戊乙醯去羥梔子誘發神經膠瘤細胞凋亡機轉之探討

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 博碩士論文 > Item 310902500/1524

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Title:	戊乙醯去羥梔子誘發神經膠瘤細胞凋亡機轉之探討 The biochemical basis of C6 glioma cell apoptosis induced by penta-acetyl geniposide
Authors:	彭瓊琿 Chiung-huei Peng
Contributors:	中山醫學大學：生物化學研究所;王朝鐘
Keywords:	戊乙醯去羥梔子;凋亡;蛋白激脢C delta;caspase;轉位;c-Jun N端激脢;c-Jun;Fas-L (AC)5GP;apoptosis;PKC delta;caspase;translocation;JNK;c-Jun;Fas-L
Date:	2003
Issue Date:	2010-06-02T06:32:01Z (UTC)
Abstract:	中草藥已沿用於各項疾病的治療多年，其中亦包含對癌症的治療。另一方面，近年來的研究在在顯示，癌細胞因無法正常凋亡而生長蔓延。因此探討其凋亡機轉，成為研究癌症治療中的一項重要課題。本研究中，我們探討戊乙醯去羥梔子（penta-acetyl geniposide, (Ac)5GP）這項中草藥的抗癌效用與其可能機轉。之前的研究結果顯示，(Ac)5GP可阻擾癌症發展、抑制腫瘤生長、以及引發細胞凋亡，然而其凋亡機轉未明。由於曾有報告指出，蛋白激脢C的某些異構物（protein kinase C isoforms, PKC isoforms）與細胞凋亡的訊息傳導（signal transduction）有關。我們因此欲探討(Ac)5GP是否可藉由活化PKC而引發細胞凋亡。結果證實，(Ac)5GP確實可導致C6神經膠瘤細胞（C6 glioma cells）凋亡，其濃度愈高所造成的DNA斷裂（DNA fragmentation）也愈嚴重。在(Ac)5GP的刺激下，PKC 與 會被轉位（translocate）至細胞膜並且活化。以流式細胞儀（flow cytometry）分析顯示，抑制PKC 可阻斷(Ac)5GP所引發的sub G1 peak細胞凋亡比率。然而以pseudopeptide抑制PKC 後，C6細胞的凋亡卻不受影響。此外，PKC 與 的mRNA總量並不受(Ac)5GP影響。上述結果顯示，(Ac)5GP所引發的細胞凋亡過程乃是透過PKC 的活化，而非PKC 的製造、合成來進行的。接著我們進一步探討PKC 的下游機轉。根據文獻顯示，c-Jun N端激脢（JNK）可能於PKC下游扮演了重要的調控角色，藉此引發細胞凋亡。因此，我們探討JNK是否與(AC)5GP所引發的細胞凋亡有關。結果顯示，(AC)5GP誘使JNK活化以及c-Jun磷酸化，進一步增加Fas-L與Fas的表現。以SP600125來阻礙JNK的活性，則(AC)5GP所引發的細胞凋亡與上述相關蛋白的表現皆受到抑制。此外，我們亦發現(AC)5GP透過JNK/ Jun/ Fas L/ Fas/ caspase 8/ caspase 3而引發細胞凋亡，但此一路徑並不牽涉到粒線體。我們又以rottlerin抑制PKC ，結果導致JNK無法活化，顯示JNK應位於PKC 下游，傳遞凋亡訊息。因此推論，(AC)5GP藉由PKC  /JNK /FasL等一連串訊息的活化而導致C6細胞死亡。綜合上述的研究成果，我們確定了PKC 以下的死亡傳訊路徑；然而其上的路徑仍不明瞭。由於我們所使用的C6細胞源自神經系統，文獻顯示，神經生長因子及其接受器（NGF、NGF R）可調控glioma cell產生凋亡，因此(AC)5GP引發C6細胞死亡，可能透過NGF R來進行。此外，某些文獻指出，神經醯氨（ceramide）可藉由調控PKC isoform而引發凋亡。因此，(AC)5GP是否透過細胞膜上NGF R/ ceramide活化PKC 而傳遞死亡訊息，成為我們接下來的工作目標。 Herbal medicine has been utilized to treat a variety of diseases, including cancer. On the other hand, disturbance of apoptosis is often observed in cancer cells. In the present study, we investigate the antitumor effect and possible mechanism of a herbal-originated product, (Ac)5GP. Since it has been reported that protein kinase C (PKC) isoforms are involved in the signaling of apoptosis, we investigate whether PKC isoforms could be involved in (Ac)5GP-induced apoptosis. We demonstrate that (Ac)5GP treatment results in DNA fragmentation of C6 glioma cells dose-dependently. Stimulated by (Ac)5GP, PKC  and  were activated and translocated to the cell membrane fraction. Flow cytometry analysis showed that PKC , but not PKC  inhibition blocks the (Ac)5GP -induced apoptosis by decreasing the cell population of sub G1 peak. However, the mRNA levels of PKC  and  were not altered by (Ac)5GP-induced glioma cell apoptosis. These results suggested that the treatment of (Ac)5GP induces apoptosis of tumor cells through the activation but not the synthesis of PKC . However, the downstream signal pathway of PKC  has not yet been investigated. It was shown that JNK may play an important role in the regulation of apoptosis and could be a possible downstream signal of PKC  isoforms. In the present study, we investigate whether JNK is involved in (AC)5GP induced apoptosis. The result reveals that (AC)5GP induces JNK activation and c-Jun phosphorylation thus stimulating the expression of Fas-L and Fas. Using SP600125 to block JNK activation shows that (AC)5GP-mediated apoptosis and related proteins expression are attenuated. Furthermore, we find that the (AC)5GP induces apoptosis through the activation of JNK/ Jun/ Fas L/ Fas/ caspase8/ caspase3, a mitochondria-independent pathway. The JNK pathway is suggested to be the downstream signal of PKC , since rottlerin impedes (AC)5GP-induced JNK activation. Therefore, (AC)5GP mediates cell death via activation of PKC  /JNK /FasL cascade signaling. In the present study, we identified the downstream signals of PKC  mediated in (AC)5GP-induced apoptosis, and the upstream signals upon PKC  will be clarified in the near future. Whether NGF R and ceramide are involved in (AC)5GP-induced apoptosis of C6 glioma cells need to be further investigated.
URI:	http://140.128.138.153:8080/handle/310902500/1524
Appears in Collections:	[生化微生物免疫研究所] 博碩士論文

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