【作者】 张学农；

【导师】 阮金兰；

【作者基本信息】 华中科技大学 ， 药理学， 2013， 博士

【摘要】 本论文主要对新型特异性抗肿瘤化合物RY10-4的部分作用机制及其抗肿瘤活性进行了研究,主要内容分为两部分：第一部分研究了RY10-4诱导人乳腺癌MCF-7细胞自噬性死亡的作用及其影响的相关信号传导通路,同时将RY10-4与原芹菜素在自噬缺陷的乳腺癌MCF-7细胞系中诱导细胞自噬活性的差异进行了对比,探讨RY10-4抗乳腺癌作用的潜在机制及其抗乳腺癌作用优于原芹菜素的可能原因；第二部分主要研究了RY10-4体外和体内抑制人肝癌HepG2细胞增殖的作用,探讨其体内外抗肝癌的活性,并对其抗肝癌活性相关的作用机制进行了初步研究。第一部分：RY10-4诱导乳腺癌细胞系MCF-7自噬性细胞死亡作用的研究目的：鉴于原芹菜素类似物RY10-4表现出更好的体外抗肿瘤作用及较低的体内不良反应,本部分探讨其可能的作用机制。研究RY10-4诱导人乳腺癌MCF-7细胞自噬的活性,并与原芹菜素的作用进行比较；另外,进一步研究RY10-4诱导的自噬活性在抑制肿瘤细胞增殖中的作用以及相关的信号传导通路。方法：通过免疫荧光法检测细胞内LC3B蛋白的表达和分布、MDC染色法、Western Blot法检测LC3B和p62蛋白的表达以及电子显微镜对细胞超微结构的分析,综合判断RY10-4与原芹菜素是否诱导MCF-7细胞自噬的发生；通过应用自噬抑制剂和干扰自噬相关基因的表达对RY10-4诱导的自噬活性在细胞死亡中的作用进行研究；通过Western Blot检测相关通路蛋白的表达水平研究RY10-4诱导自噬所涉及的信号通路。结果：RY10-4可诱导MCF-7细胞自噬的发生,而原芹菜素没有这方面的作用；自噬抑制剂3-MA或干扰自噬相关基因ATG7的表达抑制细胞自噬活性后,RY10-4处理后细胞的存活率显著提高；另外,RY10-4处理后细胞磷酸化mTOR和p70S6K的表达呈浓度依赖性的降低,磷酸化Akt的表达呈浓度和时间依赖的降低,而AMPK的活性基本无变化。结论：RY10-4通过Akt/mTOR通路抑制mTOR信号而诱导MCF-7细胞的自噬,并作为一种抑制肿瘤细胞增殖作用的机制,促进了肿瘤细胞的死亡。第二部分：RY10-4体外和体内抗肝癌作用的研究目的：研究RY10-4体外和体内抑制人肝癌HepG2细胞增殖的作用,探讨RY10-4体内外抗肝癌的活性,并对其抗肝癌活性的相关作用机制进行研究。方法：通过SRB染色法、贴壁集落形成试验研究RY10-4抑制HepG2细胞体外增殖的作用；通过PI染色法流式细胞仪分析RY10-4细胞周期阻滞作用；Hoechst33342荧光染色法、Annexin V/PI双染法流式细胞仪检测对RY10-4诱导细胞凋亡的作用进行研究；应用活性氧检测试剂盒及线粒体膜电位检测试剂盒研究RY10-4诱导活性氧生成和线粒体膜电位的变化,并使用抗氧化剂NAC进行验证；以人肝癌HepG2细胞裸小鼠异种移植瘤模型评价RY10-4的体内抗肝癌活性。结果：RY10-4显著抑制HepG2细胞体外的增殖活性,减少其体外的克隆形成率；RY10-4处理后细胞的S期和G2/M期细胞比率明显增加,出现明显的凋亡细胞的形态特征,凋亡细胞比率显著增高；RY10-4可诱导细胞活性氧的生成,降低线粒体膜电位,NAC能抑制RY10-4的上述作用；RY10-4显著抑制荷瘤裸鼠HepG2移植瘤的生长,且对动物的血液和肝肾功能无明显影响。结论：RY10-4具有良好的体外和体内的抗肝癌活性,其可能的作用机制包括：RY10-4可以阻滞细胞周期的进程；RY10-4通过增加细胞内ROS的生成,引起细胞线粒体膜电位的降低,进而诱导细胞的凋亡。更多还原

【Abstract】 The partial mechanism and anti-tumor activity of the novel compound RY10-4which have a specific anti-tumor effect were studied in this dissertation, and the main content was divided into two parts. In the first part, we studied the effect and related signal pathway of RY10-4-induced autophagic cell death in human breast cancer MCF-7cell line. And we also compared the difference between RY10-4and protoapigenone in the induction of autophagy in autophagy defect breast cancer MCF-7cell line, explored new insights for the potential mechanism of RY10-4induced cell death and the cause of RY10-4showing better antitumor activity than protoapigenone. In the second part, we studied the effects of RY10-4against human hepatocellular carcinoma HepG2cells in vitro and in vivo, and explored some related and potential mechanism of the antitumor activity of RY10-4against liver cancer.FART I:Study on the effect of RY10-4induced autophaic cell death in breast cancer MCF-7cell lineObjective: In consideration of the protoapigenone analog RY10-4showing better antitumor activity in vitro and lower side effects in vivo, the potential mechanism was explored here. The activity of RY10-4induced autophagy in human breast cancer MCF-7cell was studied, and compared it with the effect of protoapigenone. We also discussed the role of RY10-4induced autophagy in cell death and the related signaling pathways in the further researches.Methods:Employing immunofluorescence assay for microtubule-associated protein light-chain3(LC3B), monodansylcadaverine staining, western blot analyses for LC3B and p62as well as ultrastructural analysis by transmission electron microscopy, to observe whether RY10-4or protoapigenone induced autophagy in MCF-7cells. For exploring the role of RY10-4induced autophagy in cell death, inhibitions of autophagy by pharmacological and genetic approaches were performed. Moreover, the signaling pathways involved in RY10-4induced autophagy were studied through detecting the expressing of some related signal proteins by Western blot.Results:RY10-4could induce autophagy in MCF-7cells, but protoapigenone could not. When the autophagy activity was inhibited by autophagy inhibitor3-MA or siRNA specific for autophagic-related gene ATG7, the viability of MCF-7cells exposed to RY10-4significantly increased. In addition, the expression of phosphorylated-mTOR (p-mTOR) and p-p70S6K decreased in a concentration-dependent manner, the expression of p-Akt decreased in both concentration and time-dependent manners, while the activity of AMPK did not alter. Conclusion:RY10-4inhibited mTOR signal and induced autophagy in MCF-7cells via Akt/mTOR pathway, and the induction of autophagy enhanced RY10-4induced antitumor effect, promoted the cell death of tumor cells.PART Ⅱ:Study on the antitumor activity of RY10-4against liver cancer in vitro and in vivoObjective:To study the cell proliferation inhibition of RY10-4on hepatocellular carcinoma HepG2cells in vitro and in vivo, and to determine the biological activity and the underlying mechanisms of RY10-4on hepatocellular carcinoma cancer cells by using in vitro and in vivo experimental models.Methods:Sulforhodamine B (SRB) assay and clonogenicity assay were performed to determine the antiproliferation of RY10-4on hepatocellular carcinoma HepG2cells in vitro. Flow cytometry assay with propidium iodide (PI) staining was used to determine the effect of RY10-4on cell cycle distribution. Fluorescent staining with Hoechst33342and flow cytometry assay with Annexin V/PI staining were performed to evaluate the effect of RY10-4on the induction of apoptosis. Reactive oxygen species (ROS) assay kit and mitochondrial membrane potential (MMP) assay kit with JC-1were used to detect the ROS generation and changes in MMP induced by RY10-4, and these effects of RY10-4were confirmed by using the antioxidant NAC. Furthermore, the human hepatocellular tumor xenograft model established with HepG2cell line was used to investigate the antitumor activity of RY10-4in vivo.Results:The viability of HepG2cells in vitro significantly decreased after the treatment of RY10-4, and a concentration-dependent inhibition in colony formation was also observed. The percentage of cells in the S phase and G2/M phase as well as the ratio of apoptotic cells significantly increased in groups treated with RY10-4, and the typical morphological characteristics of apoptotic cell was observed. RY10-4could induce the ROS generation and the decrease of MMP, while these effects will be inhibited by NAC. Moreover, RY10-4treatment significantly inhibited the growth of HepG2tumor in tumor-bearing nude mice without significant hematological toxicity as well as hepatotoxicity and nephrotoxicity.Conclusion:RY10-4exhibited high antiproliferation and antitumor activity toward HepG2cell line in vitro and in vivo. The underlying mechanism for the antitumor activity of RY10-4included: blocking the cell cycle process, inducing ROS generation, decreasing the MMP, inducing apoptosis.更多还原