背景:己糖激酶I(HK1)在多种恶性肿瘤中高表达,调节癌细胞中的糖酵解途径,因此被认为是癌症治疗的有希望的分子靶标之一。尽管如此,针对HK1的特异性抑制剂的开发仍然难以捉摸。
目的:本研究旨在阐明冬凌草甲素抑制膀胱癌细胞增殖和免疫逃逸的机制。特别是通过抑制HK1。
方法:为了研究冬凌草甲素与HK1的半胱氨酸直接结合并抑制膀胱癌生长的机制,本研究采用了多种方法。这些包括人类蛋白质组微阵列,链霉亲和素-琼脂糖亲和试验,生物层干涉法(BLI)检测分析,质谱,细胞热转移测定,细胞外酸化率测量,和异种移植小鼠模型。
结果:正如我们目前的研究结果表明,冬凌草甲素与Cys-813形成共价键,位于HK1的葡萄糖结合域附近。这抑制了HK1的酶活性,导致糖酵解的有效减少,通过人膀胱癌细胞凋亡引发细胞死亡。重要的是,冬凌草甲素还抑制乳酸诱导的膀胱癌PD-L1表达。此外,冬凌草甲素与PD-L1抑制剂配对可增强CD8+T细胞对膀胱癌的细胞毒性.
结论:这项研究强烈表明冬凌草甲素是HK1的共价抑制剂。此外,这表明HK1的功能性半胱氨酸残基可以作为选择性抑制的可行靶标。因此,冬凌草甲素表现出通过代谢免疫调节靶向潜在治疗靶标HK1的抗癌剂进化的巨大潜力。
BACKGROUND: Hexokinase I (HK1) is highly expressed in a variety of malignancies, regulates glycolytic pathway in cancer cells, and thus considered to be one of the promising molecular targets for cancer therapy. Nonetheless, the development of a specific inhibitor against HK1 remains elusive.
OBJECTIVE: This study aims to elucidate the mechanism by which oridonin inhibits the proliferation and immune evasion of bladder cancer cells, specifically through the suppression of HK1.
METHODS: To examine the mechanisms by which oridonin directly binds to cysteines of HK1 and inhibits bladder cancer growth, this study utilized a variety of methods. These included the Human Proteome Microarray, Streptavidin-agarose affinity assay, Biolayer Interferometry (BLI) ainding analysis, Mass Spectrometry, Cellular Thermal Shift Assay, Extracellular Acidification Rate measurement, and Xenotransplant mouse models.
RESULTS: As indicated by our current findings, oridonin forms a covalent bond with Cys-813, located adjacently to glucose-binding domain of HK1. This suppresses the enzymatic activity of HK1, leading to an effective reduction of glycolysis, which triggers cell death via apoptosis in cells derived from human bladder cancer. Significantly, oridonin also inhibits lactate-induced PD-L1 expression in bladder cancer. Furthermore, pairing oridonin with a PD-L1 inhibitor amplifies the cytotoxicity of CD8+ T cells against bladder cancer.
CONCLUSIONS: This research strongly suggests that oridonin serves as a covalent inhibitor of HK1. Moreover, it indicates that functional cysteine residue of HK1 could operate as viable targets for selective inhibition. Consequently, oridonin exhibits substantial potential for the evolution of anti-cancer agents targeting the potential therapeutic target HK1 via metabolism immunomodulation.