiangiogenic properties. Previous studies have shown that pseudolarix acid B displays antiangiogenic properties in a variety of cancer cell lines including gastric carcinoma, liver, breast, colon, cervical, protate cancer and melanoma cell, and accompanied with up–regulation of p53, but the underlying mechanism has not been fully elucidated.The aim of this study was to investigate the mechanism of PLAB–antitumor in wild–type p53 and mutant–type p53 tumor cells. Since p53 mutations are often observed in human gastric carcinoma cells and rarely in melanoma cells, these kinds of cells are representative as cell line models. Firstly, we studied the antitumor effect of Pseudolarix acid B on various human tumor cells. Furthermore, we investigated PLAB mechanism of action using MGC803(mtp53), AGS(wtp53) and SK–28(wtp53) as representative cell line models, which is foundation that PLAB may be a promising chemopreventive agent anti-angiogenic properties.PartⅠAntitumor effect of Pseudolarix acid B on various human tumor cellsObjective: To evaluate the antitumor effect of PLAB on various human tumor lines in vitro, and further investigate mechanism of PLAB as a representative cell line model.Methods: The expression of PPARγwas detected by RT–PCR; the role of different concentration of PLAB on cell growth was tested by MTT in vitro.Results: 1. PLAB had a potent inhibitory effect on the growth of MGC803、AGS and SMMC7721 cells in a dose–dependent and time-dependent manner (P< 0.05), and the order of the concentration of 50% cytotoxicity (IC50) against the various human cancer cell lines for 48h was AGS (0.89μmol/L) <MGC803 (0.91μmol/L) < SMMC7721 (3.4μmol/L), but had no inhibitory effect on the growth of LOVO cells. 2. PLAB had a potent inhibitory effect on the growth of A375,SK–28 and 624mel cells in a dose–dependent and time-dependent manner (P< 0.05), and the order of the concentration of 50% cytotoxicity (IC50) against the various human cancer cell lines for 48h was SK–28 (1.12μmol/L)<A375 (1.93μmol/L) <624mel (2.86μmol/L). 3. It is of possibility that the expression of PPARγin MGC803, AGS, SMMC7721 and LOVO cell lines is responded to PLAB.Conclusion: PLAB has obviously of activity against human carcinoma in wild–type p53 and mutant–type p53 tumor cells, which suggests that PLAB possessed significantly antiangiogenic activity,may be a promising, novel agent for treating human carcinoma.PartⅡResearch on mechanisms of apoptosis induced by Pseudolaricacid B in wild–type p53 and mutant–type p53 human gastric carcinoma cells by comparsionObjective: To investigate the effects and mechanisms of PLAB-induced apoptosis in wild–type p53 and mutant–type p53 human gastric carcinoma cells by comparsionMethods: Cell cycle was analyzed by flow cytometry. Apoptotic cells were detected using Hoechst Hoechst33342/PI staining, and confirmed by DNA fragmentation assay. ELISA kit analysis was used to detect the expression of proteins. The expression of genes involved in apoptosis metabolism was analysized with RT–PCR.Results: 1.PLAB–induced apoptosis in MGC803 and AGS cells was confirmed by DNA fragmentation assay and Hoechst33342/PI staining. 2 Flow cytometry analysis showed that PLAB significantly blocked cell cycle progression in the G2/M phase in MGC803 and AGS cells. 3 In AGS cells, PLAB (10μmol/L) exhibited a time–dependent p53 protein levels after 12h of treatment, and the protein levels of p21WAF1/CIP1, a downstream target of p53, were also found to increase after treatment with 10μmol/L PLAB for 24h; In MGC803 cells, PLAB (10μmol/L) exhibited a time–dependent PPARγmRNA levels after 12h of treatment, and protein levels of p21WAF1/CIP1 were also found to increase after treatment with 10μmol/L PLAB for 24h. 4. When MGC803 and AGS cells was treated with PLAB, it induced Fas/APO–1 and caspase–3 expression, and decreased in the mRNA expression of Bcl–2, but increased in the mRNA expression of Bax.Conclusion: 1. PLAB can significantly induce G2 arrest and apoptosis in human gastric carcinoma cells. 2 PLAB–induced apoptosis of human gastric carcinoma cells is mediated through both Bcl–2–associated mitochondrial pathway and Fas–associated death receptor signaling death pathway. 3. Activation of apoptosis signaling pathway may be mediated through of p53 or PPARγ.PartⅢEffect of Pseudolaricacid B on Cell Growth in wild–type p53 human melanoma cells and its MechanismObjective: To investigate the effect of PLAB on cell growth and its mechanism in human melanoma cells.Methods: Cell cycle was analyzed by flow cytometry. Apoptotic cells were detected using Hoechst Hoechst33342/PI staining, and confirmed by DNA fragmentation assay. The expression of genes involved in apoptosis metabolism was analysized with RT–PCR. The level of the protein was detected with western blotting.Results: 1.PLAB–induced apoptosis in SK–28 cells was confirmed by DNA fragmentation assay and Hoechst33342/PI staining. 2 Flow cytometry analysis showed that PLAB significantly blocked cell cycle progression in the G2/M phase in SK–28 cells. (1) PLAB (10μmol/L) increased phosphorylation of Cdc2 and subsequently decreased expression of Cdc2. (2) PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. (3) A reduction in Cdc2 activity was partly due to induction of expression of p21waf1/cip1 in a p53–dependent manner. (4) PLAB activated the checkpoint kinase, Chk2, and increased the expression and phosphorylation of p53, and enhanced ATM kinase activity, and these effects were inhibited by caffeine, an ATM kinase inhibitor. 3. PLAB enhanced caspase–3 activity, and increased in the protein expression of Fas/APO–1, and decreased in the mRNA expression of Bcl–2, but increased in the mRNA expression of Bax.Conclusion: 1.PLAB induced G2/M arrest in human melanoma cells via a mechanism involving the activation of ATM–Chk2–Cdc25C and ATM– p53–p21WAF1/CIP1 signalling pathways. 2. PLAB–induced apoptosis of human melanoma cells is mediated through both Bcl–2–associated mitochondrial pathway and Fas–associated death receptor signaling death pathway.Summary: 1 PLAB has obviously antitumor effect against human carcinoma cells, which suggsts that PLAB may be a promising, novel agent for treating cancer. 2 PLAB–induced apoptosis of human gastric carcinoma and melanoma cells is mediated through both Bcl–2–associated mitochondrial pathway and Fas–associated death receptor signaling death pathway, which suggests it may be one of anticancer mechanisms that PLAB induces apoptosis. 3. PLAB induced G2/M arrest in human melanoma cells via a mechanism involving the activation of ATM–Chk2–Cdc25C and ATM–p53–p21WAF1/CIP1 signalling pathways, which suggests it may be one of anticancer mechanisms that PLAB induces cell cycle arrest.更多还原