Effects and inhibitory mechanisms of active components of garlic essential oil on the growth of human skin cancer and lung cancer cells

大蒜精油經證實具有抑制心血管疾病、保肝與抗癌等功效，其生理活性主要來自於含硫化合物，而我們的研究對象便是其中三種重要活性成分－二烯丙基硫化物（diallyl sulfide, DAS）、二烯丙基二硫化物（diallyl disulfide, DADS）與二烯丙基三硫化物（diallyl trisulfide, DATS）。在本研究中將探討此三種大蒜重要之含硫活性成分對人類皮膚基底癌細胞（basal cell carcinoma, BCC）、人類正常皮膚角質細胞（HaCaT cells）和人類肺癌細胞（A549 cells）之影響。BCC為人類皮膚基底角質層的癌細胞，分別以DAS、DADS和DATS處理48小時，其中2 mM之DAS對BCC的生長抑制率只有46%未達到IC50，而2 mM之DADS和DATS的IC50分別為1.55和0.85 mM。HaCaT則屬於一種正常的皮膚角質細胞株，以0~2 mM之DAS、DADS和DATS處理48小時後，其細胞存活率皆較同濃度處理下的BCC皮膚癌細胞組別高。由此可知，以DAS、DADS和DATS對BCC細胞有抑制作用的濃度處理HaCaT細胞，可觀測到其具有較高的生存率，換句話說，此三種成分對正常細胞的損害應較皮膚癌細胞小。此外，A549為一種常見的肺癌細胞株，以0~0.5 mM之DAS、DADS和DATS處理48小時後，其IC50分別為0.12、0.05和0.01 mM，由此結果可知此三種含硫活性成分對A549比對BCC具有較強之抑制效果。另外，此研究以流式細胞儀來探討DAS、DADS和DATS對三種細胞株之細胞週期的影響，結果顯示A549在DAS（0.01 mM）處理48小時後sub-G1期會增加，在DADS（0.05 mM）處理後會停滯在G2/M期的表現，而在DATS（0.01 mM）處理48小時後sub-G1期亦呈增加的趨勢。HaCaT之細胞週期在三種含硫活性成分處理下皆無明顯變化。此外，BCC經DAS（2 mM）、DADS（1 mM）和DATS（0.5 mM）處理48小時後，sub-G1期皆會增加，故推測大蒜精油中之DAS、DADS、DATS主要是藉由誘導細胞凋亡的發生來抑制BCC的生存率。接著，進一步探討DADS和DATS誘導BCC細胞凋亡之作用機制，由西方墨點法分析結果顯示，DADS和DATS皆可使抑制細胞凋亡之蛋白質Bcl-2和Bcl-xL的活性下降，促進細胞凋亡之蛋白質Bad和Bax活性上升，此外，caspase-3、PARP、caspase-9的蛋白質含量也都會上升，故可推測DADS和DATS可能是經由粒線體途徑導致BCC細胞凋亡的發生。綜合上述結果，可知對BCC存活率的抑制效果為DATS強於DADS，而DADS又強於DAS，並推測其為經由粒線體途徑導致BCC細胞凋亡的發生。而DAS、DADS、DATS在同樣濃度處理BCC和HaCaT細胞48小時的情況下，對HaCaT細胞較對BCC細胞傷害較小。由此可知此三種大蒜活性成分（DAS、DADS、DATS）或許可應用發展於預防皮膚基底細胞癌的發生或罹患皮膚基底細胞癌患者輔助治療的食療成分。Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are oil-soluble organosulfur compounds in processed garlic. They were well known to have chemopreventive activity against human cancer such as colon, lung, liver and skin cancer. Previous researches about the three compounds have shown that they have many biological activities, such as anti-oxidation, anti-inflammation, anti-tumor, anti-cancer, reduction of blood pressure and so on. In this study, DAS, DADS and DATS were used to investigate the anti-proliferative effects and mechanism on human basal cell carcinoma cells (BCC). Additionally, the effect of these three compounds on human lung cancer A549 cells and keratinocytes HaCaT cells was also studied. For BCC cells, the results indicated that DATS (IC50 0.85 mM) showed stronger anti-proliferative activity than DADS (IC50 1.55 mM) and DAS (IC50 >2 mM) after 48 hr treatment. For HaCaT cells, the results indicated that the IC50 values of DAS, DADS and DATS were all over 2 mM after 48 hr treatment. For A549 cells, it showed that DATS (IC50 0.01 mM) exhibited stronger anti-proliferative activity than DADS (IC50 0.05 mM) and DAS (IC50 0.12 mM) after 48 hr treatment. In addition, this study used flow cytometry analysis to evaluate the cell cycle effect of BCC, HaCaT and A549 cells treated with DAS, DADS and DATS. The results showed that DAS (0.01 mM) increased the percentage of sub-G1 phase; DADS (0.05 mM) induced cell cycle arrest in G2/M phase and DATS (0.01 mM) increased the percentage of sub-G1 phase after 48 hr treatment in A549 cells. For HaCaT cells, the three organosulfur compounds have no effect at these cell-cycle indicator. As for the results of cell cycle in BCC, sub-G1 phase increased after 48 hr treatment with DAS (2 mM), DADS (1 mM) and DATS (0.5 mM) in BCC cells. By the Western blot analysis, it suggested that DADS (1 mM) and DATS (0.5 mM) induced apoptosis in BCC cells which were triggered by the activation of caspase-3 and -9, upregulation of pro-apoptosis proteins Bad and Bax, downregulation of anti-apoptosis proteins Bcl-2 and Bcl-xL and degradation of PARP in BCC cells. In conclusion, DAS, DADS and DATS had greater effect on inhibiting BCC and A549 cells proliferation after 48 hr treatment than on HaCaT cell which means the three compounds effect normal skin cell lightly. And we suggested that DADS and DATS induced apoptosis in BCC cells through the mitochondrial pathway by the results of Western blot analysis. Therefore, these three garlic active components (DAS, DADS, and DATS) might be used to prevent the occurrence of BCC or to be the adjuvant constituents for therapy of BCC patients.目錄....................................................................Ⅰ 中文摘要................................................................Ⅳ 英文摘要................................................................Ⅵ 第一章 前言..............................................................1 第二章 文獻探討..........................................................3 一、大蒜的簡介...........................................................3 二、大蒜精油之簡介.......................................................4 三、大蒜的相關應用.......................................................5 四、大蒜可能之副作用.....................................................8 五、皮膚癌之簡介.........................................................8 六、Nonmelanoma類型皮膚癌的形成.........................................11 七、細胞週期的調控......................................................13 八、細胞凋亡............................................................16 九、肺癌細胞A549........................................................18 十、實驗動機和實驗架構..................................................20 第三章 實驗材料與方法...................................................23 第一節 實驗材料........................................................23 一、實驗藥品............................................................23 二、實驗儀器............................................................24 三、實驗樣品............................................................25 第二節 實驗方法.......................................................27 一、人類皮膚基底癌細胞的解凍與冷凍......................................27 二、人類皮膚基底癌細胞的培養............................................28 三、樣品之處理..........................................................28 四、以MTT分析法進行人類皮膚基底癌細胞細胞生存力之分析...................28 五、利用倒立式顯微鏡觀察DAS、DADS、DATS對細胞形態的影響.................29 六、檢測DAS、DADS、DATS對人類皮膚基底癌細胞細胞週期的影響...............29 七、利用西方墨點法（Western blot）定量參與調控細胞週期的蛋白質..........31 八、統計分析............................................................37 第四章 實驗結果.........................................................38 一、DAS、DADS或DATS對人類皮膚基底癌細胞BCC生存力之影響..................38 （一）DAS、DADS或DATS對人類皮膚基底癌細胞BCC存活率之影響................38 （二）DAS、DADS或DATS對人類皮膚基底癌細胞BCC細胞形態之影響..............39 （三）DAS、DADS或DATS對人類皮膚基底癌細胞BCC細胞週期之影響..............39 二、DAS、DADS或DATS對人類正常皮膚角質細胞HaCaT細胞生存力之影響..........40 （一）DAS、DADS或DATS對人類正常皮膚角質細胞HaCaT存活率之影響............40 （二）DAS、DADS或DATS對人類正常皮膚角質細胞HaCaT細胞形態之影響..........41 （三）DAS、DADS或DATS對人類正常皮膚角質細胞HaCaT細胞週期之影響..........41 三、DAS、DADS或DATS對人類肺癌A549生存力之影響...........................42 （一）DAS、DADS或DATS對人類肺癌細胞A549存活率之影響.....................42 （二）DAS、DADS或DATS對人類肺癌細胞A549細胞形態之影響...................43 （三）DAS、DADS或DATS對人類肺癌A549細胞週期之影響.......................43 四、DAS、DADS或DATS於不同處理時間，分別對人類肺癌細胞A549、人類皮膚角質細 胞HaCaT和人類皮膚基底癌細胞BCC生長影響之比較........................44 五、DADS或DATS誘導人類皮膚基底癌細胞BCC細胞凋亡之相關蛋白的變化.........45 第五章 討論.............................................................74 第六章 結論.............................................................81 參考文獻................................................................83 附錄....................................................................9