薑黃素(curcumin)是植物薑黃(turmeric)內主要具活性的成分,包括抗發炎、抗氧化、抗癌症轉移、誘導癌細胞計畫性死亡等活性。本篇研究探討薑黃素及其二十四個結構類似物抑制HT-29人類大腸癌細胞株增生和誘導細胞計畫性死亡的相關機制,並希望能篩選出比薑黃素更具細胞毒性之結構類似物。經由細胞存活率測試(resazurin assay),發現結構上具有一個甲氧基和三個羥基的七號結構類似物具顯著毒性,且有濃度依存性、時間依存性的現象;同時也比薑黃素較具有顯著的毒性,於是進一步探討與細胞毒性相關的研究。為了解七號結構類似物與細胞凋亡的關聯性,觀察細胞凋亡具代表性的特徵,在濃度40 ?嵱下,以光學顯微鏡觀察24小時候的HT-29細胞,觀察到細胞凋亡時會有的細胞膜突泡(blebbing)現象;利用流式細胞儀觀察細胞外膜Annexin V的結合量隨著七號結構類似物濃度的增加而增加,顯示細胞膜內的磷脂質絲胺酸(Phosphatidylserine)外翻的情形。以Hoechst 33342的染色下,可以看到染色體濃縮(condensation)的現象;在20 ?嵱下處理24 小時,觀察到HT-29細胞的G0/G1細胞週期停滯,40 ?嵱下處理24小時觀察到HT-29細胞有sub G1產生的現象。根據以上觀察到細胞凋亡時會產生的特徵,於是進一步了解細胞凋亡時之訊息傳導途徑及相關的蛋白質表現量。結果顯示,七號結構類似物會誘導caspase 3的活化,進一步活化caspase 9和caspase 8;粒線體膜電位的下降;Bcl-xl和Bcl-2的蛋白質表現量下降。而加入抗氧化劑(N-acetylcysteine, NAC)抑制ROS產生,細胞存活率明顯上升。顯示七號結構類似物可能會引起人類大腸癌細胞產生活性氧族及氧化逆境而進一步誘導走向細胞凋亡的現象。
Curcumin is a main bioactive constituent in turmeric which is used as food additives or herbs in southeastern Asia and India for a long time. It has been reported to exhibit anti-inflammatory, antioxidant, anti-metastatic, and apoptotic activities. In this study, curcumin and its 24 different kinds of analogs were selected to evaluate anti- HT-29 human colorectal adenocarcinoma and possible mechanisms. According to results of cell viable screenings (resazurin assay), it was found that analog 7 showed much higher growth inhibitory activities against HT-29 cells than that of curcumin under 20 ?嵱 and was selected as a candidate for further experiments. It was found that analog 7 showed dose-dependent and time-dependent toxicity against HT-29 cells. Therefore, the possibilities between toxicity of analog No.7 toward HT-29 cells and induction of apoptosis were investigated. Several hall marks of apoptosis were examined as followings. The blebbing of HT-29 cell membrane was observed by light microscopy under 40 ?嵱 for 24 h treatments. The annexin-V was dose-dependently bound to outer membranes of treated-HT-29 cells observed by the flow cytometry, which meant that the phosphatidylserine was externalized from inner membranes to outer membranes; the DNA condensed phenomena of HT-29 cells were found after being treated with different concentrations of analog No.7 which was stained with Hoechst 33342 fluorescent dyes and observed under UV-light microscopy; the cell cycles of G0/G1 was arrested and sub-G1 was appeared in treated HT-29 cells which were showed that analog No.7 could induce apoptosis in HT-29 cells. To study the possible mechanisms of apoptosis induced by analog No.7, the related apoptotic proteins in the intrinsic pathway and the extrinsic pathway were detected either by activity assays or by the western blotting. It was found that activities of casapase 3, caspase 8, and caspase 9 were increased, and the protein expression of PARP, Bcl-2, and Bcl-xl were dose-dependently reduced, and the mitochondrial membrane potentials were also reduced observed by JC-1 stains. To clearly understand the roles of reactive oxygen species (ROS) in apoptosis during analog No.7 treatments, the N-acetylcysteine (NAC) was used to compare the cell viability. It was found that 10 mM NAC pretreatment could increase the cell viability of HT-29 cells after treated with different concentrations of analog No.7 compared to the drug-treated one. It was proposed that analog No.7 treatment could increase ROS in HT-29 cells and then to produce oxidative stresses which in turn to induce apoptosis through intrinsic and extrinsic pathways.