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研究生:劉煥渟
研究生(外文):Huan-Ting Liu
論文名稱:有機汞與三氧化二砷抑癌作用之研究
論文名稱(外文):Anticancer Effects of Organic Mercuric Compounds and Arsenic Trioxide
指導教授:蕭水銀蕭水銀引用關係
指導教授(外文):Shoei-Yn Lin-Shiau
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:79
中文關鍵詞:黑色素瘤三氧化二砷
外文關鍵詞:phenylmercuric acetateB16F10arsenic trioxidethimerosal
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黑色素細胞瘤是常見的致死皮膚腫瘤之一,因為具有很強的轉移能力又容易產生化療的抗藥性,尋找新的藥物是刻不容緩。在本論文中,我們研究三種藥物(三氧化二砷, As2O3;thimerosal, TMHg;phenylmercuric acetate, PHgAc)對黑色素瘤細胞毒性及其作用機轉,這三種藥物在這方面的研究尚未有人報告,我們是最先發現它們具有抑制黑色素細胞瘤的有效性,我們規劃比較它們對鼷鼠黑色素細胞瘤(B16F10)的毒殺效果,可能經過的訊息傳遞路徑和抑制瘤細胞體內生長及轉移作用
As2O3 結合TMHg或PHgAc的合併作用,顯示對B16F10黑色素瘤細胞毒殺作用的加強效果。TG也增強TMHg和PHgAc對B16F10黑色素瘤細胞毒殺作用,但會減少As2O3的作用。TMHg, PHgAc 和As2O3 導致細胞週期停留在G2/M階段,並且減少G0/G1階段的比例,因三者均可增加Sub-G1階段的比例,顯示其毒殺作用可能經由細胞凋亡的過程。藉由型態改變、彗星分析、Hoechst 33258染色分析,發現三種藥物在高濃度作用下,使B16F10黑色素瘤細胞產生外觀變形、細胞核受到傷害,導致DNA受損,可由彗星分析觀察到彗星托尾程度的不同,分析其傷害程度,最後在Hoechst 33258染色分析下,三者在均會導致細胞凋亡體的出現,證明此三種藥物的毒殺作用皆經由細胞凋亡的路徑。
因為細胞凋亡具有兩種路徑:內在(粒腺體路徑)和外在(死亡受體路徑),由先前的報告指出,As2O3 主要是經由內在路徑來導致細胞凋亡過程,所以我們進而探討三種藥物導致細胞凋亡的訊息傳遞路徑。首先藉由測試細胞體內ROS的含量,在不同時間下(3h, 6h, 8h),三種藥物以不同濃度處理下,B16F10黑色素瘤細胞皆會產生高量的ROS,TMHg和PHgAc在3h和6h可以觀察到ROS急速上升,不過在8h處理下,ROS的產量變低;As2O3 作用比前兩者來的緩慢,在6h和8h處理下,才發現ROS升高,且8h的ROS產量最高,再來測試粒腺體膜電位(ΔΨm)的改變,分別以不同濃度的三種藥物8h處理下,發現TMHg和PHgAc在不同濃度作用下,B16F10黑色素瘤細胞的ΔΨm皆會升高,只有在20-30μM As2O3 才觀察到ΔΨm減少,說明三種藥物導致粒腺體不同程度的傷害。綜合上述結果,這三種藥物產生ROS使粒腺體損傷,進而釋放前細胞凋亡物質(cytochrome c, AIF, and Smac/DIABLO),形成細胞凋亡體,活化caspase,走向細胞凋亡的路徑。
FAS 在很多種人類癌細胞上有選擇性的大量表現因此可以用來當做腫瘤生長指標。本篇論文中,在細胞和動物實驗上,我們藉由西方墨點法來測量B16F10黑色素瘤細胞中FAS活性和癌細胞生長的關係。在細胞實驗結果顯示,處理TMHg, PHgAc或As2O3 後,B16F10黑色素瘤細胞的FAS蛋白質含量比未經處理的細胞表現量有意義的降低。在動物實驗結果也顯示相似的結果,以B16F10黑色素瘤細胞接種在C57Bl6鼷鼠為模式,接種後連續五天腹腔注射TMHg, PHgAc或As2O3,在第十四天犧牲,解剖取下的腫瘤,比較其重量和大小,顯示了TMHg, PHgAc或As2O3 皆會抑制腫瘤生長,尤其以TMHg, PHgAc具顯著的抑制作用。以取下的腫瘤組織,研磨成萃取液分析。FAS蛋白質含量比注射食鹽水動物的B16F10黑色素瘤細胞中FAS蛋白質表現有意義的降低,其中以注射As2O3動物的腫瘤中FAS蛋白質表現量被抑制的最多。
在Balbc鼷鼠實驗中,TMHg和PHgAc顯示極大抑制腫瘤細胞轉移到肺臟能力。綜合這些結果顯示,這三種藥物 (TMHg、PHgAc、As2O3)可能具有潛力,或野i發展為治療惡性黑色素瘤的藥物。
Melanoma is an increasingly common fatal skin cancer because malignant melanoma is highly metastatic and resistant to chemotherapeutic agents. It is an important issue to search a new effective drug for management of melanoma. In this study, we selected three kinds of chemical agents (arsenic trioxide, As2O3; thimerosal, TMHg; phenylmercuric acetate, PHgAc). We are the first to discover at their suppressive effects on the growth of B16F10 melanoma both in vitro and in vivo.
There are still no research reports about the effects of these agents on the growth of B16F10 melanoma cells. Therefore, we attempted in this theses to elucidate and also on the cell growth and metastasis to the lung in vivo.
The combined use of As2O3 with TMHg and PHgAc showed a synergistic cytotoxic effect on B16F10 melanoma cells. TG also enhanced the cytotoxic effect of TMHg and PHgAc on B16F10 melanoma cells, but decreased that of As2O3. TMHg, PHgAc and As2O3 induced cell cycle arrest at G2/M phase and decreased G0/G1 phase. These three agents all elevated sub-G1 phase and thus suggested a possibility of cytotoxic effect mediated by apoptotic pathways. Analysis of the morphological changes, comet tail formation and Hoechst 33258 staining, we observed that all of them induced morphological changes, nuclear condensation, DNA fragmentation and eventually apoptotic body formation. These results indicated that the cytotoxic effects of three agents mediated by apoptotic pathways.
In general, cell apoptosis can be executed through two different pathways: intrinsic (mitochrodrial pathways) and extrinsic (death receptors) pathways. As indicated by previous report: As2O3 induced cell apoptosis via intrinsic pathways. So, we further conferred the possible signaling pathways involved in cell apoptosis induced by these three drugs.
We measured the effects of TMHg, PHgAc and As2O3 on ROS production of B16F10 melanoma cells. TMHg and PHgAc caused a sustained generation of ROS in B16F10 melanoma cells at 3h, 6h, and 8h, maximally at 6h, which declined and showed no notable change at 8h as compared with the control. The effect of As2O3 appeared to be slower than that of TMHg and PHgAc, but sustained for a longer periods loading for 6h and 8h, maximally at 8h. Measurement of the mitochondrial membrane transition potential (ΔΨm) induced by TMHg, PHgAc and As2O3 in B16F10 melanoma cells revealed that TMHg and PHgAc at concentrations ranging 1-10μM for 8h, significantly increased ΔΨm. As2O3 (3-10μM) treated for 8h also significantly increased ΔΨm but at higher concentrations of 20-30μM reduced ΔΨm.
Fatty acid synthase (FAS) is selectively expressed in certain human cancers and is claimed to be a putative tumor marker. In this study, we measured by western blot the FAS protein expression in B16F10 melanoma cells growth either in vitro or in vivo. The results showed that FAS protein expression in B16F10 melanoma cells treated with TMHg, PHgAc or As2O3 was decreased in concentration-dependent as compared with that in untreated-control in vitro. Analysis of FAS in dissected tumor tissue extracts from the saline treated-control and As2O3-, TMHg- or PHgAc -treated C57Bl6 mice, revealed that As2O3 were more potently in reducing FAS protein expression than TMHg or PHgAc treatmment.
In the C57BL6 and Balbc study, TMHg and PHgAc significantly inhibited not only tumor size but also inhibited lung metastasis of tumor cells. Taken together all of these results, we tentatively conclude that these three agents (TMHg, PHgAc and As2O3) may possess a potential therapeutic agent for management of malignant melanoma.
目錄

Abbreviations 2
中文摘要 3
Abstract 6
Introduction 9
Materials and Methods 22
Results 31
Discussion 39
Tables 43
Figures 49
References 74
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