Cyprohepatadine 誘發人類大腸癌細胞凋亡及細胞週期停滯分子機制之研究

癌症是由於基因的缺損而造成不正常的增生而成﹐在臨床上﹐針對癌細胞的化學療法﹐大都是由影響細 胞週期的分子調控著手﹐冀望能因此影響 DNA 完整性﹐進而造成細胞週期停滯，誘發癌細胞的凋 亡 (apoptosis) 。臨床上的化療藥物卻有令人詬病的副作用﹐嚴重影響到病患的生活品質 (quality of l ife) 。本研究是針對人類大腸癌細胞 colo205﹐ 經由篩選臨床上已安全地使用於非治療大腸癌的藥 物﹐希望能找到藥物在體外誘導大腸癌細胞的凋亡。本研究是由四個部分所組成： DNA 裂解及梯 度 (DNA fragmentation and laddering) 、細胞生長曲線 (cell growth curve) 、細胞週期及凋亡體 (apopt otic body) 偵測，及西方墨蹟法 (Western Blot assay) 。有 15 項藥品被選用於初步的篩選﹐再與培養 的 colo205 細胞作用後﹐以電泳方式觀察有無 DNA laddering 現象 ( 此為凋亡之特有性表徵 ) ，接著 將篩選出的藥品﹐依濃度梯度與培養的 colo205 細胞作用一至三天﹐並於每天計算細胞總個數以求 出細胞生長曲線。再以流式細胞儀 (Flow cytometer) 分析在細胞週期中﹐各細胞週期佔所有細胞的 比率及凋亡體 (apoptotic body) 發生之比率。電泳的結果顯示﹐僅有經 Cyprohepatadine 處理過的 Col o205 細胞﹐產生 DNA laddering 的現象﹐而且其電泳結果的 laddering 清晰度隨著藥物劑量的增加而 增加。細胞生長曲線的結果亦顯示﹐即使是 20mM 的 Cyprohepatadine﹐ 也能成功地在體外抑制大 腸癌細胞的增生﹐而 80mM 以上的 Cyprohepatadine 更能在 24 小時的時間內就已完全誘殺 Colo 205 細胞。在流式細胞儀的分析結果中﹐ Sub-G1 峰的 apoptotic body 隨著 Cyprohepatadine 劑量的增加 而增加﹐ G1 峰的比例隨著 Cyprohepatadine 劑量的增加而增加 , 這顯示出 Colo205 被誘導進入停滯 於 G1 細胞週期 , 形成 apoptotic body 而進入凋亡。 Western blot assay 顯示 p53,p27, cytochrome C, c aspase, Bad,Bax, 及 PARP 隨著 Cyprohepatadine 劑量的增加而增加 , 這暗示出 Colo205 是經由 p27 抑制 cyclin E-cdk2 , 以抑制其促進 G1 轉向 S 期 , 被誘導進入停滯於 G1 細胞週期 ; 經由 p53 路徑 , 間接或直接促 mitochondria 釋放出 cytochrome c, 啟動 caspase pathway, 進而形成 apoptotic body 而進 入凋亡。本研究的結果顯示﹐ Cyprohepatadine 確可在體外誘發大腸癌細胞的凋亡。此結果可做為 日後發展化療藥物的參考。

Cyprohepatadine 誘發人類大腸癌細胞凋亡及細

胞週期停滯分子機制之研究

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Cancer is characterized by unregulated cell proliferation caused by DNA defect. Therefore, chemotherapy agents targeting at c ancer cells are almost designed to interfere regulating process of cell cycle and apoptosis. However, chemotherapy agents use d today encounter many adverse drug effects, which severely interfere patients’ quality of life. Therefore, this study was dedic ated to screening medications that are not initially designed to treat colon cancer and safely used in clinic with less cytotoxicit y in order to find out medications that might cause cell-cycle arrest and induce apoptosis of colon cancer cells in vitro. This st udy comprises four major investigations: DNA fragmentation and laddering, Cell growth curve, cell-cycle arrest and detection of apoptotic body and Western blot assay. There were 15 medications employed in the screening process. After reacting wit ﹐ h individual medications, incubated cancer cells were observed for DNA laddering through electrophoresis. The medication th at passes the screening process was then reacted with cancer cells for one to three days in various doses. Cell counts were gath ered each day for generating cell growth curves. A flow cytometery was employed to investigate the proportions of cells at ea ch stage of cell cycle and apoptotic body in sub-G1 peak. The Western blot assay was used for detecting protein of cell-cycle associated and apoptosis. The results of electrophoresis have demonstrated that DNA laddering could only be found in cancer cells treated by Cyprohepatadine. In addition, DNA laddering became more obvious as the dose of Cyprohepatadine increased . The results of cell growth curve also indicated that, even at the dose of 20mM, Cyprohepatadine has still successfully inhibit ed proliferation of colon cancer cells in vitro. Furthermore, Cyprohepatadine could significantly inhibit proliferation of colon cancer cells in 24 hours at the dose of above 80mM. The results of flow cytometry have shown that the area of sub-G1 has inc reased as the dose of Cyprohepetadin increased. The results also indicated that DNA has been induced to form the apoptotic b ody and that the cells have undergone apoptosis. Western blot assay showed that the concentration of p53, p27,Bad, Bax prote in and cytochrome c was increased. Maybe this drug induced the cell apoptoisis is through p53 ,mitochondrium, and caspase d ependent pathway and G0/G1 arrest through p27 protein inhibiting effect. The results of this study have proved that Cyprohep atadine can induce apoptosis and cell-cycle arrest of colon cancer cells in vitro. The results of this study can also contributed t o future development of chemotherapy agents.

Studies on the Molecular Mechanisms of

Cyprohepatadine-induced Apoptosis and G0/G1

cell-cycle arrest in Human Colon Cancer Cells