探討組蛋白去乙醯基抑制劑(SAHA)治療腦腫瘤幹細胞之機轉

本研究主要探討人類惡性膠母細胞瘤幹細胞的細胞特性與對於組蛋白去乙醯基抑制劑的可能產生相關對應之機轉。首先研究的部分是我們針對膠母細胞瘤幹細胞進行分析與篩選；之後進行一連串的幹細胞能力分析，包括self-renewal，differentiation、migration、invasion。另外也進行幹細胞標記的分析(CD133、nestin、Oct4、SOX2)等。最後執行致腫瘤移植實驗，發現無論是腦內或皮下腫瘤移植，皆能形成腫瘤，不過仔細分析腫瘤細胞型態與分子表現，仍以腦內移植所衍生的腫瘤較接近臨床結果。此外，同時也證實，皮下移植所形成腫瘤的團塊，其內部微血管構造的來源，極有可能是源自於腫瘤幹細胞所衍生而成的。第二部分則是針對SAHA的研究，該藥是一種組蛋白去乙醯基酶抑制劑，已經被用於癌症治療，但其作用於癌細胞的機制並不清楚。在本研究中，我們使用從惡性膠母細胞瘤(GBM)的檢體中所分離出來的腦瘤幹細胞(GSCs)為實驗的材料，發現SAHA具有專一性地誘導細胞自噬並造成該細胞存活率下降，促進GSCs的後期凋亡。SAHA在小鼠皮下異種移植腫瘤實驗發現，能有效地造成腫瘤生長減緩和誘導的自噬。SAHA足以形成細胞內酸性囊泡細胞器，西方墨點法分析證明LC3-II的自噬體的增加，加強BECN1蛋白表現，減少SQSTM1蛋白表現量。我們證實SAHA引發細胞自噬是透過降低AKT-mTOR的訊息路徑。有趣的是，當使用自噬抑制劑或是使用自噬基因沉默技術時，SAHA會促進細胞凋亡，導致細胞加速死亡，這表明SAHA所誘導自噬功能可能作為一種促生存機制，用來對抗SAHA的細胞毒性。此外，我們的結果還表明，SAHA誘導自噬使用氯喹(chloroquine)有協同效應，顯著地促進細胞凋亡。此外，我們發現，減量的SAHA可充當一個強而有力的分化促進劑，促使GSC的分化和衰老。綜上所述，我們的研究結果提供了一個新的視野，SAHA抑制腫瘤幹細胞可能透過誘導細胞自噬的方式。

We report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133+-GSC) and CD133- (CD133--GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with xenografted tumors derived from CD133--GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133+-GSC-derived tumor xenografts. Subsets of CD133+-GSC but not CD133--GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133--GSC and exclusively dispersed within the tumor tissues. Although, the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.In addition, although Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been used in clinical trials for cancer therapies, its pharmacological effects occur through a poorly understood mechanism. Here, we report that SAHA specifically triggers autophagy and reduces cell viability via promotion of apoptosis in the late phase of glioblastoma stem cells (GSCs). Using a cell line cultured from a glioblastoma biopsy, we investigated the properties and effects of GSCs under SAHA treatment in vitro. In vivo xenograft assays revealed that SAHA effectively caused tumor growth slowdown and the induction of autophagy. SAHA was sufficient to increase formation of intracellular acidic vesicle organelles, recruitment of LC3-II to the autophagosomes, potentiation of BECN1 protein levels, and reduced SQSTM1 levels. We determined that SAHA triggered autophagy through the downregulation of AKT-mTOR signaling, a major suppressive cascade of autophagy. Interestingly, upon depletion or pharmacological inhibition of autophagy, SAHA facilitates apoptosis and results in cell death at the early phase, suggesting that SAHA-induced autophagy functions probably act as a prosurvival mechanism. Furthermore, our results also indicated that the inhibition of SAHA-induced autophagy using chloroquine has synergistic effects that further increase apoptosis. Moreover, we found that a reduced dose of SAHA functioned as a potent modulator of differentiation and senescence. Taken together, our results provide a new perspective on the treatment of GSCs, indicating that SAHA is a promising agent for targeting GSCs through the induction of autophagy.