中药来源的两种抗癌药物的细胞毒活性与抑癌基因Pdcd4表达的相关性

【作者】 王汉卿；

【导师】 孙震晓；

【作者基本信息】 北京中医药大学 ， 中药制药， 2008， 硕士

【摘要】 传统中药在现代肿瘤治疗中的作用日益受到重视。在传统中药中,某些植物药与虫类药在抗肿瘤方面具有具有巨大的潜力,因此受到人们的高度重视。斑蝥(mylabris)系鞘翅目芜青科昆虫,南方大斑蝥或黑黄小斑蝥的干虫体可入药。斑蝥在我国作为药用己有两千余年的历史,具有攻毒蚀疽、破血散结的作用,用于治疗肿瘤。斑蝥中的有效成分是斑蝥素(cantharidin),可抑制细胞蛋白质的合成,影响细胞的生长分化。去甲斑蝥素( norcantharidin,NCTD)是我国自行研制生产的新型抗肿瘤药物,是斑蝥素的去甲基衍生物,具有显著的抗癌活性,其毒、副作用较低,是一种具有升高白细胞作用的抗肿瘤药物。喜树碱(camptothecin,CPT)是从珙桐科植物喜树(Camptotheca acuninata)中分离提取的五环生物碱,是唯一有选择性抑制DNA拓扑异构酶Ⅰ(TopoⅠ)作用的植物抗癌药。其后人们分离出10-羟基喜树碱(Hydroxycamptothecine, HCPT),其化学结构与喜树碱相似,仅第10位碳原子上的氢为羟基所取代。与喜树碱相比,羟基喜树碱毒性小、抗肿瘤作用更强,使喜树碱类药物在肿瘤防治中起到了重大作用。维甲酸(retinoic acid,RA)是维生素A的衍生物,又称视黄酸或维生素A酸。维甲酸包括多种同分异构体,全反式维甲酸(all-trans retinoic acid,ATRA)是其中重要的一种,全反式维甲酸是体内一种重要的生物活性物质,以往发现在胚胎发育、抑制细胞增殖、促进细胞分化和凋亡以及视觉循环中起着重要的作用。Pdcd4(Programmed cell death 4)是一个凋亡基因,因为它在各种诱导因素引起的小鼠细胞凋亡过程中均表达上调而命名。近年来研究表明它还是一个抑癌基因,它编码的蛋白质在人肺癌、乳腺癌、肠癌、前列腺癌、胰腺癌中表达丢失,提高Pdcd4在小鼠表皮中表达可以抑制致癌物质诱导小鼠上皮癌的发生。Pdcd4作为一种新抑癌物质,在今后的抗肿瘤治疗中可能成为一个新的治疗靶点。本研究的主要内容及结果:(1)选取人胃腺癌BGC-823作为转染宿主,用脂质体转染的方法,通过转染Pdcd4cDNA、空载体和Pdcd4cDNA突变体获取Pdcd4高表达癌细胞株和对照癌细胞株。选取人卵巢癌SK-OV-3作为转染宿主,用脂质体转染的方法,获取Pdcd4下调表达癌细胞株和对照癌细胞株。(2)用western blot方法,分析细胞是否被稳定转染,以及NCTD和HCPT作用转染细胞后Pdcd4蛋白的表达情况,考察两种抗癌药物NCTD和HCPT对Pdcd4表达的影响。结果确认稳定转染Pdcd4表达质粒和Pdcd4抑制质粒。结果发现60umol/LNCTD作用BGC-823三种转染细胞72h后,pdcd4蛋白表达明显下调;80umol/L HCPT作用BGC-823三种转染细胞72h后,Pdcd4蛋白表达明显上调。其机制尚不清楚。(3)用MTT方法,测定NCTD和HCPT对BGC-823转染细胞系的细胞毒活性,分析Pdcd4是否可以影响细胞对药物作用的敏感性。结果显示,NCTD低浓度长时间作用或高浓度短时间作用均能提高Pdcd4转染细胞对NCTD的敏感性。HCPT结果与NCTD相似,也是在低浓度作用长时间或高浓度作用短时间均能提高Pdcd4转染细胞对药物的敏感性。(4)用流式细胞仪测定,分析NCTD和HCPT对BGC-823转染细胞系的细胞周期及细胞凋亡的影响,探讨两种抗癌药物的细胞毒活性机制及Pdcd4的影响。分别用60umol/L NCTD和80umol/L HCPT作用三种转染细胞系0、24、48、72h,用流式细胞仪测定细胞周期和凋亡率。结果显示,NCTD对三种转染细胞均有G2-M期阻滞;NCTD作用72h后,各转染细胞的凋亡率均明显高于对照组,但其转染细胞之间并无显著差别。HCPT对三种转染细胞均有S期阻滞,HCPT作用72h后,各转染细胞的凋亡率均明显高于对照组,但其转染细胞之间并无显著差别。更多还原

【Abstract】 Traditional Chinese medicine is playing a more and more important role in the treatment of tumors. Some medicinal plants and insect drugs of traditional Chinese medicine has great potential in the anti-tumor domain. People are paying close attention to them.Mylabris has been used as a medicine for the treatment of tumors for more than2,000 years in China. The effective ingredient in mylabris is cantharidin, which can inhibits the synthesis of protein and affects cell growth and differentiation.Norcantharidin (NCTD) is a new anticancer drug developed and producted by China. NCTD is the demethyl derivative of cantharidin,which has significant anti-tumor activity and small side effects. What’s more,NCTD is the only anticancer drug that can increase the number of leucocyte.Camptothecin(CPT) is an alkaloid extracted from Camptotheca acuninata, the only selectiveinhibitor of DNA topoisomeraseⅠ(TopoⅠ). Later people abstracted 10-HCPT(Hydroxycamptothecin, HCPT) from CPT. The chemical structure of 10-HCPT is similar to CPT’s, only a hydrogen beening replaced by hydroxyl. Compared with CPT, HCPT has stronger anti-tumor effect and lower toxicity, that make camptothecin drugs play a significant role in theprevention and treatment of tumors.Retinoic acid (RA) is a derivative of vitamin A, also known as vitamin A acid. retinoic acid has a variety of isomeride. All-trans retinoic acid (ATRA) is an important one of them. ATRA is an important bioactive substances in vivo, It has been found play an important role in embryonic development, inhibiting cell proliferation and promoting cell differentiation and apoptosis, as well as in visual cycle.Pdcd4 (Programmed cell death 4) is an apoptosis gene.The up-regulated expression of Pdcd4 was seen in the apoptosis process of mice cells induced by many factors. Recent studies show that Pdcd4 is also a tumor suppressor:The expression of Pdcd4 in human lung cancer, breast cancer, colorectal cancer, prostate cancer and was down-regulated. Up-regulating Pdcd4 expression in mouse skin could inhibit epithelial cancer induced by carcinogenic substances. As a new tumor suppressor , Pdcd4 may become a new therapeutic target for anti-cancer therapy in the future.The main content of this study and the results:To obtain Pdcd4 high expression BGC-823 cell lines and the control cell lines, lipid-mediated transfection was used to transfect Pdcd4cDNA, empty carrier and mutant Pdcd4cDNA respectively into human gastric adenocarcinoma BGC-823 cells. Choosing human ovarian carcinoma SK-OV-3 cells as the host ,we use the same method to obtain Pdcd4 down-regulated expression cancer cell line and thecontrol cell line.Western blot was used to analysis the transfection results and the expression of Pdcd4 protein in the BGC-823 transfected cells treated by NCTD and HCPT. The results confirmed that we had established stable Pdcd4 up-regulated expression and Pdcd4 down-regulated expression cell lines. The results showed that after treated by 60 umol / L NCTD 72 hours, the expression of Pdcd4 protein was significantly down-regulated in the three transfected cells; after treated by 80 umol / L HCPT 72 hours, the expression of Pdcd4 protein was significantly up-regulated in the three transfected cells. The mechanism is still unclear.In the interest of whether Pdcd4 can affect the sensitivity of BGC-823 transfected cells to anti-cancer drugs, MTT method was used to detect the cytotoxic activity of NCTD and HCPT to the transfected cell lines. The results showed that low concentrations of NCTD effecting long–term and high concentrations of NCTD effecting short–term both increased the sensitivity of Pdcd4 up-regulated cells to NCTD. The results of HCPT is similar to NCTD’s.To find out the cytotoxicity mechanism of NCTD and HCPT and its relation with Pdcd4,Flow cytometry was used to analyze the effects of NCTD and HCPT to the cell cycle and apoptosis of the BGC-823 transfected cell lines. The transfected human gastric adenocarcinoma BGC-823 cell lines was treated Respectively with NCTD and HCPT in a concentration of 60 umol / L and 80 umol / L . Cells were Collected respectively after 0,24,48 and 72 hours, flow cytometry was used to analysis the cell cycle and apoptosis of the treated cells. Flow cytometry results showed that the three transfected cell lines were arrested at G2-M stage by NCTD and arrested at S stage by HCPT.But there was no significant difference between the transfected cells.更多还原