新型高温修饰柠檬果胶通过上调ROS通路促进人结肠癌细胞凋亡的机制研究

Colorectal cancer, with high morbidity and mortality, is one of the common human malignancies. Citrus pectin (CP), exists in the fruit peel of citrus, is a type of water-soluble polysaccharides. Some studies showed that CP could inhibit the proliferation of human colon cancer cells. However, CP is hard to be absorbed by human for its large molecular weight, limiting the development and application of CP. With high-temperature modification which degrades CP into samller fragments, CP possesses new biochemical and functional properties. To date, there has been few reports about the anti-tumor activities and mechanisms of high temperature modified citrus pectin (HTCP). Our previous results demostrated that HTCP had significantly anti-proliferative effect on human colorectal cancer cells, and induced apoptosis by up-regulating intracellular reacitive oxygen species (ROS). We proposed a hypothesis that GRIM-19, an important regulator of mitochondrial electron transfer chain (the respiratory chain), and/or intracellular enzymatic antioxidizing agents might be the targets of HTCP. In this project, we are going to construct GRIM-19 overexpression and invention models at human colorectal cancer cell lines. We intend to explore the functions of GRIM-19 and intracellular antioxidase through up-rerulating ROS pathway by HTCP in melecular, cellular and overall level. We will determine the influence of intracellular redox balance by HTCP, and reveal the apoptosis mechanism of HTCP. This project will provide information for exploitation HTCP as a functional food or drug with low toxicity and high specificity for human colorectal cancer therapy.

结肠癌是人类常见的恶性肿瘤之一，发病率和死亡率较高。柠檬果胶（CP）是一种广泛存在于柠檬果皮中的水溶性多糖，能抑制结肠癌细胞增殖。由于分子量大，难以被人体直接吸收，限制了人们对CP的进一步应用。高温修饰法能降低CP分子量，并获得新的生物活性。目前国际上对高温修饰柠檬果胶（HTCP）的抗肿瘤活性及机制研究较少。申请人的前期工作表明HTCP能显著抑制人结肠癌细胞增殖，并能通过上调细胞内活性氧（ROS）水平促进人结肠癌细胞凋亡。我们推测线粒体呼吸链调控因子GRIM-19和/或细胞内的抗氧化酶可能是HTCP的作用靶点。本项目拟建立人结肠癌细胞GRIM-19过表达和干扰模型，从分子、细胞和动物水平系统探讨GRIM-19及抗氧化酶在HTCP上调ROS通路中的作用，明确HTCP对细胞内氧化还原环境的影响，阐明HTCP促进人结肠癌细胞凋亡的机制，为寻找以果胶为原料的低毒性、高特异性抗结肠癌药物提供新思路。

结肠癌是人类高发的恶性肿瘤，死亡率位居全球第四。柠檬果胶（CP）是一种广泛存在于柠檬果皮中的水溶性多糖，能抑制结肠癌细胞增殖。由于分子量大，难以被人体直接吸收，限制了人们对CP的进一步应用。高温修饰法能降低CP分子量，获得新的生物活性，但具体作用及机制不清楚。申请人在前期工作中证实高温修饰柠檬果胶（HTCP）能显著抑制人结肠癌细胞增殖，并能通过上调细胞内活性氧（ROS）水平促进人结肠癌细胞凋亡，并推测线粒体呼吸链调控因子GRIM-19可能是HTCP的作用靶点。因此，本项目通过建立人结肠癌细胞GRIM-19过表达和干扰模型，明确GRIM-19 与HTCP 促进人结肠癌细胞凋亡的关系及GRIM-19 在此过程中所起的作用。并利用人源化裸鼠模型，进一步验证体外实验结果，从分子、细胞及整体水平深入探究HTCP促进人结肠癌细胞凋亡的分子机制。研究结果如下：首先，我们通过建立GRIM-19过表达和干扰模型及功能实验，证实GRIM-19在人结肠癌细胞中可作为潜在的抑癌基因发挥作用。其次，研究首次明确GRIM-19在结肠组织的定位，即在正常结肠组织中主要定位于细胞质，少量定位于细胞核，而在结肠癌组织中仅定位于细胞质。再次，研究结果表明GRIM-19的低表达与肿瘤的恶性分化、淋巴浸润、脉管浸润和器官转移密切相关，且具有更高的复发/转移率及较短的术后生存期，提示我们GRIM-19可能成为一种潜在的结肠癌预后标志物。最后，我们发现过表达GRIM-19能增强细胞对HTCP的敏感性，表现为细胞增殖率降低、ROS水平升高及细胞凋亡率提高。与之对应的，在人结肠癌细胞中干涉GRIM-19表达后，HTCP抑制细胞增殖、诱导细胞发生G2/M期周期阻滞、上调细胞内ROS水平及诱导细胞凋亡的能力，均受到抑制甚至消除。表明GRIM-19在HTCP促进人结肠癌细胞凋亡过程中作为关键靶点发挥作用。本项目的研究结果为全面了解和考察果胶的生物学活性及功能奠定基础，为寻找以GRIM-19为作用靶点的抗癌药物提供新的数据参考，为开发以GRIM-19为结肠癌预后标志物提供实验证据，为寻找以果胶为原料的低毒性、高特异性、具有抗结肠癌效果的功能性食品或药物提供新思路和数据支持。