以蛋白質體學探討雜環胺Trp-P-1處理人類肝癌細胞株HepG2

Abstract

摘要 雜環胺 ( Heterocyclic amines；HCAs ) 已被證實為高度致突變及致癌物質，而Trp-P-1為HCAs中毒性較強的種類之一。HCAs之毒性是經細胞色素 cytochrome P-450之活化後產生。本實驗是以蛋白質體學來研究以Trp-P-1處理人類肝癌細胞株 HepG2對其蛋白質表現之影響。細胞實驗結果顯示，Trp-P-1處理濃度大於2 µg/mL 時生長抑制率會隨天數增加而有明顯上升之趨勢，且以3、4、5 µg/mL Trp-P-1處理至第三天後，其生長抑制率已達90 % 以上。蛋白質體學分析顯示，HepG2以2 µg/mL Trp-P-1處理1、2、3天，蛋白質表現量增加且有50 % 以上差異者於第1天有6 個點、第2天有9個點、第3天有11個點；而表現量減少且有50 % 以上差異者於第2天有3個點、第3天有4個點。將這些表現量差異性大於50 % 之蛋白質點以胰蛋白酶 ( trypsin ) 進行膠內水解後以基質輔助雷射吸附游離離子化飛行時間 ( MALDI-Q-TOF ) 質譜儀進行分析，並經資料庫的比對以鑑定出蛋白質的種類及功能。結果在第1天處理組中有4個點被鑑定出；第2天處理組中有10個點被鑑定出；第3天處理組中有13個點被鑑定出，這些蛋白質功能分析顯示，Trp-P-1處理所造成的細胞生理變化主要以能量代謝循環及維持蛋白質結構為主。

Abstract Heterocyclic amines have been proved to be highly mutagenic and carcinogenic due to the activation of cytochrome P-450 in vivo. Among them, Trp-P-1 is one of the potent toxic heterocyclic amines. In the present study, proteomics on the protein expression of HepG2 cells following treatment with Trp-P-1 were conducted. In vitro, the growth inhibition of HepG2 cells increased in a time-dependent manner after treatment with Trp-P-1 at a level higher than 2 µg/mL. More than 90 % cells were inhibited when treated with 3, 4 or 5 µg/mL Trp-P-1 for 3 days. In proteomic analysis, 6, 9, and 11 protein spots were observed to be increased in quantity by more than 50 % if HepG2 cells were treated with 2 µg/mL Trp-P-1 for 1, 2, and 3 days, respectively. However, protein expression decreased in quantity by more than 50 % were determined to be 3 and 4 protein spots treated with the same amount of Trp-P-1 for 2 and 3 days, respectively. These protein spots were in gel digested by trypsin and analyzed by matrix assisted laser desorption ionization – time of flight ( MALDI-TOF ) mass spectrometry and 4, 10, and 13 proteins were successfully identified in day-1, -2, and -3 sample, respectively. Internet database ( Swiss-Prot ) search indicated that the changes in cell physiology induced by Trp-P-1 were mainly due to the influence of protein functions on energy metabolism cycle and protein structure maintain.