亚牛磺酸与α-酮戊二酸依赖的双加氧酶系作用与胶质瘤发生的关系研究

It is estimated that there are over 180 amino acids in the organisms. Only 20 amino acids involve in protein synthesis. Relationship between those non-protein amino acids and tumors is studied sporadically. Not only the metabolism of many amino acids changes in the tumor cells, but also the amino acids themselves can affect and/or regulate the biological traits of the tumor cells. Using metabolomic platform, we have found that hypotaurine was increased in rat glioma tissues. Computer simulation indicated that hypotaurine and α-ketoglutarate occupied the same position in prolyl hydroxylase 2 (PHD2). In this study, the mechanism of hypotaurine in the development of glioma will be explored using human glioma samples of different grades and U251 cell line.Effects of hypotaurine on the malignant biological characteristics of U251 will also be evaluated. This study intends to confirm that hypotaurine can inhibit α-ketoglutarate-dependent dioxygenases including PHD2, histone demethylases and the TET family of 5-methlycytosine (5mC) hydroxylases by competing off α-ketoglutarate. Using PHD2 as an example, a prokaryotic or eukaryotic system will be employed to express the recombinant human PHD2. Its hydroxylation ability will be explored using a synthesized peptide which is in the oxygen-dependent degradation domain of hypoxia-inducible factor-1α(HIF1α) under the presence of different concentrations of hypotaurine. Simultaneously, the recombinant PHD2 will also be utilized to calculate the Km and Ki of hypotaurine in vitro. Furthermore, by disturbing the intercellular hypotaurine concentrations, some malignant traits such as invasion ability, sensitivities to the chemical therapy agents, apoptosis abilities of U251 cells and tumor formation rates, survival time of the rats with tumors will also be studied. The success of this study will provide a new target to aid tumor therapy through intervention of non-protein amino acid metabolism.

生物体已知的180余种氨基酸，绝大多数不参与蛋白质合成。非蛋白质氨基酸与肿瘤的关系研究相对较少。申请人已利用代谢组学技术发现大鼠胶质瘤组织内亚牛磺酸含量显著升高,分子模拟提示亚牛磺酸与α-酮戊二酸在PHD2上有共同结合位点。本研究将利用各级别患者组织和U251细胞系,探究亚牛磺酸在胶质瘤发生中的作用及其与细胞恶性表型的关系。研究力图证实亚牛磺酸是通过与α-酮戊二酸竞争性结合方式，抑制包括PHD2、组蛋白去甲基化酶以及5-甲基胞嘧啶羟化酶等α-酮戊二酸依赖的双加氧酶系而促进肿瘤发生。以PHD2为例，利用表达的重组蛋白，分析亚牛磺酸对PHD2体外羟基化HIF-1α氧依赖的降解结构域脯氨酸残基的抑制效应及相应Km、Ki值，分析改变细胞内亚牛磺酸量对U251细胞侵袭性、常用化疗药敏感性、凋亡能力及动物成瘤率和生存期及其他生物学特性影响，为通过干预非蛋白质氨基酸代谢辅助胶质瘤治疗提供靶点。

本课题组的前期工作中曾发现胶质瘤患者肿瘤组织中亚牛磺酸（羟基牛磺酸）含量明显高于对照组织。在本基金的资助下，计划回答如下问题（1）揭示人胶质瘤组织亚牛磺酸含量与肿瘤WHO分级之间相关性；（2）证实亚牛磺酸能够通过与α-酮戊二酸（2-KG）竞争，抑制某些α-酮戊二酸依赖的双加氧酶系（2-KDDs）的活性；（3）利用动物和细胞实验验证，干预亚牛磺酸代谢能够影响胶质瘤的增殖和部分恶性生物学行为特征，为通过氨基酸代谢干预辅助治疗胶质瘤提供新靶点和依据。目前经过对13例WHO II级、9例III级，8例IV 级和18例对照样本的毛细管电泳-质谱（CE-MS）分析发现，肿瘤组织内亚牛磺酸的含量与胶质瘤的WHO分级之间呈显著正相关性（相关系数=0.56，p<0.05）。另外我们通过分子模拟计算显示，亚牛磺酸的确能够与2-KG竞争性结合一些2-KDDs。体外试验显示，亚牛磺酸能够导致某些2-KDDs酶系的催化活性受到抑制，对促进胶质瘤的发生有一定贡献。提高胶质瘤细胞内亚牛磺酸的含量，还可促进细胞增值和侵袭，改变细胞周期。同时利用细胞试验我们还发现，在胶质瘤细胞培养体系内加入亚牛磺酸的细胞内氧化产物-牛磺酸后，可导致肿瘤细胞的增殖受到抑制。同时还能降低细胞内亚牛磺酸水平。利用裸鼠皮下接种人源胶质瘤细胞系后，分别给予含有牛磺酸和不含有牛磺酸的水饲喂荷瘤裸鼠发现，饮用含有牛磺酸饮水的荷瘤裸鼠肿瘤的生长速度明显小于饮用不含牛磺酸水的荷瘤裸鼠皮下肿瘤的大小，说明通过牛磺酸干预细胞内亚牛磺酸的合成可以影响胶质瘤的生长。相关研究成果已经申请专利（专利受理号201410778857.6）。课题实际获批经费16万元整，已经全部按照计划使用完毕。本课题目前已经发表SCI论文2篇，中文论文1篇，投出在审SCI论文1篇。还有1篇论文在整理中。培养毕业博士研究生1名。