ABCG2调控的氧化应激对结直肠癌恶性生物学行为的影响及其机制研究

The oxidative stress plays an essential role in carcinogenesis and progression of colorectal cancer through many mechanisms, in which NF-κB signaling pathway is particularly involved. ABCG2 is an ATP-binding cassette (ABC) transporter. Previously, many studies in colon caner have focused on its relevance to multidrug resistance, however, other functions of ABCG2 remains largely unexplored . In this regard, we for the first time demonstrated that the ABCG2 is capable of protecting HEK293 cells from ROS-mediated cell damage and death. Based on these novel findings, we investigated the roles and molecular mechanisms of ABCG2-mediated protection against ROS-induced damage and signaling pathway in HEK293 cells. This analysis revealed that ABCG2 protect cells from ROS-induced cell damage by inhibiting ROS activation and enhancing antioxidant capacity of the cells which are in oxidative stress conditions. Furthormore, it also inhibits inflammatory reactions by reducing ROS-induced expression of inflammatory genes (IL-8 and GRO) and decreasing ROS-induced IL-8 cytokine secretion from cells. In addition, the NF-κB signaling pathway may be response for these effects. Our preliminary data in colon cancer cell line consistently confirmed that ABCG2 could reduce the intracelluar ROS induced by H2O2, however, the underlying mechanisms require further investigation. In normal circumstances, ABCG2 protect GI epithelium cells from toxins and loss of ABCG2 in local intestinal tract might lead to the carcinogenesis of colorectal cancer. Since ABCG2 and oxidative stress is cloesly related to bilogical characteristics of colorectal cancer, we hypothesize that ABCG2 may reduce oxdative stress and inhibit the malignant behaviour of colorectal cancer. To this end, the current proopsal is to study the biological role of ABCG2 in colorectal cancer by using clinical samples, cell and animal models, in which the possile involved signaling pathway such as NF-κB will be explored. The research may likely provide future targets for colorectal cancer therapy.

氧化应激在结直肠癌的发生发展中起重要作用，而NF-κB是其中涉及的重要信号通路之一。ABCG2是ABC基因家族编码的细胞膜转运蛋白，目前其在结直肠癌中的研究集中在耐药方面，对其他方面的研究甚少。申请人前期研究运用工具细胞HEK293首次发现ABCG2具有抗氧化应激作用，并能减少炎症因子的表达和分泌（IL-8,GRO），ABCG2可能通过NF-kB信号通路发挥上述作用。进一步在肠癌细胞的研究中亦发现ABCG2减少H2O2诱导的细胞内ROS产生，但机制有待研究。ABCG2的缺乏使多种有害因素在肠道累积，导致肠癌的发生，同时结合申请者首次发现的ABCG2的抗氧化应激功能，故推测ABCG2通过减少氧化应激影响结肠癌的发生发展。本研究将从人体标本、细胞及动物实验多层次探索ABCG2调控的氧化应激在结直肠癌中的作用，并研究可能涉及的NF-kB信号通路，为结直肠癌靶向治疗提供有价值的靶点和理论依据。

背景：氧化应激在结直肠癌的发生发展中起重要作用，而NF-κB是其中涉及的重要信号通路之一。ABCG2是ABC基因家族编码的细胞膜转运蛋白，目前其在CRC中的研究集中在多药耐药方面，其他方面研究甚少。本项目从临床标本、细胞及动物实验多层次探索ABCG2调控的氧化应激在CRC中的作用及可能机制。方法：（1）临床病例研究：收集南京大学医学院附属鼓楼医院正常肠道、结直肠腺瘤、结直肠癌组织、血清标本。免疫组化法检测标本中ABCG2和NF-κB表达情况。相应试剂盒检测不同标本中氧化应激相关指标： ROS、MDA、8-OHdG；抗氧化应激相关指标：SOD、GSH的水平。ELISA检测炎症因子：TNF-α、IL-8的分泌情况。（2）体内外实验：筛选各CRC细胞系，获得高表达ABCG2的HT-29细胞作为后续研究对象。shRNA慢病毒干扰系统下调HT-29细胞内ABCCG2的表达。将不同ABCG2表达水平的HT-29裸鼠皮下成瘤，进行后续体内研究。相关试剂盒、qRT-PCR、 ELISA等检测上述相关氧化应激指标及炎症反应指标，Western Blot检测ABCG2及p-IκB-α表达水平。划痕实验及Transwell侵袭实验检测细胞转移及侵袭能力。结果：人CRC标本中ABCG2及NF-κB均高表达，且两者具有相关性。ABCG2表达与CRC的分化程度及淋巴结转移相关，且ABCG2高表达与患者预后差成正相关。CRC患者中的ROS、MDA、8-OHdG、TNF-α、IL-8表达水平均明显升高，而SOD及GSH水平下降明显。体外研究发现ABCG2能够下调氧化应激条件下细胞内ROS水平；上调GSH水平；下调炎症因子IL-8，MCP-1和 TNF-α的表达及分泌（IL-8），同时亦能抑制NF-κB的活化。ABCG2能够促进氧化应激条件下结肠癌细胞的迁移及侵袭。体内实验发现ABCG2敲除的裸鼠移植瘤体积明显小于对照组，ABCG2的缺失导致裸鼠瘤体内ROS、MDA水平及炎症因子IL-8、TNF-α水平明显升高；GSH水平明显降低。ABCG2缺失导致NF-κB显著活化。结论：ABCG2可能是通过NF-κB 信号通路发挥抗氧化应激作用，进而促进CRC发展。本研究创新性地以氧化应激为切入点，发现ABCG2在结直肠癌中的抗氧化应激的新功能并研究其可能机制，为结直肠癌的靶向治疗提供新的理论依据。