多型性神經膠質母細胞瘤(Glioblastoma multiforme，GBM)為成人中最常見且惡性的致命性原生腦癌，被世界衛生組織(WHO)歸列為第四級神經膠質瘤。多型性神經膠質母細胞瘤(GBM)具有高度異質性與侵略性，並對於傳統的化學及放射治療有抗藥性，因此多型性神經膠質母細胞瘤(GBM)之病患即使經過治療，仍有極高機率會復發，故其平均存活率不超過16個月。而此現象可能與GBM之癌幹細胞密切相關，因此針對癌幹細胞做為治療GBM的策略為一重要之目標及方向。中草藥在中國已被廣泛使用數千年，現今則有研究指稱它具有治療某些癌症疾病的潛能。因此，我們以腦癌細胞株U251-MG及U373-MG及其衍生之tumorsphere建立篩藥平台，以篩選可抑制GBM生長之有效中草藥萃取物。我們從200種中草藥酒萃物中篩選出兩種中草藥萃取物可抑制U251及U373 tumorsphere之存活，但對於正常的老鼠星狀膠細胞毒性較低。此兩種萃取物可能藉由調控Akt或ERK之訊息傳遞路徑，並影響細胞週期而達到抑制細胞生長，另外它們可降低幹細胞標記—CD133或Sox2之表現。此外，我們發現化合物BS可能是IT229中有效對抗GBM幹細胞之有效成分，其可有效降低U373-tumorsphere之存活。綜上所述，此兩種中草藥萃取物可能為有潛能治療GBM之藥物，並期許其未來可應用於臨床治療上。

Glioblastoma multiforme (GBM), classified as the grade IV astrocytoma, is the most common, malignant and fetal primary brain cancer in adult. GBM possess the characters of heterogeneity, invasiveness and resistance to traditional therapies. The GBM patients often experienced tumor relapse after aggressive treatment, therefore the median survival of the patients is about 14.6 month. Recent studies indicated that the glioblastoma stem cells (GSCs) may play the critical role in tumor relapse, thus targeting GSCs to against GBM is one of the potential therapeutic strategies for GBM. Chinese herbal medicine has been applied widely in Chinese society for thousands of year, and it is relatively mild and has less side-effect compared to the modern western medicine. Therefore, we used human glioblastoma cell lines and their derived tumorspheres to set up a platform for screening effective herbal extracts. In this study, we have screening 200 alcoholic herbal extracts and found two of them which inhibit cell viability of U251 and U373 tumorspheres but with low toxicity to normal mouse astrocytes. Furthermore, they reduce the ability of tumorsphere formation and cell growth by inducing cell cycle arrest. In addition, the two herbal extracts inhibit Akt or ERK pathway and down-regulate the expression of stem cell marker-CD133 or Sox2. Moreover, we found that compound –BS may be the bioactive component in IT229 and it also inhibit cell viability of U373 tumorspheres. In conclusion, the two herbal extracts may have therapeutic potential in GBM treatment through targeting glioblastoma stem cells.

1. Singh, M., et al., Brain Metastasis-Initiating Cells: Survival of the Fittest. Int J Mol Sci, 2014. 15(5): p. 9117-33.
2. Chen, R., A.L. Cohen, and H. Colman, Targeted Therapeutics in Patients With High-Grade Gliomas: Past, Present, and Future. Curr Treat Options Oncol, 2016. 17(8): p. 42.
3. Ceccarelli, M., et al., Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma. Cell, 2016. 164(3): p. 550-63.
4. Folkins, C., et al., Glioma tumor stem-like cells promote tumor angiogenesis and vasculogenesis via vascular endothelial growth factor and stromal-derived factor 1. Cancer Res, 2009. 69(18): p. 7243-51.
5. Lee, J., et al., Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell, 2006. 9(5): p. 391-403.
6. Lathia, J.D., M.P. Mattson, and A. Cheng, Notch: from neural development to neurological disorders. J Neurochem, 2008. 107(6): p. 1471-81.
7. Ruiz i Altaba, A., C. Mas, and B. Stecca, The Gli code: an information nexus regulating cell fate, stemness and cancer. Trends Cell Biol, 2007. 17(9): p. 438-47.

8. Denysenko, T., et al., WNT/beta-catenin Signaling Pathway and Downstream Modulators in Low- and High-grade Glioma. Cancer Genomics Proteomics, 2016. 13(1): p. 31-45.
9. de la Iglesia, N., S.V. Puram, and A. Bonni, STAT3 regulation of glioblastoma pathogenesis. Curr Mol Med, 2009. 9(5): p. 580-90.
10. Stupp, R., et al., Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol, 2009. 10(5): p. 459-66.
11. Friedman, H.S., et al., Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. J Clin Oncol, 2009. 27(28): p. 4733-40.
12. Vredenburgh, J.J., et al., Bevacizumab plus irinotecan in recurrent glioblastoma multiforme. J Clin Oncol, 2007. 25(30): p. 4722-9.
13. Stupp, R., et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 2005. 352(10): p. 987-96.
14. Shackleton, M., et al., Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell, 2009. 138(5): p. 822-9.
15. Schneider, M., et al., A paired comparison between glioblastoma "stem cells" and differentiated cells. Int J Cancer, 2016. 138(7): p. 1709-18.
16. Xu, G., et al., Cancer stem cells: the 'heartbeat' of gastric cancer. J Gastroenterol, 2013. 48(7): p. 781-97.

17. Brabletz, S., O. Schmalhofer, and T. Brabletz, Gastrointestinal stem cells in development and cancer. J Pathol, 2009. 217(2): p. 307-17.
18. Scoville, D.H., et al., Current view: intestinal stem cells and signaling. Gastroenterology, 2008. 134(3): p. 849-64.
19. Takebe, N., et al., Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol, 2011. 8(2): p. 97-106.
20. Winquist, R.J., et al., Targeting cancer stem cells for more effective therapies: Taking out cancer's locomotive engine. Biochem Pharmacol, 2009. 78(4): p. 326-34.
21. Bao, S., et al., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature, 2006. 444(7120): p. 756-60.
22. Johannessen, T.C., et al., Highly infiltrative brain tumours show reduced chemosensitivity associated with a stem cell-like phenotype. Neuropathol Appl Neurobiol, 2009. 35(4): p. 380-93.
23. Liu, G., et al., Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer, 2006. 5: p. 67.
24. Li, W., et al., Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell, 2009. 4(1): p. 16-9.

25. Olmez, I., et al., Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth. J Cell Mol Med, 2015. 19(6): p. 1262-72.
26. Mansouri, S., et al., Sox2: regulation of expression and contribution to brain tumors. CNS Oncol, 2016. 5(3): p. 159-73.
27. Lee, J., et al., Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells. Cancer Cell, 2008. 13(1): p. 69-80.
28. Piccirillo, S.G., et al., Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature, 2006. 444(7120): p. 761-5.
29. Aung, W., et al., Anticancer effect of dihydroartemisinin (DHA) in a pancreatic tumor model evaluated by conventional methods and optical imaging. Anticancer Res, 2011. 31(5): p. 1549-58.
30. Zheng, Y.M., et al., Anti-oxidant and anti-cancer activities of Angelica dahurica extract via induction of apoptosis in colon cancer cells. Phytomedicine, 2015.
31. Wu, X., et al., Chinese Herbal Medicine for Improving Quality of Life Among Nonsmall Cell Lung Cancer Patients: Overview of Systematic Reviews and Network Meta-Analysis. Medicine (Baltimore), 2016. 95(1): p. e2410.
32. Tsai, N.M., et al., The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem, 2006. 99(4): p. 1251-62.
33. Fu, Y.S., et al., Tetramethylpyrazine inhibits activities of glioma cells and glutamate neuro-excitotoxicity: potential therapeutic application for treatment of gliomas. Neuro Oncol, 2008. 10(2): p. 139-52.
34. Libermann, T.A., et al., Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature, 1985. 313(5998): p. 144-7.
35. Roberts, P.J. and C.J. Der, Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene, 2007. 26(22): p. 3291-310.
36. Porta, C. and R.A. Figlin, Phosphatidylinositol-3-kinase/Akt signaling pathway and kidney cancer, and the therapeutic potential of phosphatidylinositol-3-kinase/Akt inhibitors. J Urol, 2009. 182(6): p. 2569-77.
37. Suresh, V., et al., In vitro anti-inflammatory and anti-cancer activities of Cuscuta reflexa Roxb. J Ethnopharmacol, 2011. 134(3): p. 872-7.
38. Liao, J.C., et al., Antinociceptive and anti-inflammatory activities of Cuscuta chinensis seeds in mice. Am J Chin Med, 2014. 42(1): p. 223-42.
39. Lahmar, Q., et al., Tissue-resident versus monocyte-derived macrophages in the tumor microenvironment. Biochim Biophys Acta, 2016. 1865(1): p. 23-34.

40. Pajouhesh, H. and G.R. Lenz, Medicinal Chemical Properties of Successful Central Nervous System Drugs. NeuroRx, 2005. 2(4): p. 541-53.
41. Nakagawa, S., et al., A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int, 2009. 54(3-4): p. 253-63.