芹菜素与PKM2相互作用抑制有氧糖酵解发挥抗肿瘤效应的分子机制

Apigenin is a flavonoid widely existed in vegetables and fruits. It has anti-tumor activity, low toxicity and no mutagenicity and can be used as a potential drug for prevention and treatment of cancer. Aerobic glycolysis is the main way of tumor energy metabolism. M2 pyruvate kinase (PKM2) is a key speed limiting enzyme. It is highly expressed in tumor cells, which can regulate the rate of glycolysis. The applicant found that apigenin selectively combined with PKM2 and inhibited its activity to block the proliferation inhibition of colon cancer cells, but the location of PKM2 binding to apigenin is not clear. Based on this study, we will explore the sites of PKM2 binding to apigenin and the molecular mechanism of apigenin exerting anti-tumor effect from the perspective of remodeling metabolism. The contents include: (1) using SILAC-MS and CETSA technology to verify the binding ability of apigenin to PKM2. (2) using genetic engineering to construct different PKM2 truncated bodies, and identify amino acid sites of apigenin interacting with target protein PKM2. (3) the antitumor effect of apigenin with PKM2 interaction was demonstrated by using PKM2 overexpressed nude mice model to inhibit the glycolysis of colon cancer cells in vivo.

芹菜素是广泛存在于蔬菜和水果中的黄酮类化合物具有抗肿瘤活性，且具低毒、无诱变性等特点，可作为预防和治疗肿瘤的潜在药物。有氧糖酵解是肿瘤能量代谢的主要方式，而M2型丙酮酸激酶(PKM2)是其中的关键限速酶，在肿瘤细胞中特异性高表达，可调控糖酵解的速率。申请人前期发现，芹菜素能选择性结合PKM2，并抑制其活性来阻断结肠癌细胞的有氧糖酵解诱导其增殖抑制，但芹菜素与PKM2结合的位点尚不明确。本课题拟在此基础上，探明芹菜素与PKM2相互作用的结合位点，从重塑肿瘤代谢的角度探明芹菜素发挥抗肿瘤效应的分子机制。内容包括: (1)采用SILAC-MS和CETSA等技术验证芹菜素与PKM2的结合能力；(2)采用基因工程手段构建不同PKM2截短体筛选并确定芹菜素与靶点蛋白PKM2相互作用的氨基酸位点。(3)采用PKM2过表达的裸鼠成瘤模型证明芹菜素在体内能以PKM2为靶点抑制结肠癌细胞糖酵解发挥抗肿瘤效应。

芹菜素因其低毒、具抗肿瘤活性且无致突变性等特点可以作为一种潜在的肿瘤防治药物。然而，目前关于芹菜素发挥抗肿瘤效应的分子靶点尚不清楚。因此，本项目主要探讨了芹菜素发挥抗肿瘤效应的细胞内靶点及分子机制。本研究发现芹菜素抑制结肠癌细胞的增殖与PKM2呈正相关，通过Pull-down实验结合银染色寻找到一条差异蛋白条带是芹菜素可能结合的靶点蛋白。采用MALDI-TOF/TOF质谱分析结合uniprot数据库鉴定该差异蛋白条带为PKM2蛋白，证明PKM2可能与芹菜素特异性相互结合。进一步通过生物层干涉技术证实芹菜素与PKM2存在中等强度的结合，PKM2是芹菜素的靶点蛋白。根据PKM2蛋白具有A1、B、A2、C四个结构域。研究报道PKM2在癌细胞中主要以二聚体形式存在且C结构域介导二聚体形成四聚体。因此，我们将PKM2蛋白构建成包含C结构域（PKM2-4）和不包含C结构域（PKM2-△4）的两个截短体蛋白，通过细胞活性实验、细胞克隆形成实验和细胞凋亡实验结果发现过表达PKM2-4截短体能够增强芹菜素对结肠癌细胞的抑制作用，表明PKM2-4是芹菜素与PKM2结合的有效结构域。在此基础上，进一步证明赖氨酸433位点(K433)是芹菜素选择性结合PKM2的关键氨基酸位点。芹菜素能够直接与PKM2的K433氨基酸位点结合，破坏肿瘤细胞内PKM2四聚体到二聚体的形成，进而在体内、外发挥抗结肠癌效应。同时，体内实验还表明芹菜素长期干预对模型组裸鼠能够显著抑制肿瘤生长，对正常组裸鼠的体重和生理活动无不良影响，揭示芹菜素是治疗结肠癌的一种潜在安全、有效药物。本项目明确了芹菜素发挥抗肿瘤效应的靶点蛋白及其相互作用的氨基酸位点，有望为芹菜素靶向抗肿瘤药物的研发提供一定的实验基础和理论依据。