AN37選擇性殺害口腔癌細胞之氧化壓力、細胞週期敏感度及DNA修復機制探討

Role of oxidative stress, sensitivity of cell cycle phase and DNA repair for the selective killing of AN37 against oral cancer cells

詹雅菁(Ya-Ching Chan)

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研究所/碩士(2013年)

摘要

之前研究指出祕魯酸漿萃取出來的純化合物AN37可能藉由活性氧化物(reactive oxygen species; ROS)調控選擇性殺害口腔癌細胞(Ca9-22)。雖然AN37誘導Ca9-22細胞的ROS含量比正常牙齦細胞(HGF-1)多，但其詳細氧化壓力(oxidative stress)機制尚不清楚。本論文發現利用N-乙醯基半胱胺酸(N-acetyl cysteine; NAC)前處理，來探討AN37處理Ca9-22細胞及HGF-1細胞的氧化壓力及細胞相關回應機制。從細胞形態及存活率得知，NAC前處理能挽救AN37所造成的Ca9-22細胞低存活率。利用流式細胞儀，發現NAC前處理能夠降低AN37誘導Ca9-22細胞的高ROS表現量，代表NAC對AN37的存活率恢復與氧化壓力的調控有關。在免疫螢光染色分析，也發現NAC前處理可以降低AN37在Ca9-22細胞及HGF-1細胞誘導的γH2AX聚集點(foci)表現量，證明NAC可以減少AN37誘發的DNA雙股螺旋斷裂的發生。即時反轉錄聚合酶鏈鎖反應顯示AN37能誘導Ca9-22細胞的抗氧化基因如榖胱甘肽還原酶(glutathione reductase; GSR)表現量比HGF-1細胞高，支持上述Ca9-22細胞的ROS誘導比HGF-1細胞高。在細胞週期對於AN37的敏感度分析，發現Ca9-22細胞的S期細胞存活率比G1期低，但不顯著，顯示細胞週期敏感度在選擇性殺害機制中扮演角色仍須釐清。最後，以γH2AX聚集點的消逝程度作為DNA修補速度來分析，發現AN37處理後的HGF-1細胞的DNA修復速度比在Ca-22細胞快。這些結果指出，ROS的確在選擇性殺害機制中為關鍵性的角色，而DNA修復在Ca9-22細胞及HGF-1細胞的差異也是重要影響因子。

關鍵字

選擇性殺害 ； 祕魯酸漿 ； N-acetylcysteine ； 活性氧化物 ； 氧化壓力 ； DNA傷害

並列摘要

Reactive oxygen species (ROS) are involved in regulating cell growth, cell differentiation and cell death. Our previous investigations had demonstrated that AN37, a Physalis peruviana (golden berry) derived-compound, had selective killing effects in oral cancer cells involving ROS. Although the AN37-induced ROS is higher in oral cancer (Ca9-22) cells than in normal gingival fibroblast (HGF-1) cells, the role of oxidative stress still needs to be further validated. Using N-acetylcysteine (NAC) pre-treatment, oxidative stress role and its corresponding cellular responses to the AN37-treated Ca9-22 and HGF-1 cells was investigated. By cell morphology and MTS assay, cell viability of both AN37-treated Ca9-22 and HGF-1 cells can be rescued by NAC pre-treatment. For flow cytometry analysis, AN37-induced ROS in Ca9-22 cells can also be reduced after NAC pre-treatment, indicating that AN37-induced selective killing can be rescued by ROS modulation of NAC pretreatment. Using immunofluorescence of γH2AX, DNA double-stand breaks was rescued in both Ca9-22 and HGF-1 cells by NAC pre-treatment. Using quantitative real-time RT-PCR, the antioxidant gene, such as glutathione reductase (GSR), the mRNA expression level of GSR is higher in Ca9-22 cells than in HGF-1 cells, suggesting that ROS induction is dominant in Ca9-22 cells. In terms of the sensitivity of cell cycle phase to AN37, cell viability of S phase-rich Ca9-22 cells was lower than G1 phase-rich cells although it was not significant. It indicated that the role of cell cycle phase to AN37 sensitivity is still needed further investigation. Last, we examined the DNA repair speed of AN37-treating Ca9-22 and HGF-1 cells in terms of the reducing speed of γH2AX foci. The repair speed in AN37-treated HGF-1 cells was faster than that in Ca9-22 cells. It indicated that ROS may play an important role in selective killing of oral cancer cells and differential repair speed between oral cancer and normal cell lines is also an important factor determining selective killing effects.

並列關鍵字

Physalis peruviana ； selective killing ； N-acetylcysteine ； ROS ； oxidative stress ； DNA damage

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