戊乙醯去羥梔子苷活化Sphingomyelinase/ NGF Receptor/ PKCδ誘發C6神經膠瘤細胞凋亡之作用

Penta-acetyl geniposide induce apoptosis in C6 glioma cells by modulating the activation of neutral sphingomyelinase-induced p75 NGF receptor and PKCδ pathway

許淑萍(Shu-Ping Hsu)

指導教授 ： 王朝鐘

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00081

摘要

過去的研究中，本實驗室探討戊乙醯去羥梔子苷（penta-acetyl geniposide, (Ac)5GP）這項中草藥的抗癌效用與其可能機轉。之前已證實，(Ac)5GP可透過活化PKCδ/ JNK/ Fas/ caspases路徑，來誘導C6神經膠瘤細胞凋謝死亡。然而，有關(Ac)5GP於PKCδ之上的訊息傳導路徑仍不明瞭。研究成果顯示，(Ac)5GP在作用15分鐘左右，即可活化C6細胞內的中性鞘磷脂酵素（neutral sphingomyelin, N-SMase）至最大效應。在處理N-SMase抑制劑（GW4869）之下，發現NGF和p75的蛋白表現都受到抑制，顯示出NGF/ p75為N-SMase/ ceramide之下游。為了研究N-SMase是否參與(Ac)5GP引發C6細胞凋亡的訊息路徑中，細胞在處理GW4869抑制劑後，發現GW4869抑制了Fas-L和caspases 3蛋白表現。同樣地，p75拮抗胜肽也阻斷了Fas-L和caspases 3蛋白表現。而(Ac)5GP所誘導的PKCδ轉位現象也被GW4869和p75拮抗胜肽所消除。為了再確認N-SMase的活化，是否在(Ac)5GP引發C6細胞凋亡中扮演很重要的角色，利用DAPI staining實驗方法來分析細胞凋亡，發現10 μM 和20 μM 的GW4869，分別降低40 % 和80 % 由(Ac)5GP引發C6細胞凋亡。因此推論，(Ac)5GP藉由N-SMase/ ceramide增加NGF/ p75的表現，進而引發PKCδ轉位與下游Fas-L/ caspase cascades等訊息的活化而導致C6細胞死亡。

關鍵字

戊乙醯去羥梔子苷SMase ； N-Smase ； A-Smase ； 神經生長因子 ； 神經生長因子接受器 ； p75 ； 蛋白激酶Cδ

並列摘要

In our previous studies, we demonstrated the apoptotic cascades PKCδ/ JNK/ Fas/ caspases induced by penta-acetyl geniposide ((Ac)5GP). However, the upstream signals mediating PKCδ activation have not yet been clarified. In the present study, (Ac)5GP showed to activate neutral sphingomyelinase (N-SMase) immediately with its maximum at 15 min. The NGF and p75 enhanced by (Ac)5GP was inhibited while added with GW4869, the N-SMase inhibitor, indicating NGF/ p75 as the downstream signals of N-SMase/ ceramide. To investigate whether N-SMase is involved in (Ac)5GP-transduced apoptotic pathway, cells were treated with (Ac)5GP added with or without GW4869. It showed that N-SMase inhibition blocked Fas-L expression and caspase 3 activation. Similarly, p75 antagonist peptide attenuated the Fas-L/ caspase 3 expression. The PKCδ translocation induced by (Ac)5GP was also eliminated by GW4869 and p75 antagonist peptide. To further confirm if N-SMase activation plays an important role in (Ac)5GP-induced apoptosis, cells were analyzed the apoptotic rate by DAPI staining. (Ac)5GP-induced apoptosis was reduced 40 % and 80 % by 10 μM and 20 μM GW4869 respectively. It indicated that N-SMase activation is pivotal in (Ac)5GP-mediated apoptosis. In conclusion, N-SMase and NGF/ p75 are suggested to mediate upstream above PKCδ, thus transducing Fas-L/ caspase cascades in (Ac)5GP-induced apoptosis.

並列關鍵字

(Ac)5GP ； SMase ； N-Smase ； A-Smase ； NGF ； NGFR ； p75 ； PKCδ

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