Ceramide經由GSK-3β及Endoplasmic reticulum stress導致細胞死亡之探討

Involvement of GSK-3β and Endoplasmic Reticulum Stress in Ceramide-induced Cell Death

葉柏成(Bo-Chen Yeh)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/碩士(2007年)

https://doi.org/10.6831/TMU.2007.00101

摘要

Ceramide (神經醯胺)為細胞膜的成份之ㄧ，且為一重要的次級訊息傳遞者 (second messenger)，參與調控眾多訊息傳遞路徑，並可能導致細胞凋亡 (apoptosis)；此外，利用放射線或化學療法處理癌細胞，亦發現ceramide的含量上升為造成細胞死亡的主要原因之ㄧ，故ceramide被認為與癌症治療相關。ceramide可因實驗模式與條件之差異而造成不同的死亡型式，包括細胞凋亡及細胞自噬 (autophagy)。因此本論文探討ceramide對惡性膠質瘤U87-MG所造成的細胞死亡型式為何？並研究可能參與之分子訊息。
實驗結果顯示，利用acridine orange染劑併用flow cytometry偵測acidic vesicular organelle (AVO)含量，代表autophagy之百分比，結果發現ceramide處理細胞24小時可達最高autophagy百分比 (62.1 ± 3.5 %, p<0.001)，36小時下降至53.0 ± 4.8 % (p<0.001)，利用Western blot亦可觀察autophagy 上游分子LC3由type-I轉變為type-II之現象；另外，利用Annexin V/Propidium iodide (PI)染劑偵測細胞膜phosphatidylserine (PS) externalization與PI uptake，於36小時測得最高apoptosis含量 (36.7 ± 1.9 %, p<0.001)，推測ceramide處理U87-MG，可在初期造成autophagy晚期產生apoptosis。
研究顯示endoplasmic reticulum (ER) stress除了造成apoptosis亦能導致autophagy，因此本論文繼而探討此系統下是否有ER stress的產生。Western blot顯示：ceramide可誘導細胞內GADD153 (growth arrest and DNA damage-inducible gene 153)表現及eukaryotic initiation factor 2α (eIF2α)磷酸化現象，代表ceramide可導致U87-MG細胞產生ER stress。亦有研究指出ER stress可誘導glycogen synthase kinase-3β (GSK-3β)的活化而導致apoptosis，且本實驗室先前之實驗結果顯示GSK-3β參與導致autophagy，故續用Western blot方式偵測此系統下GSK-3β之活性變化，實驗結果顯示GSK-3β Ser9之磷酸化表現量隨ceramide之加入而減少，代表GSK-3β活化，且利用lithium chloride抑制GSK-3β後，經由flow cytometry分析autophagy百分比由63.9 ± 2.9 %降至40.2 ± 3.7 % (p<0.001)，表示GSK-3β可能參與ceramide誘導之細胞自噬。綜合所述，本論文推測ER stress及GSK-3β的活化可能是ceramide導致細胞死亡的原因之ㄧ。

關鍵字

惡性膠質瘤 ； 神經醯胺 ； 細胞自噬 ； 細胞凋亡 ； 內質網壓力 ； 肝醣合成酶激酶

並列摘要

Ceramide is a structural constituent of cell membrane that has been recognized as an important second messenger implicated in regulating diverse signaling pathways, especially for apoptosis. Moreover, chemotherapy and radiotherapy elicit an increase in the ceramide level. Ceramide causes either apoptosis or autophagy depending on the model systems and experimental conditions. The specific aim of the present study was to investigate the molecular signaling of ceramide-induced cell death in U87-MG, a human malignant glioma cell line.
As revealed by flow cytometry staining with acridine orange to detect the acidic vesicular organelle (AVO), we demonstrated that the percentage of autophagy reached a plateau of 62.1 ± 3.5 % (p<0.001) after 24 h exposure of U87-MG cells to 30 μM C6-ceramide, using immunoblot assay we also detected the processing of microtubule-associated protein 1 light chain 3 (LC3). A parallel experiment indicated that apoptosis reached to 36.7 ± 1.9 % (p<0.001) after 36 h exposure of 30 μM C6-ceramide, using Annexin V/Propidium iodide (PI) double staining to detect cell membrane phosphatidylserine (PS) externalization and PI uptake, implying that autophagy might be a prelude to apoptosis in ceramide-treated U87-MG cells. Endoplasmic reticulum (ER) stress, which has been shown to be engaged in cell death, was induced by treatment with ceramide using immunoblot to reveal the expression of GADD153 (growth arrest and DNA damage-inducible gene 153) and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). Recent studies indicated that ER stress was able to activate the glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase. Here, we demonstrated that ceramide induced a decrease of the phosphorylated level of Ser9 of GSK-3β, resulting in elevating its enzymatic activity. Besides, lithium chloride, a GSK-3β inhibitor, was able to reduce the percentage of autophagy from 63.9 ± 2.9 % to 40.2 ± 3.7 % (p<0.001), suggesting that GSK-3β might play a pivotal role in ceramide-induced autophagy.
In conclusion, our results indicate that C6-ceramide induced both autophagy and apoptosis. Of which, autophagy was likely to be mediated by ER stress and the activation of GSK-3β.

並列關鍵字

Malignant glioma ； Ceramide ； Autophagy ； Apoptosis ； Endoplasmic reticulum stress ； Glycogen synthase kinase-3β

參考文獻

Alvarez G, Munoz-Montano JR, Satrustegui J, Avila J, Bogonez E, Diaz-Nido J (1999) Lithium protects cultured neurons against beta-amyloid-induced neurodegeneration. FEBS Lett 453:260-264.

Barone MV, Crozat A, Tabaee A, Philipson L, Ron D (1994) CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest. Genes Dev 8:453-464.

Bijur GN, De Sarno P, Jope RS (2000) Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J Biol Chem 275:7583-7590.

Bijur GN, Jope RS (2001) Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta. J Biol Chem 276:37436-37442.

Brewster JL, Linseman DA, Bouchard RJ, Loucks FA, Precht TA, Esch EA, Heidenreich KA (2006) Endoplasmic reticulum stress and trophic factor withdrawal activate distinct signaling cascades that induce glycogen synthase kinase-3 beta and a caspase-9-dependent apoptosis in cerebellar granule neurons. Mol Cell Neurosci 32:242-253.