標的導向胜肽-熱穩定溶血素重組蛋白表現 及其乳癌治療應用之研究

Abstract

熱穩定溶血素 (Thermostable direct hemolysin, TDH) 是一種穿孔毒素,由Grimontia hollisae弧菌生產。然而，這種蛋白毒素缺乏細胞選擇性，因此無法作為蛋白質藥進行應用。為了要提高對腫瘤的辨識專一性，並減少副作用的影響，我嘗試生產具有標靶腫瘤功能的熱穩定溶血素, 以作為合適的免疫毒素製劑。 由於突變後的TDHR46E生物活性以及副作用反應均降低，因此有潛力作為抗癌製劑。因此，本研究以TDHR46E的蛋白作為架構設計相應的TDH腫瘤標靶蛋白。在TDHR46E蛋白的C端接上標靶胜肽。為了將TDHR46E精確定位到腫瘤細胞上氨基肽酶P （Aminopeptidase P，APaseP）和神經氈蛋白-1（Neuropilin-1，Nrp-1）的受器，PEGA（P5）和iRGD（P6）被選定為乳腺癌標靶胜肽。 在細胞毒活性測試中使用的MDA-MB-231和MCF-7乳癌細胞株具有目標受器，且BT-474細胞株則無目標受器，進行Gh-TDHR46E-P5和Gh-TDHR46E-P6的細胞毒活性測試。結果顯示，Gh-TDHR46E-P5和Gh-TDHR46E-P6對MDA-MB-231和MCF-7表現明顯的細胞毒性，且BT-474細胞則否。與此相反，所有的腫瘤細胞系經Gh-TDHR46E處理後，皆無細胞毒性，這表示P5和P6胜肽與Gh-TDHR46E 的組合可以增強Gh-TDHR46E的細胞毒性效力。此外，P6可以提高滲透性增加治療效果。

Thermostable direct hemolysin (TDH) is a pore-forming toxin produced by Grimontia hollisae. However, this toxin protein lacks selective cytotoxicity. To specify the anti-tumor activity and reduce side effects, we tried to prepare TDH-tumor targeting toxin protein as a suitable immunotoxin. Because the Gh-TDHR46E mutation reduced bioactivities and side reactions, it has potential as an anticancer agent. In this study we used THD-based protein TDHR46E to design the corresponding TDH-tumor targeting proteins. At TDH R46E, C-terminal was added with targeting peptides. To target TDHR46E precisely to tumor cells, aminopeptidase P (APaseP) and neuropilin-1(Nrp1) receptor such as PEGA (P5), iRGD (P6) were selected as peptides that targeted breast cancer. The cytotoxic activity of Gh-TDHR46E-P5 and Gh-TDHR46E-P6 was evaluated using receptor-positive cell lines such as MDA-MB-231 and MCF7; receptor-negative tumor cell line such as BT474. The results showed apparent cytotoxic on MDA-MB-231and MCF7 cells but not on BT474 cells. In contrast, Gh-TDHR46E treatment was non-cytotoxic on all tumor cell lines, indicating enhanced cytotoxic efficacy of Gh-TDHR46E by combination of P5 and P6 peptides to Gh-TDHR46E. Furthermore, P6 can increase internalization to increase treatment effects significantly. These data may open new avenues for breast cancer therapy.