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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 - PowerPoint PPT Presentation

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PowerPoint Slideshow about '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' - dolan-blanchard

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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 life)。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(apoptotic 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Colo205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, caspase, 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。


Studies on the Molecular Mechanisms of Cyprohepatadine-induced Apoptosis and G0/G1 cell-cycle arrest in Human Colon Cancer Cells

  • Cancer is characterized by unregulated cell proliferation caused by DNA defect. Therefore, chemotherapy agents targeting at cancer cells are almost designed to interfere regulating process of cell cycle and apoptosis. However, chemotherapy agents used today encounter many adverse drug effects, which severely interfere patients’ quality of life. Therefore, this study was dedicated to screening medications that are not initially designed to treat colon cancer and safely used in clinic with less cytotoxicity in order to find out medications that might cause cell-cycle arrest and induce apoptosis of colon cancer cells in vitro. This study 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 with individual medications, incubated cancer cells were observed for DNA laddering through electrophoresis. The medication that passes the screening process was then reacted with cancer cells for one to three days in various doses. Cell counts were gathered each day for generating cell growth curves. A flow cytometery was employed to investigate the proportions of cells at each 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 inhibited 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 increased as the dose of Cyprohepetadin increased. The results also indicated that DNA has been induced to form the apoptotic body and that the cells have undergone apoptosis. Western blot assay showed that the concentration of p53, p27,Bad, Bax protein and cytochrome c was increased. Maybe this drug induced the cell apoptoisis is through p53 ,mitochondrium, and caspase dependent pathway and G0/G1 arrest through p27 protein inhibiting effect. The results of this study have proved that Cyprohepatadine can induce apoptosis and cell-cycle arrest of colon cancer cells in vitro. The results of this study can also contributed to future development of chemotherapy agents.