- 學位論文
在 Hct116 細胞中探討 Daxx 對吲哚類化合物所誘發細胞毒性之影響
Expression of Daxx Alters Cellular Toxicity Induced by Indole Compound in CRC Cells
摘要
台灣的大腸直腸癌致死率高居十大癌症的第三名，大腸直腸癌大部分是由腺瘤息肉逐漸發展而成，早期不易被診斷發現，晚期則已經發生轉移且癒後能力差。近幾年發現日常飲食與大腸直腸癌有很大的關聯性，有研究顯示天然蔬果中所含的天然化合物對於抑制腫瘤的發展有相當大的關聯性，其中包含十字花科類蔬菜，如高麗菜、花椰菜、萵苣等，都富含許多吲哚化合物，Indole-3-carbinol (I3C) 及其代謝產物3,3’-diinolylmethane (DIM) ，均已被證實會透過 MAPK (Mitogen-activated protein kinase) 路徑的訊息傳遞及粒線體凋亡路徑，促使癌細胞走向凋亡。我們實驗室先前在大腸直腸癌組織中發現 Death-associated protein 6 (Daxx) 蛋白表現量與正常組織相比之下有減少趨勢，因此我們推測在大腸直腸癌中 Daxx 蛋白的表現與癌化程度及癌細胞的轉移扮演舉足輕重的角色。本篇論文利用 shRNA 將大腸直腸癌細胞株 Hct116 的 Daxx 蛋白表現靜默，命名為 sh-Daxx，控制組為sh-Luciferase (sh-Luci)，探討 Daxx 蛋白對大腸直腸癌細胞株 Hct116 中處理吲哚化合物造成之影響及其路徑。 首先利用細胞計數方式，觀察處理 I3C 和 DIM 不同劑量及時間後，sh-Daxx 細胞較 sh-Luci 細胞之耐受性佳。結果顯示 Daxx 蛋白會促使吲哚化合物對 Hct116 細胞抑制生長。此外我們利用西方點墨法釐清，Daxx 蛋白的表現會促進吲哚化合物對 Hct116 細胞抑制生長，是透過 MAPK 及粒線體凋亡分子的活化，繼而走向 Caspase-8 的凋亡路徑。尤其在 DIM 50µM 的處理後，其 p-p38 蛋白在兩株癌細胞表現量都顯著增加，其中 sh-Luci 癌細胞株更為明顯。因此我們認為 Daxx 蛋白的表現在處理吲哚化合物造成大腸直腸癌細胞株生長抑制，透過活化 Caspase-8 傳遞路徑中扮演重要的角色。
並列摘要
Colorectal cancer (CRC) is the most prevalent cause of cancer death and incidence in Taiwan. It has been known for several decades that carcinomas mostly develop from adenomas, early symptom is not easy to diagnosis, but lately stage almost has metastasis and worse prognosis. Incidence of CRC has been considered to be related to daily food strongly. Chemoprevention by dietary compounds has received considerable attention as an effective approach for cancer prevention. Among the dietary indole compound indole-3-carbinol (I3C) present in the Brassica plants, including broccoli, cabbage and cauliflower, I3C is biologically active, and it is converted into 3,3’-diindolylmethane (DIM) under pH5-7, which is also biologically active. In various cancer cells, I3C and DIM has exhibited anti-cancer effects, including cell cycle inhibition, apoptosis. In our previous study, we suggested expression of Daxx may play an important role in CRC carcinogenesis and metastasis. It is interesting to investigate what a different cellular response with indole compound intake in different expression level of Daxx in Hct116. So we established sh-Daxx Hct116 cells (Daxx stable knock down cell line) and sh-Luci Hct116 cells (stable control cell line) to clarify this question. The cellular viability had been measured by trypan blue stain. Daxx knock down cell had a better tolerability with indole compound intake and suggested Daxx promote cellular death induced by indole compound. Furthermore, the induction of Caspase-8 mediated apoptosis in sh-Luci and sh-Daxx stable cells treated with indole compound was different and detected by western blot. We had also shown that expression of MAPK and pro-apoptotic proteins like p-p38 were increased by DIM treatment. Above of all findings, expression of Daxx might alter cellular response of indole compound through activation of Caspase-8. We demonstrate that I3C and DIM-induced apoptosis in Hct116 cells. Daxx play an important role, resulting in the induction of the Caspase-8 pathway.