| 摘要: | GSNO(S-nitrosoglutathione) 為細胞內抗氧化物GSH攜帶一氧
化氮之化合物，當它釋出一氧化氮時，可對細胞造成DNA
傷害。已有許多報告指出一氧化氮自由基可造成細胞凋亡，
但GSNO引起細胞凋亡之詳細機制尚未被研究清楚。在本實
驗中，我們使用人類腸癌細胞株，來探討GSNO對細胞之毒
性及細胞凋亡之機制。
細胞凋亡(Apoptosis)為一種細胞自殺性之死亡方式，其細
胞型態的變化包括細胞膜形成皰狀、染色質濃縮及Apoptotic
bodies之形成等等；本實驗我們依型態變化、TUNEL Assay
及DNA段片形成(DNA fragmentation) 等現象來判斷發現以
GSNO處理腸癌細胞株(HT29，Colo205) 確實會引起細胞凋亡
。我們亦發現，細胞給予GSNO處理之同時，若一起加入銅
離子(Cu++)，則細胞死亡率較單獨處理GSNO明顯減少。據
此我們亦證實了Cu++在細胞外可促使GSNO釋出NOo，由於
NOo不穩定，在極短時間內轉變為硝酸鹽及亞硝酸鹽，因而
降低進入細胞內NOo之量，而減少細胞的傷害。
NOo調控細胞凋亡過程中之基因表現，是我們想要嘗試了
解的，以西方墨點分析來偵測基因表現的變化，結果發現
Bad、Bax及c-Jun等蛋白的表現皆有增加之現象，而p27及Bcl-2
的表現卻有被抑制的現象。為了解NOo在參與細胞內訊息傳
遞所扮演之角色，我們也對PKA及PKC之表現進行探討，結
果發現PKA與PKCζ之表現會被NOo抑制，此結果告訴我們
在GSNO引起之細胞凋亡中，存在一種特殊的調節機制，而
PKA及PKCζ可能扮演一重要角色。
GSNO(S-nitrosoglutathione) is an intracellular NO donor. According
to previous studies, NO induced apoptosis was demonstrated. However,
the detail mechanism of apoptosis induced by GSNO is not yet understood.
In this report, GSNO synthesized in vitro was used to study the mechanism
of cellular toxicity and apoptosis in human colon adenocarcinoma cell lines.
Apoptosis is cells executed through a suicide program that show
distinctive morphological changes, including membrane blebbing, chromatin
condensation, and formation of apoptotic bodies. We realized that apoptosis
was induced by GSNO in HT29 and Colo205 cell lines, by examination of
morphological changes, Tunel Assay, and DNA ladder. We also found that
the mortality rate in cells treated by GSNO was obviously decreased when
simultaneous treated with copper ions. We suggest that Cu++ promote
extracellular NO release from GSNO and thus leading to decrease intracellular
NO exposure. Because NO is rapidly converted to nitrate and nitrite in the
solution phase, measurement of nitrite can serve as an indirect method to
evaluate NO production. In our study, we demonstrated that GSNO was
decomposed in the presence of Cu++ by Nitric Oxide Assay.
We wondered whether some proteins which expression induced by GSNO
could modulate the apoptosis. To test this hypothesis, we treated the
cells with GSNO in a time-dependent manner, and detected protein expression
by using western blot. In this study, we demonstrated that the Bad and Bax
protein were induced, in contrast, p27 and Bcl-2 protein were inhibited in
HT29 cells treated by GSNO. On the other hand, induction of c-Jun by GSNO
was also observed in a time-dependent manner. In order to investigate the
roles of NO involve in signal transduction in HT29 cells. We also studied
the relationship between NO-induced apoptosis and PKA or PKC protein
expression. We found that the isoenzyme of PKC--PKCζ, and PKA were down
regulated during apoptosis. Our results show that a specific modulatory
mechanism was observed in GSNO-induced apoptosis. The PKCζ and PKA may
play an important role
in this process. |
