中国科学院昆明动物研究所机构知识库

癌症是一个世界难题，是我国公民的首位死亡原因。近年来随着肿瘤生物学的发展研究发现肿瘤组织中只有极少的一类细胞具有无限增殖和自我更新的能力，称为肿瘤干细胞，是肿瘤发生发展和转移等的根本原因。肿瘤干细胞存在于血液肿瘤以及脑肿瘤、肺癌、肝癌、乳腺癌等几乎所有实体瘤中。研究显示肿瘤干细胞对放化疗具有更强的抵抗性。因此，针对肿瘤干细胞的治疗理论上将更有效，肿瘤干细胞理应是肿瘤治疗的主要靶细胞。体内研究已表明清除动物肿瘤模型中的肿瘤干细胞可以有效阻止肿瘤的发展。胶质瘤是最常见的恶性脑肿瘤，恶性程度极高，平均生存时间在12个月以内。脑胶质瘤干细胞是最早分离鉴定的实体瘤干细胞之一，也是极少数能长期体外培养仍能保持遗传特性的肿瘤干细胞之一，在肿瘤干细胞的研究中具有重要的示范意义。同时，胶质瘤干细胞除了可悬浮培养形成类似神经球样的细胞团外，也可长期贴壁传代培养，与用含血清培养基进行的胶质瘤原代细胞培养不同，胶质瘤干细胞基因组和基因表达谱在长期培养条件下都极稳定，因此胶质瘤干细胞是筛选抗肿瘤干细胞药物的理想模型。通过临床胶质瘤样本分离建立了3株EGFR，PTEN，Tp53等代表性基因突变的胶质瘤干细胞株，并通过腺相关病毒介导的同源重组将报告基因EGFP定点整合至神经干细胞分子标记Nestin基因启动子下游，通过内源基因Nestin表达变化控制报告基因EGFP的表达，从而建立了针对胶质瘤干细胞的高内涵筛选体系，可同时筛选细胞毒性药物和诱导分化药物。利用胶质瘤干细胞的高内涵筛选平台完成了小分子化合物库40675个化合物的抗肿瘤干细胞活性筛查，从中发现了35个强心苷类化合物具有选择性抑制胶质瘤干细胞的活性；以地高辛作为代表化合物，完成了其体内体外的抗肿瘤活性研究，体外实验证明地高辛能显著抑制胶质瘤干细胞的细胞增殖，阻断细胞周期进程，诱导细胞发生Caspase-3依赖的细胞凋亡，此外还可以显著的破坏胶质瘤干细胞的克隆形成；体内实验证明了地高辛可以显著的抑制脑原位移植瘤的生长，延长患瘤小鼠的生存时间，展示出了良好的体内抗肿瘤效果。此外通过建立的胶质瘤干细胞的高内涵筛选体系，还完成了360种云南特色资源植物的约7000个组分的筛查，发现来源于11种植物的11个组分具有选择性抑制胶质瘤干细胞的活性，进一步通过HPLC-MS在天门冬和龙骨节中分离得到了两个选择性抑制胶质瘤干细胞的甾体皂苷化合物WADM-3和WLRBBW-9；研究发现WADM-3能够选择性的抑制胶质瘤干细胞和其它多种类型的肿瘤细胞株，包括胶质瘤细胞，肺癌细胞，肝癌细胞，乳腺癌细胞，结直肠癌细胞，胃癌细胞，胰腺癌细胞，却对人体的多种正常组织细胞没有明显毒性，机制研究发现WADM-3是通过选择性调节胶质瘤干细胞中NAD(P)H氧化酶的两个组成亚基NCF1和NOX1表达升高，促进NAD(P)H向NAD(P)+的氧化，加快O2的消耗和电子传递，产生超氧阴离子(O2-)，对细胞施加氧化损伤压力，通过线粒体诱导细胞凋亡，而这均不会发生在正常细胞中；化合物WLRBBW-9虽然对多种类型的肿瘤细胞株：胶质瘤细胞，肺癌细胞，肝癌细胞，乳腺癌细胞，结直肠癌细胞，胃癌细胞，胰腺癌细胞，和人体正常组织细胞没有明显的抑制作用，却能选择性的抑制这些测试肿瘤细胞中的肿瘤干细胞的生长，具有广谱抑制肿瘤干细胞的活性，机制研究发现WLRBBW-9是通过选择性积累胶质瘤干细胞中的DNA损伤，引起转录因子ATF3和FOS的表达上调，进而转录抑制线粒体氧化磷酸化复合物III亚基UQCRHL和复合物IV亚基COX6B2的表达，从而抑制氧化磷酸电子传递的进行，不能正常跨膜转运H+，造成线粒体膜电位降低，释放凋亡诱导因子，诱导细胞凋亡，而这也不会发生在正常细胞中；此外体外实验还表明两个化合物抗胶质瘤干细胞活性显著，是目前广谱细胞毒抗癌药物紫杉醇活性的2-5倍；其对于胶质瘤干细胞与正常组织细胞的选择指数大于30倍，也远高于紫杉醇，具有重要的研究和开发价值。

Cancer is a major cause of death in the world, including in China. There were small population of subtype cancer cells that existed in tumor samples with the unlimited proliferative and self-renewal capacity were called cancer stem cells (CSCs). It was nearly existed in all the solid tumors, including the hematologic neoplasms, brain tumor, breast cancer, lung cancer, liver cancer, colon cancer, gastric cancer, pancreatic cancer etc. They were resistant to the present chemotherapy and radiotherapy and considered to be contribute the cancer recurrence and transfer. So the cancer stem cell should be serve as the target for the cancer treatment, and may be get a better therapeutic outcome in theory. Glioblastoma multiform (GBM), a common subtype malignant brain tumor, is a highly aggressive and frequently recurrent neurologically destructive cancer that considered to be one of the most incurable human cancers. The life expectancy of most GBM patients are within 9-12 month for its highly invasion and resistance to chemotherapy and radiotherapy. The glioma stem cells (GSCs) was one of the most early isolated and identified cancer stem cells. It could keep its genetic stability for a long time culture in vitro. Besides, the GSCs could be culture in suspended or attached medium convenient to anti-cancer drugs screening. So the GSCs was the ideal cancer stem cell model for anti-cancer stem cell drugs screening and investigation.The GSCs were established from glioblastoma multiform samples that were obtained from the Yun Nan Cancer Hospital by our lab. We selected three glioma stem cell lines with the mutation of the oncogene EGFR and the suppressor gene Tp53, pTen for the further research through the genes expression analysis. In order to identify the cytotoxic drugs and inducing differentiation drugs, the reporter gene EGFP was inserted into the neural stem cell biomarker Nestin gene promotor by the adeno-associated virus (AAV). We complished the anti-cancer drugs screening and identified 35 compounds with the selectively anti-cancer stem cell characteristic from the known small molecular compounds library contained 40675 compounds located in ShangHai. All of them belong to the cardiac glycosides and the compound digoxin was serve as a present compound for the cardiac glycosides continued to further research. It showed that digoxin could worked against the human glioma stem cell and glioma cell, lung cancer cell, colon cancer cell, breast cancer cell, but not the mouse neural stem cell. The GSCs cell proliferarion was decreased and the cell cycle was arrest significantly, and it also induced apoptosis through the cleaved-caspase-3 dependent cell apoptosis. Besides, it could damaged the GSCs colony formation and decresed the number of the spheres significantly. Furthermore, it could delay the NOD/SCID mouse survival through the tail vein injection in the intracranial xenografted tumor model, which indicated that digoxin has an effective anti-cacner efficacy in vivo.Besides, the natural products library, was consisted of 7000 extracts that were isolated from 360 medicial plants, was also finished screening and found 11 extracts with the selective anti-cancer stem cells ablility. The compounds WADM-3 and WLRBBW-9 were firstly isolated from the Asparagus spp. and Tacca spp. the compound WADM-3 could work effectively against the glioma stem cell and other cancer cells, including glioma cell, lung cancer cell, liver cancer cell, gastric cancer cell, pancreatic cancer cell, colon cancer cell and breast cancer cell, but not the human normal cell, including Embryonic kidney cells, vascular endothelial cell, gastric epithelial cells and cerebellum astrocyte cell. The cell proliferation was decreased significantly and the apoptosis was activated by the cleaved-caspase-3. The colony spheres were also dagamed by the WADM-3 treatment. Besides, it could increase the ROS level to induce the glioma stem cell mitochondrial apoptosis by selectively increasing the expression of NCF1 and NOX1 in GSCs, but it not happened in human normal cell. Other compound WLRBBW-9 could worked effectively against the glioma stem cells, but not for cancer cells and human normal cells through the cell viability assay. Futher investigation showed that WLRBBW-9 could work effectively against the cancer stem cells, including glioma stem cell, lung cancer stem cell, liver cancer stem cell, gastric cancer stem cell, pancreatic cancer stem cell, colon cancer stem cell and breast cancer stem cell at the low concentration that did not worked against the cancer cells. Besides, it also decreased the cell proliferation and increased the apoptosis by cleaved-caspase-3. Futher mechanism investigation showed that it promoted the GSCs DNA damage and activated the expressin of transcription factors ATF3 and FOS to suppress the expression of UQCRHL and COX6B2 that were consisted of complex III and complex IV respectively in the oxidative phosphorylation and decreased the mitochondrial membrance potential (MMP) to induce apoptosis. Furthermore, the WADM-3 and WLRBBW-9 worked against the GSCs was 2-5 folds than the anti-cancer drug taxol and with a more than 30 folds selection, which suggested that they may be developed as the promising lead compounds.