評估藻類萃取物對檳榔鹼誘發的口腔癌細胞的氧化傷害與機制探討

valuation of seaweed extract against arecoline-induced oxidative damage in oral cancer cells and its possible mechanisms

鍾采杏(Tsai-Hsin Chung)

指導教授 ： 張學偉

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2013年)

摘要

先前研究指出，海藻萃取物具有抗氧化的活性，有潛力可抑制檳榔鹼藉由自由基產生對細胞造成氧化性傷害。在本實驗室之前研究已經發現，海藻萃取物與檳榔鹼共同處理時，可以減少檳榔鹼造成的細胞生長抑制，但機制仍不清楚。本論文目的是利用口腔鱗狀癌細胞來比較海藻萃取物的前處理與共同處理，對於檳榔鹼造成的氧化性傷害是否有具有保護的效果。我們利用腺嘌呤核苷三磷酸試驗 (ATP assay) 來作為細胞存活率的依據，經由檳榔鹼 (0.1、0.25、0.35與0.5 mM) 處理24小時後，細胞存活率跟控制組比較，有顯著下降的趨勢。如前處理 (pre-treat) 海藻萃取物 (0.1、0.25、0.35與0.5 mg/ml) 2小時或同時處理 (co-treat) 海藻萃取物 (0.1、0.25、0.35與0.5 mg/ml) 24小時，相較於單獨處理檳榔鹼 (0.1、0.25、0.35與0.5 mM) 24小時而言，海藻萃取物確實有顯著回復細胞存活率的情形。前處理海藻萃取物 (0.1、0.25、0.35與0.5 mg/ml) 2小時，在處理24小時之間隨著劑量增加可有效降低0.35 mM以下濃度的檳榔鹼所產生的活性氧物質(reactive oxygen species, ROS) 及活性氮物質(reactive nitrogen species, RNS)。利用annexin V-FITC 配合 PI (propidium iodide) 雙染，觀察單獨處理不同濃度檳榔鹼 (0.1、0.25、0.35與0.5 mM)，細胞凋亡與壞死的情形有明顯的增加，與海藻萃取物的細胞存活率相比，海藻萃取物並不會對細胞造成傷害。於粒線體膜電位的測試，單獨處理海藻萃物的粒線體膜電位與未加藥控制組相比無差異。綜合上述結果，本研究發現僅處理海藻萃取物不會影響細胞生長；而海藻萃取物可能透過減緩氧化性傷害的機制，有效保護檳榔鹼誘發的生長抑制效果。

關鍵字

海藻萃取物 ； 檳榔鹼 ； 口腔鱗狀癌細胞 ； 活性氧化物 ； 活性氮化物 ； 粒線體膜電位 ； 細胞凋亡

並列摘要

The antioxidant activity of seaweed extract against free radicals by arecoline was validated in an Adenosine Triphosphate assay (ATP assay), ATP assay. However, the protective mechanism for extracts remains unclear. In this study, we investigated on the protective effects of seaweed extract on in vitro for a Ca9-22 cell line (oral squamous cell cancer cell line, OSCC) in vitro. The DNA damage agent arecoline was used and tested by an ATP assay. ATP content of the arecoline (0.1、0.25、0.35、0.5 mM) treatment was reduced in a dose-dependent manner, but increased by which pre-treated with seaweed extract (0.1、0.25、0.35、0.5 mg/ml). Added arecoline (0.1、0.25、0.35、0.5 mM) to the Ca9-22 cell culture showed that the doses-dependent manner in reactive oxygen species (ROS) and reactive nitrogen species (RNS) were reduced by which pre-treated with seaweed extract (0.1、0.25、0.35、0.5 mg/ml). In treatment with arecoline (0.1、0.25、0.35、0.5 mM), the results of PI with annexin V double stain showed that cell apoptosis and necrosis increased remarkably. The extract (0.1、0.25、0.35、0.5 mg/ml) was effective protected arecoline (0.1、0.25、0.35、0.5 mM) induced annexin V positive that indicated the cell death rate. We expected that arecoline (0.1、0.25、0.35、0.5 mM) induced cell damage prevented by seaweed extract (0.1、0.25、0.35、0.5 mg/ml) was available. Thus, the crude extract of seaweed extract (0.1、0.25、0.35、0.5 mg/ml) has the potential of cell protective abilities against the arecoline (0.1、0.25、0.35、0.5 mM) induced cell damage and become a potential natural product for human health.

並列關鍵字

seaweed extract ； arecoline ； oral squamous cell cancer cell line ； ROS ； NOS ； MMP ； apoptosis

參考文獻

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