相思樹胰蛋白酶抑制劑誘導人類結腸腺癌細胞（SW480）凋亡之研究

Induction of Apoptosis by Acacia confusa Trypsin Inhibitor in Human Colon Adenocarcinoma Cancer Cells (SW480)

本研究主要探討相思樹（Acacia confusa）種子所分離出的胰蛋白酶抑制劑（Acacia confusa trypsin inhibitor|簡稱ACTI）誘導人類結腸腺癌細胞（SW480）凋亡。首先從相思樹種子中純化出ACTI，分子量約20kDa，是屬於Kunitz-型抑制劑。以不同的ACTI濃度處理SW480細胞，利用trypan blue dye exclusion assay偵測細胞存活率，發現隨著ACTI濃度及處理的時間增加，細胞的存活率逐漸減少，在10μM ACTI處理48小時後，細胞存活率下降至83.6%，處理72小時後細胞存活率下降至70%|利用annexin V來檢測細胞凋亡（apoptosis），隨著ACTI處理SW480細胞的濃度及時間增加，細胞進行凋亡有明顯增加的趨勢|利用西方轉漬法（western blotting）分析，發現ACTI處理SW480細胞後會導致Bcl-2蛋白質表現量降低，Bax、cleavage-caspase-9、cleavage-caspase-3及cleavage PARP增加，cytochrome c由粒線體釋出至細胞液的量增加。因此，ACTI抑制SW480細胞生長，是藉由誘導SW480 cells走向細胞凋亡路徑。

The main specific aim of this study is to study the properties of Acacia confusa trypsin inhibitor（ACTI）isolated from the seeds of Acacia confusa. First, ACTI was purified from seeds of Acacia confusa. The molecular weight of ACTI is about 20 kDa. It belongs to Kunitz-type inhibitor. The human SW480 colon adenocarcinoma cells were treated with different concentrations of ACTI and the cell survival rates were analyzed by trypan blue dye exclusion assay. The cell survival rate decreased as the ACTI concentrations increased at 48 hours. The cell survival rate decreased to 83.6％ at 10 μM and to 70％ after 72-hour incubation. The apoptosis of SW480 cells was detected with Annexin V. In treatments of SW480 cells with different ACTI concentrations, apoptotic effects were detected. As the time of ACTI treatments increased, the Annexin-positive cells increased significantly. ACTI was able to induce SW480 cells to apoptosis. On the other hand, by western blotting, it was found that ACTI induced Bax activation, Bcl-2 down regulation, cleavage- caspase-9 activation, cleavage- caspase-3 activation, cleavage PARP activation and the increased cytochrome c releasing from mitochondria, as well as indicated that ACTI drove cell to apoptosis. Therefore, ACTI inhibits the human colorectal cancer cell growth by inducing cells to apoptotic pathways.