| 摘要: | 多型性神經膠母細胞瘤 (glioblastoma multiforme, GBM) 屬於第四級的神經膠質瘤，也是原發型腦瘤病例中最常見且惡性的腦瘤類型。 目前腦瘤的第一線用藥為 temozolomide (TMZ)，但絕大多數病人會對 TMZ 產生抗藥性且使腦瘤再度復發。 因此對腦瘤治療而言，尋找合適的治療用藥是刻不容緩的議題。 Sunitinib 是一種小分子標靶藥物，可抑制 VEGFR 等多種酪胺酸激酶之活性且正應用於臨床腦瘤治療。 但仍未達到良好的治療效果。 因此本研究欲探討造成腦瘤細胞對 sunitinib 藥物敏感性降低之可能分子機制，並試圖尋找能有效增強 sunitinib 毒殺效果之改善方式。 我們發現 sunitinib 劑量越高時，越可顯著降低細胞存活率並誘發細胞凋亡 (apoptosis)，但卻會提高細胞內細胞自噬 (autophagy) 比例以及增加 LC3-II，ATG12，Beclin-1 之蛋白質表現量。 我們透過不同的 autophagy inhibitors包括 chloroquine (CQ)、hydroxychloroquine (HCQ)、Lys05、cycloheximide，搭配 sunitinib 處理，證實 sunitinib 所誘導的是腫瘤保護型 autophagy，且抑制 autophagic flux 可增強 sunitinib 細胞毒性。 為了釐清 sunitinib 與 autophagy 間的基因網絡，我們將 sunitinib 調控之基因表現圖譜和與 autophagy 有關的基因群進行交集，再進一步分析 TCGA 腦瘤病人數據，尋找出與病人低存活率相關之危險因子為 Growth arrest and DNA damage-inducible protein (GADD34)。 隨後也確認 sunitinib 劑量越高時，GADD34 的基因與蛋白質表現量越高，進而導致 autophagy 的發生。 此外，改變內生性 GADD34 表現量會顯著影響 sunitinib 細胞毒性，並確認 p38-MAPK 訊息途徑參與 sunitinib 調控 GADD34 的表現。 過去文獻指出 guanabenz 為一種 alpha2-selective adrenergic agonist，做為臨床降血壓用藥且能抑制 GADD34 活性展現。 以 guanabenz 搭配 sunitinib 處理後，發現 guanabenz 可抑制 GADD34 所促進之保護型 autophagy 進而增強 sunitinib 毒殺效果。 兩藥物共同處理也能有效毒殺 TMZ 抗藥性，1% 低氧培養及 tumorsphere 型態之腦瘤細胞。 未來，guanabenz 搭配 sunitinib 或許能做為臨床腦瘤治療之新策略。 企盼此研究結果能對腦瘤之精準醫療有所貢獻，以有效提升病人福祉。
Glioblastoma multiforme (GBM) is the most common and malignant grade IV glioma. Temozolomide (TMZ) is used as a first-line treatment drug. However, limited therapeutic efficacy of TMZ results in recurrence of GBM patients. Nowadays, finding out the replaced drugs in GBM treatment is still a critical topic. Sunitinib, a small molecular multitargeted receptor tyrosine kinase inhibitor, is currently used in therapy for GBM. Unfortunately, sunitinib still has insufficient activity to cure recurrent GBM. Our aim is to determine the molecular mechanisms in counteracting sunitinib drug sensitivity to GBM, and find out the effective therapeutic strategies to enhance the sunitinib cytotoxicity. Here, we found that sunitinib dose-dependently induced apoptosis, reduced cell viability, increased autophagy ratio, and influenced the protein levels of LC3-II, ATG12, beclin-1 in U-87 MG and A172 cells. By combined treatment with sunitinib and different autophagy inhibitors including chloroquine (CQ), hydroxychloroquine (HCQ), Lys05, and cycloheximide, we identified that sunitinib induced a protective autophagy. Inhibition of autophagy enhanced sunitinib cytotoxicity. By intersecting the sunitinib-upregulated transcriptome profile, autophagy-related genes, and The Cancer Genome Atlas (TCGA) risk genes, Growth arrest and DNA damage-inducible protein (GADD34) was selected as a candidate gene. Higher GADD34 expression occupied poor survival of GBM patients. Moreover, sunitinib significantly increased GADD34 expression, resulting in autophagy formation. Changes in endogenous GADD34 expression obviously influenced sunitinib cytotoxicity. The p38-MAPK signaling pathway was also identified to participate in sunitinib-upregulated GADD34 expression. Guanabenz, an alpha2-selective adrenergic agonist used as an antihypertensive agent, could inhibit the function of GADD34. We found that guanabenz enhanced the efficacy of sunitinib by targeting GADD34-induced protective autophagy in GBM cell lines, TMZ resistant cells, 1% hypoxia cultured cells, and tumorsphere cells. In the future, combination therapy with sunitinib and guanabenz may provide a new strategy toward precision medicine in GBM therapy. |
