晚期糖化终末产物AGEs促进细胞增殖的机理研究

Epidemiological studies indicate that patients with diabetes mellitus have an increased risk of several cancers including liver and colorectal cancer. Diabetic patients have increased level of advanced glycation end products (AGEs) which might play a role in tumorigenesis. However,the underlying mechanism remains unclear. Our preliminary work showed that AGEs promoted liver and colon cancer cell proliferation. Western blot analysis indicated that the expression of ChREBP, a glucose responsive transcription factor regulating tumor cell metabolism and proliferation, is increased in AGE-treated cancer cells. Based on published work and our study, we aim to find out the molecular mechanism by which AGEs induce ChREBP expression and the role of ChREBP in tumor cell proliferation promoted by AGEs. We have four specific aims: 1) study whether AGEs act through binding its receptor RAGE; 2) study whether reactive oxygen species are involved in ChREBP upregulation by AGEs; 3) locate the promoter element and transcription factor responsible for ChREBP upregulation by AGEs; 4) determine whether ChREBP mediates increased tumor cell proliferation induced by AGEs. Our proposed work will not only help understanding the molecular mechanism by which diabetics have an increased risk of several cancers, but also may provide new strategies for preventing and treating tumors in diabetic patients.

流行病学研究表明，与非糖尿病患者相比，糖尿病患者某些肿瘤如肝癌和结肠癌发生更常见。已知糖尿病患者体内糖化终末产物（AGEs）浓度上升并可能参与肿瘤发生，但机制不明。课题组前期研究证示，AGEs可促进HepG2和HCT116肿瘤细胞的增殖，且AGEs可显著上调细胞中调节代谢和增殖的转录因子ChREBP的表达。本项目将进一步探讨肿瘤细胞中AGEs诱导ChREBP转录的机制以及ChREBP是否在AGEs促进细胞增殖中发挥重要作用。我们进行四方面研究 1）研究AGEs是否通过结合受体RAGE而诱导ChREBP表达；2）研究活性氧ROS是否参与调节AGEs诱导的ChREBP表达；3）定位受AGEs调控的ChREBP启动子区域和鉴定相应的转录因子；4）研究AGEs促进细胞增殖是否依赖于ChREBP。本研究将深化对糖尿病患者肿瘤发病机制的理解，并为延缓糖尿病患者肿瘤发生发展开辟新的途径。

流行病学研究表明，与非糖尿病患者人群相比，糖尿病患者罹患某些肿瘤如肝癌和结肠癌发生更为常见。已知糖尿病患者体内糖化终末产物（AGEs）浓度上升并可能参与肿瘤发生，但是具体机制不明。本文将探讨AGEs促进肿瘤细胞增殖的具体分子机制。.目的：明确AGEs是否可促进结肠癌HCT116和肝癌HepG2两种肿瘤细胞的增殖；明确调节代谢和增殖的转录因子碳水化合物反应元件结合蛋白（ChREBP）是否在AGEs促进肿瘤细胞增殖中发挥重要作用；明确AGEs是否通过结合受体RAGE而诱导ChREBP表达；明确活性氧ROS是否参与调节AGEs诱导的ChREBP表达和肿瘤细胞增殖。.方法：运用细胞计数和MTS法检测AGEs促肿瘤细胞增殖能力；采用Western blot、核质分离、免疫荧光和荧光定量PCR等方法检测AGEs处理的肿瘤细胞中ChREBP表达；采用封闭型抗RAGE抗体阻断RAGE检测AGEs是否通过结合受体RAGE而诱导ChREBP表达和肿瘤细胞增殖；采用siRNA技术阻断ChREBP检测AGEs诱导的肿瘤细胞增殖是否依赖于ChREBP；运用ROS捕获剂孵育细胞，通过流式细胞仪和荧光显微镜检测AGEs刺激后肿瘤细胞内ROS水平，同时用N-乙酰半胱氨酸（NAC）干预氧化应激状态，观察是否影响AGEs诱导的活性氧ROS 增加、ChREBP表达增加及细胞增殖。.结果：AGEs可促进结肠癌HCT116和肝癌HepG2两种肿瘤细胞的增殖；AGEs处理的肿瘤细胞中转录因子ChREBP表达都明显增加；封闭型抗RAGE抗体可以降低AGEs所诱导的ChREBP表达增加和肿瘤细胞增殖；ChREBP被抑制后，AGEs诱导的细胞增殖被明显抑制；AGEs刺激后肿瘤细胞中ROS的生成增加；上述细胞经抗氧化剂NAC处理后，AGEs刺激所致ROS 生成增加、ChREBP表达增加和促细胞增殖能力都被明显抑制。.结论：AGEs-RAGE途径通过激活氧化应激反应来上调ChREBP的表达，从而增加结肠癌和肝癌细胞的增殖，提示AGEs-RAGE-ROS- ChREBP通路在肿瘤细胞增殖中具有重要作用。本研究不仅有助于深化对糖尿病患者易患肿瘤发病机制的理解，并为延缓糖尿病合并肿瘤的发生及进展开辟新的思路与途径。