2-AAPA诱导硫醇氧化应激逆转结肠癌5-FU耐药的机制研究

5-fluorouracil (5-FU) is a conventional chemotherapeutic drug for colon cancer. Drug resistance is one of the main reasons that restrict the efficacy of chemotherapy for colon cancer. Therefore, it is particularly important to find strategies to reverse the chemoresistance of colon cancer. Our previous studies have found that 2-AAPA, as an irreversible competitive inhibitor of glutathione reductase (GR), can accumulate GSSG in tumor cells，resulting in a significant decrease in the ratio of GSH/GSSG. 2-AAPA induces thiol oxidative stress, reverses the EMT of drug-resistant cells and increases the apoptosis of drug-resistant cells, thus reversing the 5-FU resistance of colon cancer. Therefore, we speculate that 2-AAPA induces thiol oxidative stress to reverse 5-FU resistance in colon cancer. On this basis, the effect of 2-AAPA-induced thiol oxidative stress on reversing 5-FU resistance of colon cancer and its regulatory mechanism will be studied by flow cytometry and liquid chromatography-mass spectrometry. By using xenograft model and immunohistochemistry, the effects of 2-AAPA on the therapeutic efficacy of 5-FU resistance of colon cancer will be investigated in vivo. Through this study, our finding will provide a new therapeutic target and approach for drug resistance reversal in colon cancer, laying a solid theoretical foundation for clinical application of 2-AAPA.

5-氟尿嘧啶(5-FU)是结肠癌常规化疗药物，而耐药性是制约结肠癌化疗效果的主要原因之一。因此，寻找逆转结肠癌化疗耐药的策略尤为重要。本课题组前期研究发现2-AAPA作为一个谷胱甘肽还原酶(GR)不可逆的竞争性抑制剂，能够使GSSG在细胞内大量累积，造成GSH/GSSG的比值显著降低，诱导硫醇氧化应激，抑制耐药细胞EMT并促进凋亡，从而逆转结肠癌5-FU耐药。因此，我们推测：2-AAPA诱导硫醇氧化应激逆转结肠癌5-FU耐药。本项目拟在此基础上采用流式细胞术、液质联用等技术来研究2-AAPA诱导硫醇氧化应激在逆转结肠癌5-FU耐药中的作用及其调控机制；采用人结肠癌耐药细胞皮下移植瘤裸鼠模型和免疫组化等实验来研究2-AAPA对结肠癌5-FU耐药体内治疗疗效的影响，探讨2-AAPA逆转结肠癌5-FU耐药的体内调控机制，为2-AAPA的临床转化运用打下坚实的理论基础。

5-氟尿嘧啶(5-Fu)是结肠癌常规化疗药物，而耐药性是制约结肠癌化疗效果的主要原因之一。转谷氨酰胺酶2 (TGM2)是转谷氨酰胺酶家族的一员，可通过阻止肿瘤细胞凋亡而形成肿瘤耐药，但机制不明。近期我们发现谷胱甘肽还原酶抑制剂2-AAPA，使GSSG在细胞内大量累积，造成GSH/GSSG的比值显著降低，从而诱导硫醇氧化应激，增加TGM2的谷胱甘肽化，下调TGM2的蛋白水平，逆转耐药细胞EMT并增加细胞凋亡，从而逆转结肠癌5-Fu耐药。基于此我们提出2-AAPA诱导硫醇氧化应激通过下调TGM2蛋白水平逆转结肠癌5-Fu耐药假说。本课题以细胞、动物和人体肿瘤组织为研究对象，采用Western blot、免疫共沉淀、流式细胞术、液质联用等一系列体内和体外实验，对TGM2进行深入的功能及其通路调控的研究，确立TGM2与5-Fu耐药的调控关系，探明硫醇氧化应激通过下调TGM2的蛋白水平逆转结肠癌5-FU耐药的机理。结果有望深入认识硫醇氧化应激和TGM2在肿瘤耐药过程中的作用，并为逆转结肠癌5-Fu耐药提供新的靶点。