相思樹胰蛋白酶抑制劑藉由抑制ERK 1／2磷酸化和活化caspase-3誘導HT-29人類結腸癌細胞凋亡

Acacia confusa Trypsin Inhibitor Induced Apoptosis of HT-29 Human Colon Cancer Cells via Inhibition of ERK 1/2 Phosphorylation and caspase-3 Activation

相思樹胰蛋白酶抑制劑（ACTI）是屬於Kunitz型胰蛋白酶抑制劑家族，由兩條多肽鏈組成，分子量為19.4 kDa。ACTI對人類結腸癌細胞（HT-29）表現出的細胞毒性作用確切機制尚不清楚。我們分別利用MTT和流式細胞儀分析證實ACTI能夠抑制HT-29細胞的生長和誘導細胞凋亡，隨著處理ACTI的濃度及時間增加而愈顯著。在濃度為5 μM ACTI處理HT-29細胞48小時後，caspase-3活性增加，加入Z-VAD-fmk（一種廣效型caspase抑制劑）能阻止ACTI誘導的細胞凋亡；而以PD98059（一種ERK 1／2抑制劑）處理，則能促進ACTI誘導的細胞凋亡。另外，以ACTI處理HT-29細胞會抑制ERK 1／2的磷酸化並促進細胞色素c釋放到細胞液中。此外，先以PD98059處理HT-29細胞後，能促進ACTI誘導caspase-3活性。這些結果顯示，ACTI可藉由抑制ERK 1／2磷酸化，使得位於粒線體中的細胞色素c釋放到細胞液，進而活化caspase-3而引起HT-29細胞凋亡。

Acacia confusa trypsin inhibitor (ACTI) is a Kunitz-type family two-chain trypsin inhibitor with an MWr of 19.4 kD. ACTI exhibits cytotoxic effects on human colon cancer cells (HT-29) although the exact mechanism of this action of ACTI is unknown. We report here that ACTI strongly inhibits the growth and induced apoptosis of HT-29 cells in both a time- and concentration-dependent manner, determined by MTT and flow cytometric analysis, respectively. Caspase-3 activity increased 48 h after treatment with ACTI at 5 μM concentration. Caspase inhibitor, Z-VAD-fmk, prevented ACTI-induced apoptosis, while PD98059, an extracellular signal-regulated kinase 1/2 (ERK 1/2) inhibitor, promoted ACTI-induced apoptosis. Additionally, treatment with ACTI inhibited the phosphorylation of ERK and promoted the release of cytochrome c into cytosol. Furthermore, pretreatment with PD98059 further promoted ACTI-induced caspase-3 activity. These results show that ACTI causes the apoptosis of HT- 29 cells by inhibiting ERK 1/2 phosphorylation, releasing cytochrome c into the cytosol and, in turn, activating caspase-3.