臺灣博碩士論文加值系統

Benzyloxycarbonyl-Val-Ala-Asp (ZVAD)是一種非選擇性細胞凋亡蛋白酶(caspase) 抑制劑，廣泛用於抑制細胞凋亡 (apoptosis)。有趣的是，近來研究發現ZVAD本身在mouse L929 fibrosarcoma 會導致細胞壞死 (necrosis)，並伴隨著自噬性細胞體(或稱細胞自噬小體) (autophagosome) 的形成，也被稱為自噬性細胞死亡 (autophagic cell death)。他們發現ZVAD誘發的自噬性細胞死亡仰賴caspase 8抑制，RIP和 JNK的活性，以及氧化自由基的堆積。至今這些分子或媒介物之間的連結及詳細的訊息傳遞的機制並不清楚，因此本實驗的目的在於闡明ZVAD誘發自噬性細胞死亡的分子機轉，找出當細胞凋亡蛋白酶抑制下，JNK，ROS，PARP，Src及鈣離子在此訊息傳遞路徑所扮演的角色，並進一步釐清其和細胞自噬形成的上下游關係。
首先，我們確認ZVAD處理L929細胞可以促進LC3 cleavage，beclin 1基因表現，細胞自噬小體形成，及胞內氧化自由基堆積。利用對於粒腺體具專一性的螢光染劑 (MitoSox) 及抑制劑 (Rotenone，FCCP，BHA)，我們發現ZVAD產生的氧化自由基主要是來自於粒腺體。抗氧化劑，beclin 1 silence，第三型PI3K 抑制劑 (3-MA, 其已知為細胞自噬小體形成的抑制劑) 皆能有效的阻斷氧化自由基的產生及細胞死亡，表示氧化自由基的堆積在細胞自噬的下游並導致細胞壞死。另外，我們的結果顯示ZVAD能夠刺激PARP的活化，且PARP抑制劑 (DPQ) 能夠顯著地降低ZVAD造成的細胞死亡，但並不影響氧化性自由基的產生。以上結果顯示氧化自由基增加會造成PARP的活化，並進一步導致細胞的死亡。除此之外，我們發現ZVAD能夠刺激細胞內鈣離子的增加，而給予BAPTA/AM 或是除去鈣離子的培養液，都能夠抑制氧化自由基的產生及細胞死亡。
當我們用特定的蛋白激酶抑制劑去分析其參與ZVAD的訊息傳遞路徑時，發現JNK，ERK及Src和ZVAD引發的細胞死亡及氧化自由基的增加有關。此外，結果亦顯示ZVAD能夠快速地活化JNK，ERK，而此作用受Src抑制劑所拮抗。這些結果促使我們提出一個假設: ZVAD 導致的自噬性細胞壞死是經由抑制caspase 8 /Src活化 /JNK和ERK的活化 /產生細胞自噬/ 經由粒腺體產生氧化自由基 /PARP活化，最後造成細胞壞死性的死亡。
這些發現提供了我們更多關於caspase 8 在細胞凋亡之外作用的資訊。除了之前已知參與在典型caspase cascade造成細胞凋亡外，在caspase 8抑制下反而會造成另一種新穎的訊息傳遞路徑導致細胞自噬並進一步造成細胞壞死。

Benzyloxycarbonyl-Val-Ala-Asp (ZVAD), a pancaspase inhibitor has been widely used to abolish apoptotic cell death. Interestingly, previous reports showed that ZVAD alone induces necrosis accompanying with autophagosome formation, which termed as autophagic cell death, in L929 fibrosarcoma cells. They found that ZVAD-induced autophagic cell necrosis relies on caspase 8 inhibition, RIP1, JNK activity, and ROS accumulation. Until now the connection of these molecules and signaling mechanisms in details, however, are unclear. Therefore the aim of this study is to elucidate the molecular mechanisms of ZVAD-induced autophagic cell death, and find out the sequential roles of JNK, ROS, poly (ADP-ribose) polymerase (PARP) , calcium and Src in the signaling pathways triggered upon caspase inhibition and their action levels either upstream or downstream of autophagosome formation.
First, we confirm ZVAD indeed can stimulate LC3 cleavage, beclin 1 gene expression, autophagosome formation, and cytosolic ROS accumulation in L929 cells. Further study with mitochondria specific fluorescence dye (MitoSox) and inhibitors (rotenone, FCCP and BHA), we suggest that the ROS production by ZVAD is generated from mitochondria. Antioxidants, beclin 1 silence, class III PI3K inhibitor (3-MA) all effectively block ROS production and cell death, implying ROS accumulation downstream of autophagy contributes to cell necrosis. Moreover, our results reveal that ZVAD can stimulate PARP activation and PARP inhibitor DPQ significantly reduces ZVAD-induced cell death, but does not affect ROS production, suggesting ROS increase leading to PARP activation, and in turn causing cell death. Besides, we find that ZVAD stimulates intracellular calcium elevation, and ZVAD-induced ROS production and cell death are abolished by either BAPTA/AM or Ca-free medium. When using specific kinase inhibitors to analyze their involvement in ZVAD-elicited events, we find that JNK, ERK and Src are involved in ZVAD-induced cell death and ROS increase. Biochemical data evidence ZVAD rapidly induces JNK and ERK phosphorylation, and both signaling activations are sensitive to Src inhibitor PP2 and its siRNA treatment. All these results prompt us to propose that ZVAD-induced autophagic cell necrosis is mediated sequentially through caspase 8 inhibition/Src activation/JNK and ERK activation/autophagy formation/ROS generation from mitochondria/PARP activation, and eventually leads to cell death.
Theses finding provide more information to understand the apoptosis- independent role played by caspase 8 in cell death. In addition to initiate classical caspase cascade leading to cell apoptosis, caspase 8 inhibition in contrast trigger a novel signaling pathway leading to cell autophagy, and in turn necrosis cell death.

Abbreviations 5
Abstract 7
Abstract in Chinese 9
Introduction 11
Specific aims 24
Materials and Methods 26
Results 34
Discussion 42
Figures 53
Appendix 69
References 74

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