中藥砒霜對口腔癌細胞毒殺作用機制之探討

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題名:	中藥砒霜對口腔癌細胞毒殺作用機制之探討 The Cytotoxic Effects of Arsenic Trioxide on Oral Cancer Cells
作者:	蔡佳紋;Chia-Wen Tsai
貢獻者:	中醫學院中國醫學研究所碩士班
關鍵詞:	三氧化二砷;dithiothreitol;毒殺作用;口腔癌;檳榔鹼;arsenic trioxide;dithiothreitol;cytotoxicity;oral cancer;arecoline
日期:	2010
上傳時間:	2010-09-29 12:00:02 (UTC+8)
摘要:	三氧化二砷俗稱砒霜，被作為藥物“以毒攻毒”治療許多人類的疾病，尤其因可促進血癌細胞凋亡而被應用在急性血癌的治療上，此成功的機制或許也可以應用在其他癌症的治療上，本篇論文即為探究其運用於口腔癌治療可行性之先導性研究。張乃文教授利用了200 ?慊/ml 4-nitroquinoline 1-oxide (4NQO) 以及 500 ?慊/ml 檳榔鹼 (arecoline) 進行八週的同步飲水處理，可以誘使C57BL/6J品系小鼠在停藥後二十週內全部得到口腔癌。我們將處理藥物八週後及停藥後十週及二十週的小鼠(分別稱為8-w, 18-w及28-w的NA組小鼠)進行犧牲取樣，也同步針對不含此兩藥物的溶劑對照組小鼠進行相同的操作(對照組)。在每個時間點至少六隻小鼠的條件下，我們將個別長出口腔癌的細胞區及遠端較正常的細胞區進行初代細胞培養，以進行後續實驗。我們發現以2 ?嵱三氧化二砷進行二十四小時的處理對於8-w, 18-w及28-w的NA組及8-w, 18-w及28-w的對照組小鼠細胞，分別有27.1%, 28.5%, 34.4%, 23.2%, 26.6%, 及24.3%的毒殺力。然而這個毒殺作用對於對照組的結果並不符合臨床用藥的需求精神，所以我們選擇了更低的0.5 ???? 三氧化二砷處理條件下(此時對於28-w NA組口腔癌初代細胞及對照組細胞之毒殺作用為15.6%及9.1%)，來進行藥物之共同毒殺作用實驗。在這個0.5 ?嵱低劑量的三氧化二砷處理模式中，10及20 ?嵱的 dithiothreitol 可以分別增進其原本對於28-w NA組口腔癌初代細胞15.6%之毒殺作用到 43.3%及62.1%, 效果遠較 4 J/m2 的短波紫外光、20 ?嵱的雙氧水或 100 ?嵱 buthionine sulfoximine 分別只能夠增進到21.3%, 13.2% 及14.2% 為佳。同時10及20 ?嵱的 dithiothreitol 加上三氧化二砷的處理對於對照組細胞只會造成 12.3%及15.2%的細胞毒殺作用。dithiothreitol 原本是一個用來減輕重金屬毒性的抗氧化分子，在這邊卻反而增進了砷的毒殺作用，它的作用是否與結構中的兩個巰(SH)基有關呢?為了驗證這個假設，我們共用了四個含有單巰基的藥物分別是 2-mercaptoethanol (2ME), Glutathione (GSH), diethyl-dithiocarbomate (DEDTC), D-penicillamine (DPC) 以及三個雙巰基的藥物分別是meso-2,3-dimercaptosuccinic-acid(DMSA)2,3-dimercaptopropane-1-sulfonate (DMPS)和 2,3-dimercapto-propanol (BAL)，來進行對當的實驗，卻發現除了2ME外沒有同樣對於三氧化二砷促進其毒殺作用的現象。故應有其他與巰基作用無關的胞內機制來負責執行此共同毒殺作用。在未來的研究方向中，我們除了以初代培養人類口腔癌病患的口腔癌細胞來驗證在小鼠口腔癌模式中的上述發現外，也會進一步地探討這個共同毒殺作用的胞內分子機制。深深希望本論文中所有的先導性發現，都能夠對於口腔癌的臨床應用與治療有所貢獻(如以潄口水的方式協助病人防止口腔癌之轉移與再復發)。 The anti-tumor properties of arsenic trioxide have attracted extensively attention after its successful inducing apoptosis of acute promyelocytic leukemia cells. However, the therapeutic spectrum should not be only restricted to acute promyelocytic leukemia, but also should extend into other tumor cells. In this study, we aimed at investigating its potential application to clinical therapeutics in oral cancer. In this preclinical animal test, primarily cultured cells from the tumor sites and normal sites of two drugs, 200 ?慊/ml 4-nitroquinoline 1-oxide (4NQO) plus 500 ?慊/ml arecoline, induced oral cancer C57BL/6J Narl mice model were examined of their viabilities after the treatments of arsenic trioxide with/without other drugs. In this model, the mice were treated with 4NQO plus arecoline (NA) in their drinking water for eight weeks (8-w), and then removed the drugs for another 10 or 20 weeks (18-w and 28-w, respectively). The results showed that although 2 ?嵱 of arsenic trioxide 24-h treatment can suppress the viabilities of cells primarily cultured from the tumor sites of 8-w, 18-w and 28-w NA-treated mice to 72.9%, 71.5% and 65.6%. However, it can also suppress the viabilities of cells from the sham-treated mice of 8-w, 18-w and 28-w to 76.8%, 73.4% and 75.7%, respectively. Therefore, 0.5 ?嵱 of arsenic trioxide treatment for 24 h, which suppressed the viabilities of cells primarily cultured from the tumor sites of 28-w NA-treated and sham-treated mice to 15.6% and 9.1%, was examined of its synergistic fluencies on the two primarily cultured cell lines with other drugs. The results showed that 10~20 ?嵱 dithiothreitol can enhance the cytotoxic effects of arsenic trioxide to 43.3~62.1%, better than those of 4 J/m2 UVC, 20 ?嵱 H2O2 or 100 ?嵱 buthionine sulfoximine (21.3%, 13.2%, and 14.2%, respectively). At the same time, 10~20 ?嵱 dithiothreitol plus 0.5 ?嵱 arsenic trioxide treatments caused only 12.3% and 15.2% of cell death in the control group. Last, four mono-thiol agents, 2-mercaptoethanol (2ME), Glutathione (GSH), diethyl-dithiocarbomate (DEDTC) and D-penicillamine (DPC), together with three di-thiol agents, meso-2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane-1-sulfonate (DMPS) and 2,3-dimercaptopropanol (BAL), do not have similar synergistic effects with arsenic trioxide as dithiothreitol does, with the only exception of 2ME. The results suggested that the effect of dithiothreitol is not via the SH antioxidant group. The co-cytotoxicity of dithiothreitol and arsenic trioxide on primarily cultured cells from oral cancer mice should be confirmed in oral cancer cell lines before its application in clinical therapy, and the detail mechanism is worth further study in the future.
顯示於類別:	[中國醫學研究所] 博碩士論文

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