Ceramide 經由 GSK-3β 誘導大 鼠腦神經膠質瘤細胞 Autophag y 之探討

Autophagy ( 細胞自噬 ) 為一演化下高度保留之蛋白質代謝機制，但過度表現亦會導致細 胞死亡。阿茲海默症成因為 amyloid-beta (Aβ ，乙型澱粉樣蛋白 ) 之沉積導致腦神經細胞 死亡。 ceramide ( 神經醯胺 ) 為真核細胞膜的成份之ㄧ，為 Aβ 之下游代謝物，因此本論 文利用 ceramide 模擬 Aβ 之細胞毒性。為探討 ceramide 對大鼠腦神經膠質瘤細胞 (C6 gli oma cells) 之細胞毒性，以 acridine orange 染色法併用流式細胞儀偵測，發現 autophagy 百 分比隨 ceramide 劑量及加藥時間而上升之趨勢，其中以 30 μM ceramide 加入 24 小時後達 到最高 47.24 ± 2.73 % (p<0.001) 的 autophagy ，隨即降低，但 apoptosis 則隨之升高，於 3 6 小時達到 27.3 ± 5.5 % (p<0.01) 。以西方墨漬法可看出 autophagy 上游蛋白分子， LC3 蛋白之 processing 及 Beclin 1 蛋白的表現量隨時間點往後推移而上升；利用穿透式電子顯 微鏡亦可於細胞質觀察到雙層膜之 autophagosomes 構造，顯示 ceramide 可導致 C6 glioma cells 進行 autophagy 。此外，若同時加入 autophagy 抑制劑 vacuolar-ATPase inhibitor ， ba filomycin A1 (BafA1) ，可將 ceramide 所誘導之 apoptosis 由 5.35 ± 0.72 % 提高至 23.21 ± 6 .87 % (p<0.05) ，因此推測 autophagy 為一保護性機制。 GSK-3β ( 肝醣合成酶激酶 ) 為一 s erine/threonine protein kinase ，已被證實會參與細胞死亡之調控，但於 autophagy 方面之研 究則尚無探討。以西方墨漬法發現 GSK-3β 之 Ser-9 位置磷酸化現象隨時間增加而降低；

另外，發現加入 GSK 抑制劑， SB216763 ，可將 autophagy 由 56.51 ± 4.05 % 降至 40.42 ± 3.83 % (p<0.001) 。另ㄧ方面，細胞加入 ROS ( 活性氧分子 ) 清除劑， NAC ， autophagy 之比例亦由 51.68 ± 0.85 % 降至 34.92 ± 2.64 % (p<0.001) 。實驗結果顯示， ROS 與 GSK- 3β 之訊息傳遞路徑可能參與 ceramide 所誘導之 autophagy 。

Ceramide 經由 GSK-3β 誘導大 鼠腦神經膠質瘤細胞 Autophag

y 之探討

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Autophagy, an evolutionarily conserved mechanism for degradating and recycling of long-lived proteins, is considered as a type II programmed cell death (type II PCD). Aggregation of amyloid-beta (Aβ) induces br ain cells death is one of the main causes of Alzheimer’s disease (AD). Ceramide, a ubiquitous constituent o f eukaryotic membranes, is a downstream metabolite of Aβ, which was often exploited to mimic the toxicit y of Aβ. In this study, using C6 glioma cells as a cell model, we investigated the cytotoxity of ceramide. A s revealed by flow cytometry staining with acridine orange, we observed that the percentage of autophagy and apoptosis reached a plateau of 47.24 ± 2.73 % (p<0.001) and 27.3 ± 5.5 % (p<0.01) after 24-h and 36- h exposure of the C6 glioma cells to 30 μM ceramide. Using immunoblot assay, we detected the processing of microtubule-associated light chain 3 (LC-3) and the expression of Beclin 1 in a time-dependent manner, which have been found to promote autophagy. Moreover, transmission electron microscopy (TEM) analysi s demonstrated that the formation of double-layer autophagosomes were observed in the cytosol, indicating that ceramide is able to induce autophagy in C6 glioma cells. In addition, using vacuolar-ATPase inhibitor, bafilomycin A1 (BafA1), to suppress the ceramide-induced autophagy, we found that the percentage of apo ptosis was increased from 5.35 ± 0.72 % to 23.21 ± 6.89 % (p<0.05), suggesting that autophagy might play a protective role in C6 glioma cells treated with ceramide. In the study of glycogen synthase kinase-3β (GS K-3β), a serine/threonine kinase, we demonstrated that ceramide induced a decrease of the phosphorylated level of Ser9 of GSK-3β which implied that the activation of GSK-3β. Besides, GSK-3β inhibitor, SB2167 63, and a reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), was able to reduce the per centage of autophagy from 56.51 ± 4.05 % to 40.42 ± 3.83 % (p<0.001) and 51.68 ± 0.85 % to 34.92 ± 2.6 4 %, respectively. It suggests that GSK-3β and ROS are involved, at least partially, in autophagy. We concl ude that ceramide-induced autophagy was regulated by ROS and GSK-3β signal transduction pathway.

The Study of Ceramide-Induced Autophagy in C6

Glioma Cells-the Role of GSK-3β