双氢青蒿素靶向PKM2/LDHA干预糖酵解抑制黑色素瘤干性表型的机制研究

Cutaneous melanoma is one of the most lethal malignancies in the world due to its invasion and metastasis. Melanoma-initiating cells (MIC) energized by glycolysis play a vital role in these processes. To our interest, recent studies have demonstrated that dihydroartemisin (DHA) inhibits glycolysis. Therefore, we have explored the function of DHA on glycolysis of melanoma cells and MIC self-renewal. It is discovered that DHA significantly inhibited the self-renewal of MIC. More importantly, the treatment of DHA suppressed the produce of lactic acid and ATP. Furthermore, by bioinformatics analysis we found tha DHA molecule convergently targeting to the active structures of PKM2 and LDHA proteins. Moreover, we have identified DHA as a repressor of the enzyme activity of PKM2 and LDHA in melanoma and found that depression of PKM2 inhibited MIC self-renewal. . Based on these results, we hypothesize that DHA regulates self-renewal of human melanoma-initiating cells by targeting PKM2/LDHA related glycolysis. In this study, we intend to further explore the possible mechanisms of DHA reducing the stem-like function of melanoma-initiating cells, with the technolegies of spheres culture, FACS sorting, chemical fluorescent tags fusion, protein active site mutation and so on. And to provide scientific evidence for DHA in melanoma treatment.

黑色素瘤死亡率高，复发转移及放化疗抵抗是主要致死原因，具干性表型的黑色素瘤起始细胞（MIC）在其进程中起关键作用。糖酵解是MIC必需供能来源。我们研究发现，双氢青蒿素（DHA）可抑制黑色素瘤细胞糖酵解；抑制细胞侵袭与MIC自我更新。进一步研究发现，DHA处理显著降低糖酵解关键酶PKM2及LDHA酶活性。生物信息学分析提示：PKM2及LDHA蛋白结构中均存在DHA共价结合位点，提示DHA可能直接靶向PKM2和LDHA。由此，我们提出DHA治疗黑色素瘤可能的工作机制：DHA直接靶向抑制PKM2和LDHA，干预糖酵解，阻断细胞能量来源，抑制MIC干性表型。本项目拟进一步采用肿瘤球培养、MIC流式分选、化学荧光标记及蛋白活性位点突变等技术，旨在获得DHA靶向抑制PKM2/LDHA的可靠证据，阐明DHA抑制黑色素瘤干性表型的分子机制，为青蒿类药物治疗黑色素瘤提供科学依据。

研究背景：黑色素瘤死亡率高，复发转移及放化疗抵抗是主要致死原因，具干性表型的黑色素瘤起始细胞（MIC）在其进程中起关键作用。以PKM2及LDHA为关键酶的糖酵解途径是MIC必需供能来源。研究发现，提取自草本植物黄花蒿，具有过氧桥结构的倍半萜内酯化合物——双氢青蒿素（Dihydroartemisinin，DHA）参与肿瘤细胞代谢调节。.研究目的：探究双氢青蒿素通过调控PKM2/LDHA相关糖酵解途径抑制黑色素瘤起始细胞（MIC）的作用及机制。.研究方法：采用MTS法、平板克隆法及EdU染色法检测双氢青蒿素处理后黑色素瘤细胞的体外增殖活性；流式细胞仪检测细胞周期；裸鼠肿瘤移植瘤实验检测双氢青蒿素的体内处理效果；通过干细胞相关球的培养和实验检测MICs的自我更新；利用BIAcore (biomolecular interaction analysis)分析DHA与PKM2/LDHA的相互作用；进一步利用mRNA表达谱芯片筛选黑色素瘤中DHA作用的下游相关靶基因。所有统计分析均采用SPSS软件（13.0版）进行，p≤0.05的分析具有统计学意义。.研究结果：双氢青蒿素可抑制黑色素瘤细胞的增殖，并阻断细胞周期过程；同时，抑制黑色素瘤细胞侵袭与转移运动；重要的是，双氢青蒿素可抑制黑色素瘤起始细胞（MIC）自我更新，诱导MIC分化。进一步研究发现，DHA处理可显著减少黑色素瘤细胞中ATP及乳酸合成，下调PKM2和LDHA的活性，而不调节黑色素瘤细胞中PKM2和LDHA蛋白的表达。利用BIAcore技术，深入研究发现：双氢青蒿素可通过其倍半萜内酯结构与PKM2和LDHA的蛋白骨架共价结合，结合表达谱及miRNA芯片研究证实，DHA处理可并抑制黑色素瘤细胞的糖及其他能量物质代谢进程。.结论及临床前景：双氢青蒿素可通过靶向PKM2/LDHA，干预糖酵解进程，抑制黑色素瘤干性表型。作为上市药物，双氢青蒿素在黑色素瘤的临床治疗中具有广阔的应用前景。