【作者】 晋佳路；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 黑色素瘤高表达抗凋亡蛋白Bcl-2,是黑色素瘤对化疗药物产生耐药的原因之一。Apoptin(凋亡素)是来自鸡贫血病毒的小分子蛋白质,能选择性地诱导肿瘤细胞凋亡而对正常细胞无毒副作用,其诱导凋亡的作用不依赖抑癌基因p53亦不被抗凋亡蛋白Bcl-2所抑制,Bcl-2增高甚至能够增强其凋亡诱导效应。PTD4-apoptin融合蛋白是一种具有透膜能力的能特异性诱导肿瘤细胞凋亡的融合蛋白。达卡巴嗪(Dacarbazine, DTIC)是最早由美国FDA批准的治疗恶性黑色素瘤的一线药物,至今仍作为评价其他药物对黑色素瘤疗效的标准。本实验旨在探讨PTD4-apoptin融合蛋白与化疗药物达卡巴嗪联用对小鼠黑色素瘤细胞B16-F1,人黑色素瘤细胞系A875, SK-MEL-5的细胞毒效应以及对荷黑色素瘤小鼠C57BL／6的抗肿瘤效应,并初步探讨其联用机制。MTT法检测达卡巴嗪联合PTD4-apoptin融合蛋白对小鼠黑色素瘤细胞系B16-F1,人黑色素瘤细胞系A875, SK-MEL-5的生长抑制作用,CDI分析药物相互作用的特点。DAPI染色观测药物孵育B16-F1细胞后细胞核形态的变化,AnnexinV-FITC/PI双染法检测细胞凋亡率。Western blotting检测药物孵育B16-F1细胞后Bcl-2和CDK2的表达变化,初步探索两药联合作用的机制。皮下注射B16-F1细胞建立C57BL／6小鼠荷黑色素瘤模型,观察PTD4-apoptin融合蛋白及其与化疗药物达卡巴嗪联用对黑色素瘤的生长抑制作用。肿瘤标本制成石蜡切片后分别做原位免疫组化以及TUNEL染色,观察PTD4-apoptin融合蛋白分布情况及肿瘤细胞凋亡情况。对无瘤C57BL/6小鼠施药后,进行血常规检测,分析药物的骨髓抑制作用。体外实验结果表明,PTD4-apoptin融合蛋白与达卡巴嗪均可抑制小鼠黑色素瘤细胞系B16-F1,人黑色素瘤细胞系A875, SK-MEL-5的增殖并呈剂量依赖关系。三种不同浓度的PTD4-apoptin融合蛋白分别与不同浓度的达卡巴嗪联用对三种不同的黑色素瘤细胞的生长抑制作用比达卡巴嗪单药明显增强,CDI分析两药相互作用的性质,CDI<1,表明两者联合有协同效应；在某些浓度联合时CDI<0.7,具有显著的协同效应。DAPI染色和AnnexinV-FITC/PI双染法检测两药联合孵育B16-F1细胞的细胞凋亡事件,结果表明,联合用药后,细胞凋亡程度加强,细胞凋亡率显著升高。Western blotting检测到联合用药后B16-F1细胞中CDK2和Bcl-2的表达均有所升高。采用C57BL／6荷瘤小鼠模型进行药物的体内实验,结果表明,PTD4-apoptin融合蛋白能有效抑制小鼠黑色素瘤生长,而PTD4-EGFP融合蛋白与PBS不能。PTD4-apoptin融合蛋白与达卡巴嗪以不同比率联合治疗小鼠黑色素瘤,结果表明“半剂量”联合治疗组或“全剂量”联合治疗组对黑色素瘤的抑制效应显著高于PTD4-apoptin融合蛋白单药治疗组或达卡巴嗪单药治疗组,且“半剂量”或“全剂量”联合治疗组其抑瘤效应无明显差异。TUNEL染色以及原位免疫组化结果显示,PTD4-apoptin融合蛋白单用能够有效透过皮肤进入肿瘤组织,两者联合能引起大量的肿瘤组织细胞凋亡。血常规检测,分析药物的骨髓抑制作用,结果显示,与对照组相比,达卡巴嗪单药治疗组有骨髓抑制作用,PTD4-apoptin融合蛋白及“半剂量”联合治疗组没有骨髓抑制作用,而“全剂量”联合治疗组骨髓抑制作用也明显小于达卡巴嗪单药组。PTD4-apoptin融合蛋白能够有效抑制黑色素瘤的生长,与达卡巴嗪联用后对黑色素瘤的生长有协同抑制效应。PTD4-apoptin融合蛋白能够加强达卡巴嗪的抑瘤效应且无骨髓抑制作用。PTD4-apoptin融合蛋白和达卡巴嗪的协同效应可能与CDK2和Bcl-2有关。本研究为临床针对黑色素瘤个体特性进行治疗提供了一个新的思路。更多还原

【Abstract】 Bcl-2 is an antiapoptotic protein that is generally expressed in melanoma and high level of Bcl-2 expression has been associated with resistance to chemotherapy in melanoma. Apoptin is a small molecule protein derived from chicken anemia virus (CAV), which can selectively induce cell death in tumor cells and transformed cells, but not in normal cells. Apoptin induces cell death independently of the tumor suppressor p53 and can be stimulated by Bcl-2. Recently, it has been identified that CDK2 phosphorylates apoptin’s C-terminal Thr-108, which is crucially required for apoptin-induced cell death.PTD4-apoptin protein harbors penetrating membrane capacity and tumor-selective cell death activity. Dacarbazine (DTIC) is mainstay of treatment for malignant melanoma. In this study, we investigated the cytotoxic effect of PTD4-apoptin protein alone and combined with dacarbazine on mouse B16-F1 and human A875 and SK-MEL-5 melanoma cells in vitro and by means of an mouse B16-F1 melanoma model in vivo, and preliminarily investigated the mechanism of the combination.B16-F1, A875 and SK-MEL-5 cells were cultured in PTD4-apoptin protein or/and DTIC in vitro. Cell viability was analyzed by MTT assay and the nature of drug interaction was analyzed by the coefficient of drug interaction (CDI). B16-F1 cells were incubated with PTD4-apoptin protein or/and DTIC, cell morphological changes, flow cytometric analysis and the expression of Bcl-2 and CDK2 were analyzed. For in-vivo therapeutic experiments, C57BL/6 mice bearing B16-F1 melanoma were treated with PTD4-apoptin protein or/and DTIC in different strategies. The inhibitory effect on the tumor growth was observed and the apoptosis were investigated. The experiments for determining possible side effects by PTD4-apoptin and/or dacarbazine were carried out in tumor-free C57BL/6 mice. The blood parameters were measured and compared with normal values.In vitro, PTD4-apoptin protein alone could inhibit the growth of B16-F1, A875 and SK-MEL-5 cells in a dose-dependent manner, and it combined with DTIC produced synergistic inhibitory effect on the growth of the three cell lines (CDI<1). The DAPI staining and the Annexin-V/PI staining assay indicated that PTD4-apoptin protein combined with dacarbazine achieved the most prominent apoptotic-inducing effect in B16-F1 melanoma-derived tumor cells. The level of CDK2 and Bcl-2 expression in B16-F1 cells increased after incubation with 2-drug combination.In vivo, PTD4-apoptin protein could penetrate into the tumors via the epidermal tissue of the mice and effectively inhibited the growth of tumor in C57BL/6 mice. The inhibitory effect were enhanced in "half dose" or "full dose" combination treatment group, and the apoptotic cells in combined treatment group were more than that in either single treatment group. Analyzing the bone marrow suppression of dacarbazine and/or PTD4-apoptin protein in C57BL/6 tumor-free mice, the results showed that the dacarbazine treatment alone revealed bone-marrow suppression. PTD4-apoptin protein alone and "half-dose" drug combination both had no detectable bone marrow suppression. In contrast, "full dose" drug combination had, but less in comparison to the treatment of dacarbazine alone. One of the potential mechanisms may be that the apoptosis-induced activity of apoptin was enhanced by high CDK-2 and Bcl-2 expression and the high Bcl-2 expression exerting resistance to chemotherapy in melanoma was avoided by changing the properties of Bcl-2 from an anti-apoptotic to a proapoptotic molecule.In conclusion, PTD4-apoptin protein combined with the chemotherapy drug dacarbazine showed synergistic inhibitory effects on the growth of mouse melanoma B16-F1 cells in vitro and in vivo, and of human melanoma A875 and SK-MEL-5 cells in vitro. As important, PTD4-apoptin was shown to enhance the anti-tumor activity induced by dacarbazine without side effects on bone marrow suppression. One of the potential mechanisms may be related with Bcl-2 and CDK2. Our preclinical observations indicate that a combinatorial therapy based on PTD4-apoptin protein and dacarbazine is a promising novel anti-cancer strategy.更多还原