松蘿地衣的甲醇萃取物透過誘發氧化壓力抑制口腔癌細胞的增殖並導致凋亡和DNA損傷

Methanol Extract of Usnea barbata (MEUB) Inhibits Proliferation and Induces Apoptosis and DNA Damage against Oral Cancer Cells through Oxidative Stress

吳光涵(Kuang-Han Wu)

指導教授 ： 張學偉老師

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2020年)

摘要

口腔癌是世界上常見的癌症之一，亦為台灣死亡率最高的癌症。化學療法使用的臨床藥物經常對正常組織顯示副作用，並限制藥物對癌症治療的效用。天然萃取物含有多種具抗氧化物特性的生物活性化合物，這些化合物可能在不同的機制中起關鍵作用，從而產生協同的抗癌作用，並減少對正常細胞的傷害。地衣松蘿(Usnea barbata; U. barbata)的萃取物顯示出高抗氧化能力，因此，我提出假說，methanol extract of U. barbata (MEUB)具有抗口腔癌的潛力。為驗證此假說，我執行以下六個目標：目標1）使用MEUB處理人類口腔癌(Ca9-22，HSC-3，CAL 27，OECM-1和SCC9)和正常口腔(HGF-1)細胞，以MTS分析細胞存活率。目標2）評估MEUB影響口腔癌細胞的細胞週期，使用(7-aminoactinomycin D; 7AAD)為染劑，再由流式細胞儀檢測分析。目標3）評估MEUB處理口腔癌細胞的凋亡作用，使用流式細胞技術分析Annexin V、Pancaspase的活性。並透過Western blotting測定cleaved form of Poly (ADP-ribose) polymerase (c-PARP)表現量。目標4）透過流式細胞儀檢測MEUB在口腔癌細胞中氧化壓力的調控，尤其是活性氧化物(Reactive Oxygen Species; ROS)的功能。目標5）評估MEUB處理口腔癌細胞的粒線體超氧化物(Mitochondrial Superoxide; MitoSOX)含量和粒線體膜電位(Mitochondrial Membrane Potential; MMP)變化，個別使用MitoSOX和MMP為染劑，再由流式細胞儀檢測結果。目標6）評估MEUB對口腔癌細胞中DNA損傷的影響，進行雙股斷裂(γH2AX)和氧化性損傷(8-oxo-2'-deoxyguanosine; 8-oxodG)檢測，用流式細胞儀分析。結果表明，MEUB可以毒殺口腔癌細胞，但不影響正常的口腔細胞。對MEUB表現出最高敏感性的口腔癌Ca9-22和OECM-1細胞，將選擇為後續所有實驗的細胞株，用於評估濃度和時間依賴性反應，以了解其細胞毒性機制。由流式細胞儀檢測和目標蛋白質變化發現，細胞週期中sub G1積累，Annexin V檢測和c-PARP裂解，最終MEUB誘導口腔癌細胞凋亡。此外，藉由流式細胞儀分析結果，可以證實MEUB誘導口腔癌細胞中，氧化壓力的增加會造成DNA損傷，例如ROS/MitoSOX的產生，MMP的下降以及γH2AX和8-oxodG的過度表現。再者，使用活性氧化物清除劑N-acetylcysteine (NAC)對細胞進行前處理，結果證實MEUB所誘發口腔癌細胞的變化都是由氧化壓力造成。因此，我首先證明MEUB對口腔癌細胞具有選擇性殺死癌細胞的能力，藉由調控細胞內的氧化壓力，造成口腔癌細胞凋亡和DNA損傷。

關鍵字

地衣 ； 天然藥物 ； 選擇性殺死 ； 氧化壓力 ； 細胞凋亡 ； DNA損傷

並列摘要

Oral cancer, a common cancer worldwide, is the malignancy with the highest mortality in Taiwan. For chemotherapy, clinical drugs frequently show side effects on normal tissues and limit its efficiencies to cancer therapy. Natural products containing a lot of bioactive compounds with antioxidant property may play a crucial role in different targeting mechanisms, resulting in synergistic anticancer function and showing less side effects on normal cells. Since extracts of lichens Usnea barbata (U. barbata) show high antioxidant capacity, I hypothesize that methanol extract of U. barbata (MEUB) has a potential for antioral cancer treatment. To test this hypothesis, I proposed six aims as follows: Aim 1) Cell viability of human oral cancer (Ca9-22, HSC-3, CAL 27, OECM-1, and SCC9) and normal oral (HGF-1) cell lines after MEUB-treated were analyzed by MTS assays. Aim 2) Cell cycle disturbance of oral cancer cells was analyzed by flow cytometry for 7-aminoactinomycin D (7AAD). Aim 3) The effects of apoptosis in MEUB-treated oral cancer cells were analyzed by flow cytometry for annexin V expression and pancaspase activity and by western blotting for cleaved form of Poly (ADP-ribose) polymerase (c-PARP). Aim 4) The function of oxidative stress caused by MEUB in oral cancer cells was detected by flow cytometry for reactive oxygen species (ROS). Aim 5) The function of oxidative stress induced by MEUB in oral cancer cells was also detected by flow cytometry for mitochondrial superoxide (MitoSOX) and mitochondrial membrane potential (MMP). Aim 6) The changes of DNA damages after MEUB-treated oral cancer cells were evaluated by flow cytometry for γH2AX and 8-oxo-2'-deoxyguanosine (8-oxodG). After finishing these aims, I found that MEUB can sensitively kill several types of oral cancer cells rather than normal oral cells. Oral cancer Ca9-22 and OECM-1 cells showing the highest sensitivity to MEUB were used to evaluate the concentration and time course responses to address its cytotoxic mechanisms. Mechanically, MEUB increases apoptosis of oral cancer cells with the evidence of flow cytometric assays and western blotting, such as sub G1 accumulation, annexin V detection, and pancaspase activation. MEUB overproduces oxidative stress of oral cancer cells including ROS/MitoSOX productions and MMP depletion. MEUB induces DNA damages such as γH2AX and 8-oxodG in oral cancer cells. Pretreatment with antioxidant N-acetylcysteine (NAC) validates that all MEUB-induced changes in oral cancer cells are triggered by oxidative stress. I firstly demonstrate that MEUB shows preferential killing potential by mediating oxidative stress to apoptosis, and DNA damage of oral cancer cells in a ROS-dependent manner.

並列關鍵字

Lichen ； Natural product ； Preferential killing ； Oxidative stress ； Apoptosis ； DNA damage

參考文獻

參考文獻

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