探討Cardiotoxin III對Colo 205大腸癌細胞株細胞凋亡的作用機制

Cardiotoxin III( CTX III )由60個胺基酸殘基組成，從台灣眼鏡蛇毒( Naja naja atra venom )中分離。本實驗研究主要的目的是在探討台灣眼鏡蛇心臟毒素Cardiotoxins III( CTX III )對大腸癌細胞株Colo 205 cells的毒殺作用，並研究作用機制。首先利用MTT分析試驗，來檢測CTX III對於人類大腸癌細胞株Colo 205 cells 的影響。研究結果顯示：CTX III劑量遞增，細胞存活率會遞減，呈現明顯劑量-效應關係，並在MTT分析法中測出大腸癌Colo 205 cells之IC50 為4 ug/ml。本研究亦利用流式細胞儀技術，偵測細胞內DNA的分布，及其細胞週期停滯情形。結果顯示：CTX III有明顯抑制細胞週期停滯在S期( S phase )，且隨著劑量的增加sub G1 fraction的比例亦會明顯的增加，利用膠體電泳( Agarose gel electophoresis )測定DNA斷裂情形( DNA fragmentation )，結果顯示出加入CTX III確實會造成DNA ladder的產生，以上結果顯示Colo 205 cells經由細胞凋亡的途徑使細胞死亡。經由流式細胞分析儀，西方點墨法，及Caspase活性測定，顯示以CTX III處理Colo 205癌細胞後，粒線體膜電位下降Cytochrome C釋出，Caspase-3, -8, -9被活化，以及Bax蛋白質表現增加，但Bcl-2的表現不受影響。以流式細胞分析儀測定細胞內ROS的表現並不增加，同時以抗氧化劑N-acetylcysteine或Catalase預先處理，並不改變細胞的存活率。顯示CTX III並不經由ROS的作用機制，以Z-VAD-FMK，Caspase抑制劑預先處理，再加入CTX III，會抑制細胞Sub G1 fraction的增加。綜合以上結果顯示，本研究發現台灣眼鏡蛇心臟毒素Cardiotoxins ( CTXs )中CTX III對大腸癌細胞株Colo 205 cells的存活率有抑制情形，且呈現劑量-效應依存關係；此外，實驗結果明顯得知，CTX III的作用機制經由粒線體，Caspase-3, -8, -9及Bax/Bcl-2比率之增加而引起Colo205細胞凋亡現象。

關鍵字

細胞凋亡；眼鏡蛇毒心臟毒素III ；大腸癌細胞

並列摘要

Cardiotoxin III ( CTX III ) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human Colo 205 cells . CTX III was found to inhibit the growth of Colo 205 cells in a time-and dose-dependent manner with IC50 value of 4μg/ml, and displayed several features of apoptosis including apoptotic body formation, increase of sub G1 fraction, DNA fragmentation. Investigation of the mechanism of CTX III-induced apoptosis revealed that the treatment of Colo 205 cells with CTX III resulted in the loss of mitochondrial membrane potential( △Ψm ), cytochrome c release from mitochondria into cytosol, and activation of caspase-9, caspase-8, caspase-3 and also markedly enhanced the expression of Bax but not Bcl-2 protein in the cells. However, CTX III did not generate reactive oxygen species( ROS ), and antioxidants including N-acetyl cysteine and catalase could not block CTX III-induced apoptosis in the Colo205 cells. Moreover, the CTX III-induced apoptosis was markedly blocked by the broad-spectrum caspase inhibitor, Z-VAD-FMK. Taken together, the findings suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells. Key words: Cardiotoxin III, Apoptosis, Caspase, Colo205 cells