- 學位論文
軟珊瑚衍生的海洋天然物DHS透過氧化壓力和細胞凋亡優先殺死口腔癌細胞
Soft coral-derived DHS preferentially kills oral cancer cells showing oxidative stress and apoptosis
摘要
文獻指出軟珊瑚衍生天然物sinularin可誘導以活性氧化物(reactive oxygen species; ROS)為媒介的乳癌細胞生長抑制與細胞凋亡。而DHS是sinularin的相近化學活性成分，但DHS的抗癌作用與詳細機制，尚未完全被釐清。由於兩者結構相似，因此我假設DHS能調節氧化壓力和細胞凋亡，進而選擇性殺死口腔癌細胞。為了驗證假說，我執行4個目標: (目標1)評估DHS處理多種口腔癌細胞與正常口腔細胞的增生變化。(目標2)評估DHS處理口腔癌細胞的細胞凋亡變化。用annexinV/7AAD與pancaspase等流速細胞儀方法檢測細胞凋亡。(目標3)評估DHS處理口腔癌細胞的氧化壓力變化。使用流式細胞儀檢測活性氧化物(ROS)、粒線體超氧化物(mitochondrial superoxide; MitoSOX)與粒線體膜電位(mitochondrial membrane potential; MitoMP)。(目標4)評估DHS處理口腔癌細胞的DNA傷害變化。使用流式細胞儀檢測DNA傷害(γH2AX與8-oxo-2'-deoxyguanosine (8-oxodG))。結果發現: DHS會選擇性地殺死多種口腔癌細胞(Ca9-22、SCC-9、OECM-1、CAL 27、OC-2、HSC-3)，但對正常口腔細胞(HGF-1)無毒性副作用。同時，DHS會呈劑量關係與時間關係地誘導口腔癌細胞Ca9-22的細胞凋亡(如annexin V與pancaspase檢測)。DHS會呈劑量關係與時間關係地誘導氧化壓力(如ROS產生、MitoSOX製造與MitoMP下降)。DHS會呈劑量關係與時間關係地誘導DNA傷害(如γH2AX與8-oxodG)。最後發現以上所有DHS誘導的改變(如選擇性殺死、細胞凋亡、氧化壓力與DNA傷害)，均可由ROS捕捉劑N-acetyl-L-cysteine; NAC)前處理回復。因此，本論文貢獻在於首次報導天然海洋衍生物DHS對口腔癌的生長抑制作用，探索其選擇性殺死口腔癌細胞的機制，並釐清氧化壓力對DHS抗口腔癌生長所扮演的角色。
並列摘要
Literature reported that soft coral-derived marine natural product sinularin may induce reactive oxygen species (ROS)-mediated growth inhibition and apoptosis in breast cancer cells. DHS is a similar compound to sinularin; however, the antioxidant effect and mechanism of DHS remain unclear. Due to the similar structure between each other, I hypothesize that DHS may regulate oxidative stress and apoptosis leading to selectively inhibit cell growth of oral cancer cells. To test this hypothesis, I preformed four aims as follows: Aim 1) To evaluate the change of proliferation in DHS-treated several types of oral cancer and normal oral cell lines. Aim 2) To evaluate the change of apoptosis in DHS-treated oral cancer cells. Aim 3) To evaluate the change of oxidative stress in DHS-treated oral cancer cells. Flow cytometric analysis for reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX), and mitochondrial membrane potential (MitoMP) were performed. Aim 4) To evaluate the change of DNA damage in DHS-treated oral cancer cells. Flow cytometric analysis for γH2AX and 8-oxo-2'-deoxyguanosine (8-oxodG) were performed. In cell viability assay, DHS shows selective growth inhibition against several types of oral cancer cell lines (Ca9-22, SCC-9, OECM-1, CAL 27, OC-2, and HSC-3) but shows no cytotoxic side effect to normal oral cells. DHS also shows dose- and time-dependent induction of apoptosis with the evidence of annexin V overexpression and pancaspase activation. DHS shows dose- and time-dependent induction of oxidative stress with the evidence of ROS/MitoSOX generation and MitoMP depletion. DHS shows dose- and time-dependent induction of DNA damage with the evidence of γH2AX and 8-oxodG overexpression. Finally, all these DHS-induced changes are able to be partly recovered by ROS scavenger N-acetyl-L-cysteine (NAC) pretreatment. Therefore, the contribution of this thesis is to firstly report that marine natural product DHS can selectively inhibit cell growth of oral cancer cells. Moreover, the selective growth inhibition mechanism is explored and the role of oxidative stress involving this mechanism is clarified.