Capsaicin induces apoptosis and autophagy in B16F10 cells

Abstract

Melanoma is a malignant tumor of melanocytes. If melanoma is found at early stage, it can be removed completely by surgery and the chance of cure is high. Unfortunately, if it is diagnosed late it may lead to skin cancer-relatred death. The treatments to melanoma these days include chemo- and immunotherapy, and radiation therapy. Capsaicin has an anti-proliferative effect in vitro on prostate, colon, gastric, hepatic and leukemic cancer cell lines. It is demonstrated that capsaicin inhibited melanoma cacncer cell lines, however, most of the mechanisms are still unclear. In this study, we used rat melanoma cell line, B16F10, for the evaluation of the anticancer effect of capsaicin . We found that the percentage of apoptosis and changes of mitochondrial membrane potential are notably increased in high concentration of capsaicin (400 μM). Western blot also shows that high concentration of capsaicin decreases Bcl-2 protein. Low concentrations of capsaicin are found to not to activate PARP (Poly ADP ribose polymerase) resulting in no apoptosis. While oxidative stress is not significantly different among different concentrations of capsaicin. We suggest that high concentration of capsaicin might induce apoptosis in B16F10 through mitochondrial dependent pathway. We also found that low concentrations of capsaicin (10, 100 μM) result in autophagy induction in B16F10 cells. Western blot also shows that high concentration of capsaicin increases the level of mTOR phosphorylation. On the other hand, low concentrations of capsaicin increase beclin-1 and autophagy. We suggest that capsaicin-mediated autophagy may involved in cell survival mechanism in B16F10 cells. Thus, we propose to utilize inhibitors of autophagy to examine the percentage of apoposis in B16F10. Similarly, inhibitors of apoptosis will be used to study the autophagy mechanism in B16F10 cells, with the hope to find out the relationship between autophagy and apoptosis in capsaicin-exposed B16F10 cells.中文摘要 …………………………………………………………… i 英文摘要 …………………………………………………………… iii 目錄 ………………………………………………………………… v 圖表目錄…………………………………………………………… vii 壹、緒論…………………………………………………………… 1 一、辣椒素 (Capsaicin) …………………………………… 1 二、黑色素瘤細胞 (Melanoma) …………………………… 1 三、細胞凋亡 (Apoptosis)………………………………… 2 四、細胞自噬 (Autophagy) ………………………………… 5 貳、實驗材料……………………………………………………… 9 參、實驗方法 ……………………………………………………… 13 一、細胞培養 (Cell culture) …………………………… 13 二、西方墨漬蛋白表現偵測 (Western blotting) ……… 13 三、細胞凋亡測試 (Apoptosis assay) …………………… 15 四、過氧化物含量分析 (ROS measurement ) …………… 15 五、細胞自噬測試 (Autophagy assay)…………………… 16 六、粒線體膜通透性測試 (Mitochondrial membrane potential assay) 16 七、細胞數計數 (Trypan Blue Assay) …………………… 17 八、xCELLgence System儀分析 (xCELLgence System) … 17 九、統計分析 (Staistical analysis) …………………… 18 肆、實驗結果 ……………………………………………………… 19 一、辣椒素具有抑制B16F10細胞生長的效果……………… 19 二、辣椒素主要引發粒線體路徑的細胞凋亡……………… 20 三、辣椒素在較低濃度會引發細胞自噬…………………… 20 (圖一) B16F10在辣椒素作用下的細胞生長曲線………………… 22 (圖二) B16F10在辣椒素作用下的細胞計數……………………… 23 (圖三) B16F10在辣椒素作用下的細胞凋亡偵測………………… 24 (圖四) B16F10在辣椒素作用下的細胞凋亡偵測 (統計圖表) … 25 (圖五) B16F10在辣椒素作用下的西方墨點法偵測 (細胞凋亡代表性蛋白) 26 (圖六) B16F10在辣椒素作用下的粒線體膜通透性測試………… 27 (圖七) B16F10在辣椒素作用下的超氧化物 (ROS)測試………… 28 (圖八) B16F10在辣椒素作用下的細胞自噬測試………………… 29 (圖九) B16F10在辣椒素作用下的西方墨點法偵測 (細胞自噬代表性蛋白) 30 (表一) B16F10在辣椒素作用下的西方墨點法偵測 (細胞自噬代表性蛋白)數據化 31 伍、討論 …………………………………………………………… 32 陸、參考資料 ……………………………………………………… 3