相思樹胰蛋白酶抑制劑抑制大腸直腸癌細胞侵入轉移能力

Inhibition of Invasion in Human Colon Adenocarcinoma Cells by ＂Acacia confusa＂ Trypsin Inhibitor

洪志宏(Chih-Hung Hung)；吳佳貞(Chia-Chen Wu)

相思樹胰蛋白酶抑制劑 ； ACTI ； Human colon cancer cells ； Cell invasion ； MMP-2 ； MMP-9

健康管理學刊

12卷1期（2014 / 06 / 01）

49 - 60

繁體中文

本篇是由相思樹（Acacia confusa）種子所分離的胰蛋白酶抑制劑（Acacia confusa trypsin inhibitor），簡稱ACTI，屬於Kunitz-type的胰蛋白酶抑制劑。以不同濃度的ACTI處理HT-29大腸直腸癌細胞，利用MTT試驗觀察細胞的存活率，以12.5 μM ACTI處理細胞48小時後，細胞的存活率下降至43.3 ± 3%，在72小時後更可以下降至34.6 ± 2.5%。所以HT-29細胞的存活率隨著處理ACTI的劑量及時間的增加而下降。在軟性瓊膠群落生長試驗 （soft agar colony growth assay）中，處理5 μM ACTI兩星期後，觀察群落之生成與型態，發現細胞群落聚集的現象有明顯的降低，只剩下38 ± 3%。利用侵入能力試驗（invasion test），ACTI在濃度5 μM處理細胞48小時後，抑制HT-29侵入性的能力達到48 ± 5%，再利用明膠蛋白酵素電泳法（gelatin zymography）可明顯觀察到在濃度5 μM ACTI處理細胞48小時後，可以抑制HT-29 cells分泌表現MMP-2及MMP-9的量，特別是MMP-9更明顯。因此ACTI抑制HT-29大腸直腸癌細胞侵入性的能力，可能經由抑制MMP-2及MMP-9的分泌表現量。故本研究闡釋ACTI有潛力發展成臨床抑制細胞轉移能力的藥劑。

Acacia confusa trypsin inhibitor (ACTI) is a Kunitz family 2-chain trypsin inhibitor with anMr of 19.4 kD. The cytotoxic effects of ACTI on human colon cancer cells (HT-29) have been demonstrated, but the exact mechanism by which ACTI induces these effects is not completely understood. In this study, we found that ACTI inhibited the growth of HT-29 cells in a time- and concentration-dependent manner based on MTT test results. A soft agar colony growth assay revealed that the number of colonies decreased with increasing concentrations of ACTI. To confirm the effects of ACTI, an invasion assay was conducted. Treating HT-29 cells with up to 5 μM of ACTI decreased cell invasion significantly-by 48% ± 5% for 48 h. Gelatin zymography assay results revealed that ACTI exerted a significant inhibitory effect on the MMP-9 activity of HT-29 cells, but only slightly reduced MMP-2 activity. The results suggested that ACTI suppression of the invasive ability of colorectal cancer HT-29 cells might involve its inhibition of the expression of MMP-2 and MMP-9. Our results indicated that ACTI has potential in the clinical treatment of cancer metastasis.