APE/Ref-1参与戒酒硫抗肝癌作用的机制研究

Hepatocellular carcinoma (HCC) is one of the most aggressive neoplasms. Excess Cu facilitates production of reactive oxygen species (ROS), and oxidative stress related cellular disorders, such as cancer. It is well documented that the elevated copper and oxidative stress levels are hallmarks of range of malignancies. Disulfiram (DSF), a drug used to treat alcoholism with low toxicity to human, has attracted much attention as an anti-cancer agent by binding with tumor cellular copper and forming an active complex. In our initial study, inhibitory ability was observed on the proliferation of HCC cells exposed to DSF, not normal hepatocytes whose proliferation was strenghened by copper. Additionally, we also uncovered a synergistic effect of DSF and sorafenib on HCC cells. However, the underlying mechanism is poorly understood. The human apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) is a master regulator of cellular responses to oxidative stresses. Moreover, APE/Ref-1 facilitates the DNA binding activity of several transcription factors via redox-dependent and redox-independent mechanisms. APE/Ref-1 is involved in different stages of carcinogenesis (initiation, promotion and progression) mainly through maintenance of intracellular redox balance and activation of a cell survival signal and repair of damaged DNA lesions. We ever demonstrated for the first time the existence of a functional feedback loop contributing to progression and metastasis of melanoma cells mediated by APE/Ref-1.In addition, elevated expression of APE/Ref-1 compared to normal cells and tissues have been demonstrated in HCC by us. Copper also could induce the expression of APE/Ref-1.Herein, we are going to investigate the role of APE/Ref-1 involved in the anti-HCC treatment with DSF by using HCC cell lines and human HCC xenografts. Moreover, pathologically confirmed HCC specimens were from patients undergoing curative resections for further detection to make sure if possible relationship existed between APE/Ref-1 expression pattern and clinicopathologic features such as poor differentiation, recurrence-free survival and overall survival. As a result, it is of great importance to define a novel role of APE/Ref-1 in HCC as being an important mediating and potentiating molecule, and also provides a basis for further investigations utilizing DSF or other appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment. Additionally, it is possible to support a prognostic role of APE/Ref-1 in the outcome of HCC.

肝癌是最常见的恶性肿瘤之一，Cu以及氧应激在其发生发展过程中有着重要作用。申请人前期研究发现Cu制剂促进肝细胞增殖,而铜螯合剂戒酒硫（DSF）抑制肝癌细胞增殖，并与索拉非尼有协同作用，但具体机制尚未明确。人类脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1(APE/Ref-1)是细胞对氧应激反应的关键性调节因子，通过维持细胞内氧化还原平衡、激活转录因子、增强DNA损伤修复而参与到肿瘤发生发展过程中。前期研究亦证实肝癌细胞中ROS水平升高，肝癌细胞及组织中APE/Ref-1表达显著增强，而Cu制剂能诱导APE/Ref-1表达增强。在此基础上，本课题拟进一步探讨DSF对肝癌的治疗价值，深入研究APE/Ref-1在肝癌发生发展中的作用以及与肝癌分化程度、转移及复发等特征相关性，阐明APE/Ref-1参与DSF抗肝癌作用机制，为肝癌治疗提供科学依据。

原发性肝癌简称肝癌（HCC），是指由肝细胞或肝内胆管上皮细胞发生的恶性肿瘤，死亡率在恶性肿瘤中居第二位。全世界每年平均约有25万人死于肝癌，而我国约占其中的45%。在临床实践中，80%以上的肝癌患者在确诊时已存在肝内播散、远处转移或伴有严重的肝硬化而失去手术机会，甚至是介入等治疗也存在限制，肝癌预后仍然未能得到明显改善，因此进一步研究肝癌发病机制对发现新的治疗靶点、判断预后具有重要意义。铜是常见的一种金属元素，但在肿瘤发生发展过程中有着重要意义，铜和氧应激水平升高已被认为是肿瘤恶性程度的标志。肝癌发展与铜水平升高始终密切相关。金属转录因子MTF-1除调节金属稳态平衡外，还参与细胞对氧应激、缺氧的反应调节，可在转录水平激活氧应激、缺氧诱导的靶基因表达，促进肿瘤细胞增殖、血管生成以及药物、放疗抵抗，此外，其目的基因a-fetoprotein是肝癌特异性标志物，提示MTF-1亦可能参与到肝癌的发生发展过程中。APE / Ref-1在不同类型的肿瘤中表达增强，包括肝癌。铜治疗可诱导MTF-1、APE / Ref-1及其下游靶标表达升高，因此我们假设，MTF-1、APE / Ref-1的可能通过介导铜的作用而参与了肝癌进展。研究结果显示肝癌细胞中MTF-1表达水平显著升高。铜诱导MTF-1激活并伴有肝细胞增殖潜能增强；而铜螯合剂戒酒硫抑制肝癌细胞增殖，但却对肝细胞无明显影响，此外戒酒硫与肝癌靶向治疗药物索拉非尼有协同作用。肝癌细胞和肝癌组织中APE / Ref-1表达明显增强，而胞浆中APE / Ref-1的浓集与肿瘤低分化以及更具侵略性相关。 APE / Ref-1信号通路可以促进细胞增殖，增强其抗凋亡能力，促进细胞转移。综上结果证实APE / Ref-1、MTF-1在肝癌进展中有着重要意义，为我们今后研究肝癌治疗新靶点的可能性及可行性奠定基础。