神經膠質母細胞瘤 (Glioblastoma)是人類腫瘤中，惡性程度最高以及最具侵略性的原發性腦瘤。神經膠質母細胞瘤中的缺氧微環境與腫瘤的生長、進展和對傳於傳統治療的抗性相關。在缺氧的壓力下，缺氧誘導因子 HIF1-α 的增加會改變侵入相關的分子，對於活化神經膠質瘤細胞的移動性而言是相當重要的。許多報導都指出缺氧會促進許多癌細胞由上皮形態轉變為間質形態，其中也包含神經膠質母細胞瘤。鳥糞嘌呤核苷酸交換因子 BIG1 和 BIG2 (Brefeldin A-Inhibited Guanine Nucleotide-Exchange Protein 1 and 2, BIG1 and BIG2)藉由其 Sec7 domain
來活化腺嘌呤核苷二磷酸核糖化因子 (ADP-ribosylation factors, ARFs)，促使ARFs 由 GDP 構型的失活態轉換為 GTP 構型的活化態，進而調控真核細胞的細胞膜轉運、蛋白質運輸和細胞骨架重建。最近有研究顯示，BIG1 和 BIG2 與細胞遷移時細胞極性的調節與肌動蛋白的動態有關。然而，BIG1 和 BIG2 在神經膠質母細胞瘤中扮演的角色以及神經膠質瘤細胞在氧化壓力下 BIGs 表現的調控，目前仍不清楚。在本篇研究中我們發現，氯化鈷誘導的氧化壓力會影響神經膠質母細胞瘤 U251 的細胞形態、影響細胞上皮-間質形態轉換 (EMT)之指標基因mRNA 和蛋白的表現量、改變細胞增殖指標基因 mRNA 的表現量以及提升磷酸化 AKT 的水平。藉由 Western blot 來分析在氯化鈷處理下，U251 細胞內 BIG1和 BIG2 的蛋白表現。我們使用 siRNA 及 shRNA 專一性標的 BIG1 和 BIG2 來降低 U251 細胞內 BIG1 和/或 BIG2 的表現量。我們也探討 U251 細胞在 BIG1 和/或 BIG2 降低下，對於細胞形態、細胞增殖以及訊息傳遞路徑的影響。在此所得的初步結果，提供我們有用資訊以進一步研究 BIG1 和 BIG2 於腫瘤生長及進展中所扮演角色。

Glioblastoma, is the most malignant and aggressive primary brain tumor in humans. Hypoxia in glioblastoma and its microenvironment is associated with the tumor growth, progression and resistance to conventional therapy of cancers. The amount of invasionrelated molecules altered by the increase of HIF-1α expression under hypoxic stress is
an essential event in the activation of glioma cell motility. Several reports indicated that hypoxia is also a well-known inducer of the epithelial to mesenchymal transition (EMT) program in many cancers, include glioblastoma. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 and 2 (BIG1 and BIG2) are well known to activate, via its ~200-amino-acid Sec7 domain, the replacement of ADP-ribosylation factors (ARFs)-bound GDP with GTP to regulate membrane transport, protein trafficking and cytoskeleton remodeling in eukaryotic cells. Recent studies showed that BIG1 and BIG2 are implicated in the regulation of cell polarization and actin dynamics for cell migration. However, little is known about the role of BIG1 and BIG2 in glioblastoma and the regulation of BIGs expression in glioma cells under oxidative stress condition. In this study, we found CoCl2-induced oxidative stress influence cell morphology, affect
the expression of mRNA and protein levels of epithelial-mesenchymal transition (EMT) markers, change the mRNA expression of cell proliferation markers and elevate the
pAKT protein level in glioblastoma U251 cells. Protein expression levels of BIG1 and BIG2 in U251 cells after CoCl2 treatment were determined by Western blotting. We
also used siRNA and shRNA that specific targeting BIG1 and BIG2 to deplete expression of BIG1 and/or BIG2 in U251 cells. Effects of BIG1 and/or BIG2 depletion on U251 cell morphology, cell proliferation, and signaling pathways were examined. The preliminary results obtained here will provide us useful information for further studies of roles of BIG1 and BIG2 in tumor growth and progression.

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