| 摘要: | 台灣海域蘊藏著多樣性且豐富的珊瑚資源，提供廣泛的研究題材，因此對於海洋天然化合物的研究，值得進一步探討。先前的文獻中顯示，Sinularia屬軟珊瑚萃取物對於癌細胞具有毒殺活性，但是對於其機轉並沒有詳細之研究。因此本論文以探討台灣海域中九種Sinularia屬軟珊瑚粗萃物(S. grandilobata、S. parva、S. triangular、 S. leptoclados、S. scabra、S. depressan、S. nanolobata、S. inflata和S. gibberosa)對於人類頭頸部鱗狀細胞癌細胞株(SCC25)及人類皮膚角質株化細胞株(HaCaT)之細胞毒殺作用(cytotoxicity)、調控細胞週期(cell cycle)和細胞凋亡(apoptosis)之作用機制。
在細胞貼附性試驗中發現，九種軟珊瑚粗萃物皆具抑制SCC25和HaCaT細胞株之細胞貼附性，結果顯示相較於其他八種萃取物，S. parva萃取物對於SCC25和HaCaT細胞株之細胞貼附性有顯著抑制效果。於細胞毒殺性試驗中發現九種軟珊瑚萃取物對於SCC25和HaCaT細胞株皆具細胞毒殺作用，由濃度IC50與IC80顯示S. parva和S. nanolobata的細胞毒殺活性最為明顯。從光學顯微鏡觀察細胞型態變化和細胞核螢光表現中發現，經九種粗萃物作用後，細胞呈現細胞凋亡特有的表現(碎片化、半月形、皺縮…等)。流式細胞儀分析試驗發現九種粗萃物會引起SCC25和HaCaT兩株細胞株細胞凋亡，細胞週期中G0/G1和S-G2/M期下降，而sub-G1比例均明顯的增加。而S. leptoclados、S. depressan和S. inflata萃取物對於SCC25和HaCaT細胞株之細胞週期調控不同，由SCC25細胞株中發現，經九種軟珊瑚萃取物作用後，細胞週期中G0/G1和S-G2/M期下降，由HaCaT細胞中發現經三種軟珊瑚萃取物(S. leptoclados、S. depressan和S. inflata)作用後，S-G2/M期有增加的趨勢，因此將這三種粗萃物進一步針對P53和Caspase-3做免疫螢光染色試驗分析，結果發現三種粗萃物皆有顯著誘導P53和Caspase-3蛋白質表現的效果。
由本研究中的實驗結果證明，九種Sinularia屬軟珊瑚粗萃物會抑制人類鱗狀癌細胞的細胞貼附性與增生，引起細胞週期中sub-G1比例增加和誘發細胞凋亡，因此九種Sinularia屬軟珊瑚粗萃物為具有潛力的細胞週期阻斷劑。對於九種Sinularia屬軟珊瑚粗萃物是否可當作抗癌藥物需要再作更深入的評估其抗癌的幼纂C
Marine soft corals of the genus Sinularia are being increasingly adopted to treat a wide variety of disease processes, including anti-inflammatory, anti-microbial, anti-HIV activities and various cancers. However, the mechanism underlying its anti-oral cancer effect is poorly understood. This study evaluates the antiproliferative effects of ethyl acetate extracts of soft corals of the genus Sinularia (S. grandilobata, S. parva, S. triangular, S. leptoclados, S. scabra, S. depressan, S. nanolobata, S. inflata and S. gibberosa) towards human head and neck squamous cell carcinoma cells (SCC25) and premalignant keratinocytes (HaCaT). The cell adhesion assay indicates that nine extracts reduce the number of cell attachments with rising treatment concentrations (0-100 g/ml). The S. parva extract is revealed to be the most effective inhibitor of cell adhesion. The genus Sinularia extracts exhibit dose-dependent cytotoxicity effect using MTS assay. The IC50 and IC80 values demonstrate that the S. parva and S. nanolobata extracts are the best inhibitors of cell proliferation. Treatment of extracts (IC50) to observe the morphological alterations in cells, membrane blebbing, the typical nuclear condensation, nuclear fragmentation and apoptotic bodies of cells are demonstrated with a phase contrast inverted microscope and Hoechst staining. Flow cytometry indicates that nine extracts sensitized SCC25 cells in the G0/G1 and S-G2/M phases with a concomitant dose-dependently increased sub-G1 peak. However, cell cycle analysis reveals that distinctly different from HaCaT cells, cells were locked in an S phase by S. leptoclados extracts, and the S-G2/M phase were arrested by S. depressan and S. inflata extracts. This apoptosis process was accompanied by up-regulation of p53 and activation of caspase-3 expression after SCC25 and HaCaT cells were treated with S. leptoclados, S. depressan and S. inflata extracts. Extracts of the genus Sinularia thus apparently cause apoptosis of SCC25 and HaCaT cells, and could be utilized as an anti-oral cancer cells activity drug for future studies. |
