【作者】 张海燕；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 第一章姜黄素在宫颈癌动物模型中选择性抑制宫颈癌细胞生长研究证实姜黄素有治疗各种肿瘤的潜力,但是姜黄素在宫颈癌中的效果一直不明显。本研究评价了姜黄素对SiHa,Caski,HeLa-S3和HeLa229四种宫颈癌细胞核和SiHa小鼠体内移植瘤模型的细胞凋亡,选择性,效能及安全性的影响,并探讨了其机制。体外实验结果表明姜黄素明显抑制了宫颈癌细胞的增殖和迁移,还会引起细胞的死亡,姜黄素的效应发生在p53和PTEN诱导的细胞突变时期,但是姜黄素并不影响正常宫颈上皮细胞的生存,这就表明姜黄素选择性的作用于转移细胞,在SiHa和Caski细胞中,姜黄素组合型抑制了PI3K/AKT和NFkB存活途径的激活,下调由NFkB调节的抗凋亡蛋白bcl-xl并引起线粒体的功能性障碍还会导致细胞凋亡,细胞分裂到G2/M期时会引起sub-G1的细胞凋亡及细胞凋亡小体的形成,Caspase-3的激活发生在p53正常的细胞SiHa中,不会发生在p53突变的细胞Caski,除了对细胞凋亡的影响,姜黄素能够调控化疗药物多柔比星和顺铂共同引起宫颈癌细胞的死亡。在植入SiHa细胞的小鼠体内。与经过处理组相比,腹膜内的姜黄素降低了小鼠宫颈的肿瘤数,未引起组织成分改变,细胞代谢,氧化反应和血液毒性都没受到影响,本研究结果表明姜黄素是一种能够有效抑制宫颈癌细胞生长增殖的潜在天然药物。第二章姜黄素增强阿霉素对宫颈癌细胞的增殖抑制作用及其机制研究宫颈癌化疗中使用的多种药物降低了患者的生活质量。研究已经证实癌症患者接受化疗过程中,在食物中添加抗肿瘤辅助药物能显著提供患者生活质量。姜黄素在食品中广泛作为着色剂和矫味剂使用。本研究探讨了姜黄素对阿霉素抑制宫颈癌细胞作用的影响。阿霉素单独作用并没有降低肿瘤质量,而阿霉素和姜黄素联合作用降低肿瘤质量至56.5%。与姜黄素联合作用降低了细胞生存率,加强了癌细胞凋亡。姜黄素阿霉素联合作用对caspase3,8,9的抑制作用也大于阿霉素单独作用。本研究还证实姜黄素增强阿霉素抗宫颈癌活性的机制主要是通过非caspase依赖的细胞死亡途径。姜黄素还能通过抑制正常组织的脂质过氧化而减少阿霉素诱导的副作用。第三章姜黄素作为去甲基化药物在宫颈癌治疗中的应用宫颈癌是威胁女性健康的恶性肿瘤之一。虽然,宫颈癌本身可以通过手术和放射疗法的到有效治疗,但是肿瘤的转移也是致命的。最近的研究表明,人类宫颈癌与后天的基因突变及遗传缺陷有关。近来,我们的研究发现,在Caski、鼠长生前列腺肿瘤的过程中,细胞抗氧化防御系统的一个关键调控因子Nrf2逐渐沉默表达。本文的目的旨在研究姜黄素(CUR)的功能,姜黄素是一种DNA低甲基化物质,存在于人们的日常饮食中,能够抑制Caski小鼠前列腺肿瘤的发生。通过重亚硫酸盐基因组测序(BGS)发现,Caski Cl小鼠Nrf2基因启动子区域的前5个CpGs的甲基化状态能被CUR逆转。甲基化DNA免疫共沉淀(MeDIP)分析显示,CUR明显减弱了甲基化抗体与Nrf2基因启动子区域前5个CpGs之间的结合,这也证实了之前的BGS的结果。Nrf2的甲基化能够在mRNA和蛋白水平修复Nrf2基因及其下游靶标分子NQQ-1的表达,NQQ-1是一种重要的抗氧化应激反应酶。综上所述,我们目前的研究表明CUR对宫颈癌有化学防御作用,这种作用可能通过对Nrf2基因的修饰及之后引发的Nrf2介导的抗氧化应激通路来实现。更多还原

【Abstract】 Charpter I The curry spice curcumin selectively inhibits cancer cells growth in vitro and in preclinical model of cervical carcinomaPrevious studies suggested that curcumin is a potential agent against cervical carcinoma. However, the in vivo efficacy of curcumin in cervical carcinoma remains not established. In this work, we examined the mechanisms underlying apoptosis, selectivity, efficacy and safety of curcumin from in vitro (SiHa,Caski,HeLa-S3and HeLa229cell lines) and in vivo (SiHa implants) models of cervical carcinoma. In vitro, curcumin markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of cervical carcinoma growth. Curcumin effects occurred irrespective of the p53and PTEN mutational status of the cells. Interestingly, curcumin did not affect viability of primary astrocytes, suggesting that curcumin selectivity targeted transformed cells. In Caski and SiHa cells, curcumin decreased the constitutive activation of PI3K/Akt and NFkappaB survival pathways, down-regulated the antiapoptotic NFkappaB-regulated protein bcl-xl and induced mitochondrial dysfunction as a prelude to apoptosis. Cells developed an early G2/M cell cycle arrest followed by sub-G1apoptosis and apoptotic bodies formation. Caspase-3activation occurred in the p53-normal cell type SiHa, but not in the p53-mutant Caski. Besides its apoptotic effect, curcumin also synergized with the chemotherapeutics cisplatin and doxorubicin to enhance cervical carcinoma cells death. In SiHa-implanted rats, intraperitoneal curcumin (50mg kg-1/d-1) decreased cervical tumors in9/11(81.8%) animals against0/11(0%) in the vehicle-treated group. Importantly, no evidence of tissue (transaminases, creatinine and alkaline phosphatase), metabolic (cholesterol and glucose), oxidative or hematological toxicity was observed. In summary, data presented here suggest curcumin as a potential agent for therapy of cervical carcinoma. Charpter Ⅱ Beneficial effects of curcumin on antitumor activity and adverse reactions of doxorubicinMany medicines used in cancer chemotherapy decrease the quality of life (QOL). It is believed that an increase in food intake during cancer chemotherapy may produce an improvement in QOL. Curcumin is widely used as a coloring and flavoring agent in food. The effects of curcumin in relation to the chemotherapeutic drug doxorubicin (DOX) were examined. While DOX alone did not decrease tumor weight, the combination of DOX and curcumin significantly reduced tumor weight to56.5%(p<0.05) of that of the control group. The combined curcumin enhanced apoptosis by DOX and decreased cell viability. The curcumin-DOX combination also suppressed activation of caspase-3,-8, and-9compared to DOX alone. It is presumed that combining curcumin increased DOX-induced antitumor activity by suppressing the main caspase pathway and activating the main caspase independent pathway. The combination of curcumin and DOX suppressed the reduction of glutathione peroxidase activity and increased lipid peroxide levels in the heart. Therefore, it is expected that curcumin may reduce the adverse reactions associated with DOX. Our results suggest that curcumin can be used as a modulator to enhance the therapeutic index of cancer patients and improve their QOL Charpter III Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2via promoter CpGs demethylationCervical carcinoma is one of the most deadly malignancies among women in the world. Although localized Cervical carcinomar can be effectively treated via surgery or radiation, metastatic disease is usually lethal. Recent evidence suggests that the development and progression of humanCervical carcinoma involves complex interplay between epigenetic alterations and genetic defects. We have recently demonstrated that Nrf2, a master regulator of cellular antioxidant defense systems, was epigenetically silenced during the progression of Cervical carcinoma tumorigenesis in Caski mice. The aim of this study is to investigate the potential of curcumin (CUR), a dietary compound that we have reported to be able to prevent the development of Cervical carcinoma in Caski mice, as a DNA hypomethylation agent. Using bisulfite genomic sequencing (BGS), treatment of TRAMP C1cells we showed that CUR reversed the methy lation status of the first5CpGs in the promoter region of the Nrf2gene. Methylation DNA immunoprecipitation (MeDIP) analysis revealed that CUR significantly reduced the anti-mecyt antibodybinding to the first5CpGs of the Nrf2promoter, corroborated the BGS results. Demethylation of Nrf2was found to be associated with the re-expression of Nrf2and one of its downstream target gene, NQO-1, one of the major anti-oxidative stress enzymes, both at the mRNA and protein levels. Taken together, our current study suggests that CUR can elicit its prostate cancer chemopreventive effect, potentially at least in part, through epigenetic modification of the Nrf2gene with its subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway.更多还原