滇天冬提取化合物抗肿瘤干细胞活性及分子机制研究

Cancer stem cells (CSCs) are considered as the main driver for the initiation, development, metastasis and recurrence of tumors. Drugs targeting CSCs have been the focus of anti-tumor drug development. We screened about 6,000 components of more than 300 Yunnan herbal medicines by using the previously established high-throughput drug screening system. The compound LAF-74 extracted from Asparagus filicinus was proved to have high activity against CSCs and no obvious cytotoxicity to all tested normal cell lines. Further studies showed that LAF-74 could significantly promote cell apoptosis and prolong the survival of glioma mice. In addition, LAF-74 showed a significant inhibitory effect on the colony formation of multiple cancer cell lines in addition to glioma cell lines. This project will isolate CSCs from common tumors such as lung cancer, liver cancer, and breast cancer, and test the effect of LAF-74 on CSCs in vitro and in vivo to confirm its broad spectrum of CSCs toxicity. This project will also identify CSC-specific LAF-74 binding proteins and elaborate the molecular mechanism of LAF-74 against CSCs, taking advantage of commonly used technologies such as surface plasmon resonance（SPR），HPLC-MS/MS and computer-aided simulation. The results of this study will provide early support for the new drug discovery of LAF-74.

肿瘤干细胞被认为是肿瘤发生、发展、转移和复发的主要原因，针对肿瘤干细胞的治疗药物已成为抗肿瘤药物研发重点。我们对300多种云南中草药的6千余个组分进行筛选，发现来自滇天冬的化合物LAF-74具有良好的抗胶质瘤干细胞活性，同时对所有测试的正常细胞无明显毒性。进一步研究表明LAF-74可显著促进细胞凋亡以及延长胶质瘤小鼠的生存期；此外LAF-74可抑制多种肿瘤细胞系的克隆形成，提示可能对其他肿瘤干细胞也有良好效果。本项目将从肺癌、肝癌、乳腺癌等常见肿瘤中分离肿瘤干细胞，并在体内外检测LAF-74对肿瘤干细胞的作用以验证其抗肿瘤干细胞广谱性；以及利用表面等离子共振、液相色谱-质谱、计算机辅助模拟等技术对肿瘤干细胞特异性LAF-74结合蛋白进行鉴定和功能分析，阐明LAF-74抗肿瘤干细胞的分子机制。研究结果将为LAF-74的成药开发提供前期支持。

肿瘤干细胞被认为是肿瘤发生、发展、转移和复发的主要原因，针对肿瘤干细胞的治疗药物已成为抗肿瘤药物研发重点。然而，现有的肿瘤干细胞靶向药物十分匮乏，并且临床效果有限。天然产物具有悠久的药用历史和广泛的化学多样性，是抗肿瘤干细胞药物筛选和开发的理想资源。在本研究中，我们利用基于胶质瘤干细胞建立的高通量药物筛选平台，对来自386种云南中草药的8344组分进行筛选，发现来自滇天冬的化合物LAF-74可有效且特异性地消除肿瘤干细胞；分析其化学结构，发现LAF-74为呋甾皂苷taccaoside A。由于在C-22位的官能团差异，taccaoside A两个同源物对肿瘤干细胞、肿瘤细胞系和永生化细胞系均有显著细胞毒性，表明在taccaoside A中C-20和C-22之间的碳碳双键是其特异性杀伤肿瘤干细胞的关键结构。分子作用机制研究表明，与RAS蛋白的直接结合是taccaoside A抑制肿瘤干细胞增殖和诱导肿瘤干细胞凋亡的必要条件。我们的研究结果提供了一种筛选肿瘤干细胞特异性药物的方法，并为taccaoside A成药开发提供前期支持。. 在LAF-74抗肿瘤干细胞的分子机制研究中，我们发现NKG2D配体在胶质瘤干细胞表面表达显著上调，而在正常组织中低表达或者不表达，是CAR-T清除胶质瘤干细胞的理想靶点。因此我们以NKG2D胞外序列为靶点识别域，4-1BB和CD3ζ为共刺激信号构建了CAR-T细胞。实验结果表明NKG2D CAR-T细胞在体外可以高效地杀死胶质母细胞瘤细胞系和肿瘤干细胞，释放高浓度的细胞因子、穿孔素和颗粒酶。在小鼠体内，NKG2D CAR-T完全消除移植瘤，并且无明显治疗相关毒性。此外，慢病毒介导的CAR表达对T细胞的增殖、凋亡和基因组稳定性无显著作用。以上结果证明了NKG2D CAR-T细胞以NKG2D配体依赖的方式高效地靶向杀死胶质母瘤细胞和肿瘤干细胞，为NKG2D CAR-T的临床应用奠定了基础。.