神經膠母細胞瘤 (Glioblastoma, GBM) 是成人腦瘤當中最常見且最惡性的，使用現今標準治療神經膠母細胞瘤的病人的中位存活期只有15個月，在癌症治療臨床研究上，組蛋白去乙醯酶 (histone deacetylase, HDAC) 抑制劑具有很不錯的抗癌效果，然而或許是廣泛性抑制組蛋白去乙醯酶的抑制劑造成嚴重的副作用，而導致在 GBM 的臨床研究失敗，本篇論文研究目標就在於使用新的選擇性抑制不同亞型的組蛋白去乙醯酶抑制劑 (HDAC inhibitor) 或許可以增加臨床應用的成功機率，並探討 GBM 的機制和基因調控的路徑。研究結果發現使用選擇性 HDAC4/5 的抑制劑LMK235 0.5 μ 能夠顯著的降低 GBM 病人取下來的腫瘤細胞 P#5 TMZ-R 和另一株 GBM 細胞 U87-MG 細胞增生跟細胞聚落之形成。從西方墨點法 (Western blotting) 的結果可以看到 LMK235 0.5 μM 處理會增加 P#5 TMZ-R 細胞的乙醯化 α-tubulin ，證明確實有抑制 P#5 TMZ-R 細胞的 HDAC ，而 LMK235 0.5 μM 處理沒有增加 cleaved-PARP ，也沒有增加 Annexin V-positive/PI-negative 的細胞，推測 LMK235 沒有引起 P#5 TMZ-R 細胞凋亡。 從LC3-II 蛋白的增加還有 GFP-LC3 點狀聚集分佈，可以推測 LMK235 0.5 μM 可以引起 P#5 TMZ-R 細胞自噬。此外，利用分析核糖核酸定序 (RNA-sequencing) 的結果，發現 LMK235 處理後高度的調控細胞黏附分子 (cell adhesion molecules) 這條途徑，而分析差異表達並且與劑量相關的基因有 299 個基因上調， 253 個基因下調，最後使用西方墨點法 (Western blotting) 的結果找到上皮鈉離子通道 α 次單元的編碼基因 SCNN1A 或許在 LMK235 治療 GBM 的結果中扮演著重要的角色。根據這個結果，利用 LMK235 來選擇性抑制組蛋白去乙醯酶在對抗神經膠母細胞瘤的治療當中，或許能成為新的臨床應用治療方法。

Glioblastoma (GBM) is a primary brain tumor in adults characterized as the most common and malignant. For GBM patients received standard treatment, the median survival is less than 15 months. Many histone deacetylase inhibitors (HDACis) have already showed effective in clinical cancer treatment; however, the severely side effects caused by pan-HDAC inhibition are currently critical issues. The present study aimed to evaluate the anticancer effects of novel selective HDACis, which may have tolerable toxicities for the treatment of GBM, and to study the mechanisms of selective HDACis on GBM. We found that a HDAC4/5 selective inhibitor LMK235 at 0.5 μM significantly reduced cell proliferation and colony formation in GBM cell lines U87-MG and P#5 TMZ-R, a patient-derived, TMZ-resistant GBM cells. Also, α-tubulin acetylation, which represent HDAC inhibition, was significantly increased by treatment of LMK235 at 0.5 μM in P#5 TMZ-R cell. LMK235 at 0.5 μM did not enhance PARP-1 cleavage form and Annexin V-positive/ PI-negative cell accumulation, suggesting LMK235 did not induce apoptosis in P#5 TMZ-R cell. Immunoblotting revealed a significant increase of LC3-II, and the fluorescent image of GFP-LC3 puncta suggesting that LMK235 treatment induce autophagosome formation in P#5 TMZ-R cell. Furthermore, from the data of RNA-sequencing, we performed KEGG pathway analysis and found that the “Cell Adhesion Molecules (CAMs)” pathway was enriched in the LMK235 treated group (both 0.5μM and 2μM). 299 genes were up-regulated and 253 genes were down-regulated in the LMK235 treated group in a dose-dependent manner. The potential role of SCNN1A might be important in LMK235 treatment on GBM cells. In conclusion, the selective inhibition of class IIa HDACs by LMK235 might have therapeutic utility for GBM.

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