新型糖酵解抑制剂3-BrOP逆转胃肠肿瘤细胞多药耐药性的作用及其机制

Cancer stem cell is an important factor in causing multi-drug resistance in gastrointestinal tumor cells, it is of great significance to reverse its drug resistance. Our previous study found that cancer cells especially cancer stem cells are highly active in and dependent on aerobic glycolysis for metabolism than normal tissue. Based on this finding we developed a novel highly selective glycolytic inhibitor 3-BrOP to targets cancer cells, and synergistically sensitize anti-cancer effects of chemotherapeutic agents (Stem Cells. 2013). However, which steps of glycolysis 3-BrOP target and its regulatory mechanism are not yet fully elucidated. We found that the drug resistant colorectal cancer cell lines are sensitive to 3-BrOP, and have high expression of GAPDH, furthermore 3-BrOP can inhibit purified GAPDH in vitro. Thus, we speculate that 3-BrOP targets GAPDH, resulting in ATP depletion in glycolysis and finally energy crisis in cancer cells bearing deficient mitochondrial respiratory function, which might render cancer cells incapable of repairing DNA damage induced by chemotherapeutic agents, and finally lead to necrosis of the cancer cells. This study is to further verify how 3-BrOP impairs DNA repair and the site and way of enzyme modification of GAPDH, and verify its efficiency in inhibiting or killing the tumor in inoculated NOD/SCID mice when combined with chemotherapeutic agents, and explore its possibility as a promising agent in clinics.

肿瘤干细胞是引起胃肠肿瘤多药耐药的重要原因，逆转其耐药很具临床意义。我们研究发现，肿瘤细胞尤其是肿瘤干细胞有氧糖酵解代谢明显活跃，由此研发了高选择性的新型糖酵解抑制剂3-BrOP，可高效抑制细胞内ATP产生，逆转常规化疗药物BCNU的耐药性(Stem Cells.2013),但3-BrOP逆转肿瘤多药耐药的具体机制及其作用靶点尚未明确。我们发现肠癌耐药细胞系对3-BrOP敏感，且高表达糖酵解代谢酶- - 甘油三磷酸脱氢酶（GAPDH），此外3-BrOP可抑制GAPDH纯化蛋白，由此我们推测3-BrOP作用于GAPDH，抑制糖酵解通路ATP的产生，引起细胞内能量危机，导致常规化疗药物引起的DNA损伤无法正常修复，从而杀灭肿瘤细胞。本研究将阐明3-BrOP影响DNA修复的机制及其抑制GAPDH酶的修饰位点和方式，并验证其协同化疗药物有效抑制或杀灭荷瘤小鼠体内肿瘤的作用，探索其作为临床用药的前景。

临床中普遍存在胃肠肿瘤对化疗药物耐药的现象。如何发现引起胃肠肿瘤细胞多药耐药的重要原因，并逆转其耐药有重要的临床意义。我们研究发现，肿瘤细胞有氧糖酵解代谢较正常细胞明显活跃，而我们研制的新型糖酵解抑制剂，可高效抑制细胞内ATP产生，逆转常规化疗药物的耐药性，提高化疗药物的疗效。本研究中我们联合糖酵解抑制剂3-BrPA、3-BrOP、PEITC及胃肠肿瘤常用化疗药物奥沙利铂、顺铂、5-FU、阿霉素、MMC可协同产生更强的杀灭肿瘤细胞作用， 逆转胃肠肿瘤细胞系MGC803、BGC823、SGC7901、HGC27、HCT116的多药耐药性。本研究也对肿瘤耐药及糖酵解抑制剂逆转耐药的机制进行了初步的探索，我们发现耐药细胞系中多药耐药蛋白MDR1、MRP1、ABCG2均有明显的高表达，其药泵作用造成细胞内化疗药物水平下降，化疗药物的杀细胞效应下降。而糖酵解抑制剂逆转耐药的关键机制为高效抑制肿瘤细胞内ATP及还原型谷胱甘肽（GSH）的生成，同时可提高肿瘤细胞内的铂离子水平，增强铂类药物的杀肿瘤细胞效应。糖酵解抑制剂在细胞内有多个靶点及发挥多种功能，其中甘油三磷酸脱氢酶（GAPDH）是3-BrOP的有效关键作用靶点。我们在结肠癌细胞系及结肠癌患者手术标本的原发癌灶、转移癌灶均可检测到高表达的GAPDH。本研究验证了研究假设：糖酵解抑制剂靶向GAPDH，抑制糖酵解通路ATP，引起肿瘤细胞内能量危机，导致常规化疗药物引起的DNA损伤无法正常修复，从而杀灭肿瘤细胞。本研究拟进一步探索其作为临床用药的前景。