【作者】 陆成伟；

【导师】 宋鄂； 胡诞宁；

【作者基本信息】 吉林大学 ， 眼科学， 2010， 博士

【摘要】 脉络膜黑色素瘤(choroidal melanoma, CM)是成人最常见的眼内恶性肿瘤。CM的治疗方法尽管已有一些新进展,除外科手术外,目前多采取综合性治疗,如放疗、化疗、冷冻、激光、免疫及光动力学等,但其疗效有限。目前的研究结果,尚不足以阐明肿瘤发生的机制。姜黄素作为天然多酚类物质且对多种疾病尤其是癌症具有抑制和治疗作用,因此倍受研究者的关注。虽然有实验表明姜黄素对鼠和人类的皮肤黑色素瘤细胞有毒性作用,但是姜黄素对人眼脉络膜黑色素瘤细胞的作用尚无相关报道。本实验是通过对人眼脉络膜黑色素瘤细胞与成纤维细胞和脉络膜黑色素细胞进行比较,研究姜黄素对人眼脉络膜黑色素瘤细胞的毒性作用。通过对姜黄素诱导肿瘤细胞产生毒性作用的相关生物学机制如线粒体细胞色素c的释放和caspase-9与caspase-3的激活的研究,进一步阐明姜黄素诱导细胞毒性作用的信号通路。本实验结果表明姜黄素能诱导脉络膜黑色素瘤细胞死亡且呈剂量依赖效应和时间依赖效应。姜黄素诱导脉络膜黑色素瘤细胞的死亡是通过线粒体细胞色素c的释放并激活caspase-9-caspase-3通路实现的。本实验首次将姜黄素应用于人眼脉络膜黑色素瘤的研究,这个令人振奋的体外实验结果为进一步研究姜黄素对脉络膜黑色素瘤的治疗奠定了基础,并且随着姜黄素生物利用率的提高,很可能使这一天然的药物在将来成为治疗脉络膜黑色素瘤有效的治疗药物,为脉络膜黑色素瘤患者的治疗提供一种新方法。更多还原

【Abstract】 Uveal melanoma is the most common primary intraocular tumor in adults and eventually metastasizes to the liver in up to 50% of patients within15 years after the initial diagnosis. Despite advances in diagnosis and treatment of uveal melanoma in the last decades, the overall mortality rate remains high, and systemic uveal melanoma metastases are generally not amenable to treatment. Based on the Collaborative Ocular Melanoma Study Group’s report, only 45% of uveal melanoma patients were alive and clinically cancer free 12 years after treatment.Curcumin, a yellow colored polyphenol isolated from the plant Curcuma longa, the principal curcuminoid of the popular spice turmeric, is a member of the ginger family (Zingiberaceae). It was first discovered in 1815, isolated in crystalline form in 1870. Curcumin has a long history of being used as a safe herbal medicine in India and China.Curcumin is a pleiotropic molecule that possesses various biological activities. Curcumin has been shown to have inhibitory effects relevant to all three stages of carcinogenesis: initiation, promotion, and progression.It has been widely studied for its anti-inflammatory, anti-angiogenic, antioxidant and wound healing effects. Curcumin has been demonstrated recently to possess anti-tumor effects both in vitro and in vivo by a variety of mechanisms. It inhibits proliferation and induces apoptosis in a variety of cancer cell types in vitro, including cells from cancers of the prostate, breast, lung, pancreas, bladder, cervix, head and neck, ovary, bone marrow, brain, kidney, and skin. A broad spectrum of anticarcinogenic activity of curcumin has been demonstrated in experimental animal model systems of duodenal, colon, oral, liver, breast, and skin cancers. Whereas curcumin may be relatively nontoxic to normal cells, in vitro studies in tumor cells suggest that curcumin is cytotoxic at low concentrations. Moreover, at least 12 active clinical trials of curcumin for the treatment of various diseases are ongoing in the United States, Israel, and Hong Kong. The cytotoxic effect of curcumin on tumor cells is attributed to induction of cell apoptosis mediated by a mitochondria passway. Mitochondria play an important role in cell proliferation and apoptosis processes both in normal and malignant cells. An excess of pro-apoptotic over anti-apoptotic signals initiates increased mitochondrial outer membrane permeability, which leads to the release of cytochrome c from the mitochondrial intermembrane space to the cytosol. Human somatic cytochrome c is a 15 kDa, 104 amino acid polypeptide that participates in apoptosis. Once cytochrome c is released, it activates pro-caspase-9 into caspase-9, which in turn activates the effector caspase-3, allowing apoptosis to proceed.It has been reported that curcumin is cytotoxic in murine and human cutaneous melanoma cells in vitro. Curcumin inhibits lung metastasis of cutaneous melanoma cells in mice. However, the effects of curcumin on uveal melanoma cells has not been reported previously. The purpose of the present study was to investigate the cytotoxic effects of curcumin on human uveal melanoma cells in vitro with comparison to normal human scleral fibroblasts. Curcumin induced biological changes in tumor cells, such as release of cytochrome c from mitochondria and the activation of caspase-9 and caspase-3, were also studied in uveal melanoma cells to elucidate the signaling pathway involved in curcumin-induced cytotoxicity in this neoplasm.Methods:Two human uveal melanoma cell lines (M21 and SP6.5), scleral fibroblasts and choroidal melanocytes were treated with curcumin. The effects of curcumin on cell viability were assessed by using the MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay. In dose-response studies, 24 hr after seeding, curcumin was applied to the cultures at final concentrations of 3, 10, 30 and 100μM and cultured for another 48 hr. In time-response studies, curcumin was added to the culture at a final concentration of 30μM and cultured for 3, 12, 24 and 48 hr. Cytosol cytochrome c levels and the activities of caspase-9 and caspase-3 were measured by using an enzyme-linked immunosorbent assay.Results:Curcumin induced cell death of cultured human uveal melanoma cells in a dose-dependent manner and time-dependent manner, with IC50 at 19.05μM and 22.39μM in M21 and SP6.5 cell lines, respectively. The difference in cell viability between melanoma cells treated with or without curcumin was significant (P<0.05) at 3-100μM levels. Curcumin at lower concentrations (10-30μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of fibroblasts and choroidal melanocytes. The difference in cell viability between melanoma cells treated with or without curcumin was significant (P<0.05) at 12-48 h. Curcumin significantly increased the level of cytosol cytochrome c (2 fold greater than the controls after 2 h treatment), caspase-9 and caspase-3 activities (approximately 4.5 and 6 fold greater than the controls after 2-6 h treatment, respectively) in a dose-dependent manner. The difference in the cytosol cytochrome c level between the curcumin-treated group (10, 30, and 100μM) and the controls was statistically significant (P<0.05). The difference in the caspase-9 and -3 levels between curcumin treated groups (3, 10, 30 and 100μM) and the controls was statistically significant (P< 0.05).Conclusions:Curcumin induces uveal melanoma cell death in a dose- and time-dependent manner. Curcumin induces cell death of the human uveal melanoma cell lines M21 and SP6.5 in a dose- and time-dependent manner. The effects of curcumin on uveal melanoma cell M21 and SP6.5 were compared with scleral fibroblasts. Curcumin at 10μM and 30μM levels induces cell death of uveal melanoma cell whereas the viability of fibroblasts is not affected. Our results suggested that anticancer specificity presents in curcumin for uveal melanoma cells. In the present studies, release of cytochrome c into the cytosol occurred early (2 hr after curcumin treatment). Then significant increase of caspase-9 and caspase-3 was detected after 2–6 hr treatment, followed by significantly decreased cell viability after 12 hr. Curcumin induces cell death by initiating the release of mitochondrial cytochrome c, thereby activating the caspase-9-caspase-3 apoptosis pathway in uveal melanoma cells. These encouraging in-vitro results support further investigation of the use of curcumin in the treatment of uveal melanoma.更多还原