開發具有ROS調節的選擇性殺害口腔癌細胞的藥物

Identification of ROS-modulating and selective killing drug against oral cancer cells

謝哲瑜(Che-Yu Hsieh)

指導教授 ： 張學偉

高雄醫學大學/生命科學院/生物醫學暨環境生物學系碩士班/碩士(2016年)

摘要

癌細胞被報導跟正常細胞相比有較高表現量的活性氧化物質(ROS)。AMA是粒線體電子傳遞鏈的抑制劑，在生物系統中具有ROS產生作用。這個研究的目的是為了檢驗AMA在口腔癌細胞可能造成的選擇性殺害影響，為了達到這個目標，透過MTS檢測和流式細胞儀評估經過AMA處理的口腔癌細胞的細胞存活率、ROS產生和細胞凋亡的影響。在MTS檢測中，AMA對口腔癌細胞(Ca9-22 and CAL 27)的生長有劑量依存關係的抑制但是對於正常口腔細胞(HGF-1)只有些微的影響。AMA能有效的誘導口腔癌細胞的細胞凋亡，藉由流式細胞儀的檢測證實sub-G1的累積、annexin V的結合和pan-caspase的活化。這些細胞凋亡的過程還伴隨著粒線體膜電位(MitoMP) (ΔΨm)的損失、細胞內ROS和粒線體的superoxide表現量增加。此外，ROS的角色透過N-acetylcysteine (NAC)的前處理被進一步的檢驗，藉由MTS檢驗，NAC的前處理(2 mM for 1 h)可以明顯的抑制AMA誘發的口腔癌細胞和正常口腔細胞的死亡。NAC前處理也可以預防細胞凋亡、粒線體膜電位的損失和氧化壓力的產生，顯示氧化壓力對於AMA所誘導的口腔癌細胞死亡扮演著重要的角色。總結來說，AMA誘導產生的氧化壓力與口腔癌細胞的細胞凋亡有緊密的關係，而且，AMA是一個有潛力的ROS調節藥物，藉此特性對口腔癌細胞選擇性誘發凋亡。

關鍵字

氧化壓力 ； 細胞凋亡 ； 口腔癌 ； 選擇性殺害

並列摘要

Cancer cells were reported to have higher level of reactive oxygen species (ROS) than that of normal cells. AMA, an inhibitor of electron transport in mitochondria, has been used as a ROS generator in biological systems. The purpose of this study is to examine the possible selective killing effect of AMA on oral cancer cells. To address this aim, the cell viability, ROS generation, and apoptosis effects of AMA-treated oral cancer cells were evaluated using MTS assay and flow cytometry. In MTS assay, the growth of oral cancer (Ca9-22 and CAL 27) cells was dose-responsively inhibited by AMA but just a little effect to oral normal (HGF-1) cells. AMA efficiently induced apoptosis of oral cancer cells, as evidenced by flow cytometric detection of sub-G1 accumulation, annexin V staining, and pan-caspase activation. These apoptotic processes were accompanied by the loss of mitochondrial membrane potential (MitoMP) (ΔΨm), the increase of intracellular ROS and mitochondrial superoxide levels. Moreover, the role of ROS level was further examined by the pretreatment with N-acetylcysteine (NAC). MTS assay revealed that NAC pretreatment (2 mM for 1 h) could strongly inhibit AMA-induced death of oral cancer and normal oral cells death. NAC pretreatment also reduced apoptosis, MitoMP loss and oxidative stress induction of AMA-treated cells, suggesting that oxidative stress played an important role in AMA-induced death of oral cancer cells. In conclusion, AMA induced apoptotic death of oral cancer cell is tightly correlated with its ability to elicit ROS generation. Thus, AMA could selective kill oral cancer cells via it ROS-modulating effect.

並列關鍵字

oxidative stress ； apoptosis ； oral cancer ； selective killing

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