| 摘要: | 本研究探討軟珊瑚Sinularia leptoclados之純化物5-epi-Sinuleptolide及Sinuleptolide在人類頭頸部鱗狀癌細胞(Human head and neck squamous cell carcinoma, SCC25)之細胞毒殺作用、細胞週期調控和細胞凋亡機制。以MTT(3-[4,5-cimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)分析法評估人類上皮癌細胞(Human epithelial carcinoma cell, A431)、基底細胞癌(Basal cell carcinoma, BCC)、人類頭頸部鱗狀癌細胞(SCC25)、人類頭頸部鱗狀癌細胞(Human head and neck squamous cell carcinoma, SCC9)，人類皮膚角質株化細胞(Human skin immortalized keratinocyte, HaCaT)與人類皮膚纖維母細胞(Human skin fibroblast, Hs68)之毒殺效果，並計算出其抑制細胞存活度百分之五十之濃度(Concentration of 50% inhibition, IC50,)。5-epi-Sinuleptolide之IC50分別為18.6、26.4、13.2、17.8、22.9和41.9 μM，而Sinuleptolide之IC50分別35.0、53.1、46.8、29.8、26.1和大於57.4 μM。結果顯示5-epi-Sinuleptolide抑制細胞存活度的效果比Sinuleptolide顯著。而其對於SCC25比起其餘五株細胞株細胞的毒性較具顯著。以5-epi-Sinuleptolide作用SCC25細胞後，由細胞型態觀察出其典型細胞凋亡特徵包含：細胞皺縮、染色質濃縮、凋亡小體的產生等典型的細胞凋亡型態變化。以流式細胞儀分析5-epi-sinuleptolide調控細胞週期之影響，結果顯示SCC25細胞對於G2/M時期較為敏感，且在sub-G1的比例有顯著的增加。在反轉錄聚合酵素連鎖反應(Reverse transcription polymerase chain reaction, RT-PCR)與西方墨點法(Western Blot)分析結果發現，Cyclin B1和CDC2之基因與蛋白質表現量皆有下降而p53與p21則有上升的情形與控制組相比較之下可發現此現象。因而推測p53以及p21基因與蛋白質表現量的增加可能是5-epi-Sinuleptolide引起細胞週期停滯於G2/M期的其中一個主要的因子。
在細胞凋亡機制探討中，5-epi-Sinuleptolide活化了TNF-α (Tumor necrosis factor-alpha)、FasL並別與TNF-R1(Tumor necrosis factor recptor 1)、TNF-R2(Tumor necrosis factor recptor 2)以及Fas結合，向下調控TRADD (Tumor necrosis factor receptor associated death domain)和FADD (Fas-associated death domain)的鍵結產生死亡訊號傳遞下去，進而影響Caspase (Cysteine asparate protease)的活化。然而更藉以活化Caspase-8，增加Bid表現形成活化態t-Bid，促進粒線體相關蛋白Cytochrome c、Bax與下游因子Caspase-9和Caspase-3的活化以及降解了Bcl-2，導致細胞凋亡發生。因此推測其具誘導調控與粒線體凋亡途徑相關的蛋白活化。另一方面，在探討5-epi-Sinuleptolide誘發粒線體凋亡途徑中，以RT-PCR與西方墨點法分析，結果顯示Cytochrome c、Bax表現量提升，Bcl-2表現量下降，Caspase-9與Caspase-3被活化其表現量也有增加的趨勢。結果推論5-epi-Sinuleptolide誘導SCC25產生細胞凋亡是藉由通過死亡受體路徑與粒線體路徑。因此期望5-epi-Sinuleptolid能夠更進一步評估用於活體及其應用，並發展成一有效之抗皮膚癌藥劑。
The apoptosis effects of 5-epi-Sinuleptolide and sinuleptolide from Sinularia leptoclados on human epithelial carcinoma (A431), basal cell carcinoma (BCC), human head and neck squamous cell carcinoma (SCC25 and SCC9), human skin immortalized keratinocyte (HaCaT) and human skin fibroblast (Hs68) cells were demonstrated. The half-inhibitory concentrations (IC50) for A431, BCC, SCC25, SCC9, HaCaT and Hs68 cells were 18.6, 26.4, 13.2, 17.8, 22.9, 41.9 μM for 5-epi-Sinuleptolide, and 35.0, 53.1, 46.8, 29.8, 26.1 and surpassed 57.4 μM for sinuleptolide, respectively, as determined by MTT assay. 5-epi-sinuleptolide exhibited higher potency than sinuleptolide in inhibiting cell growth. 5-epi-Sinuleptolide exhibited the greatest cytotoxicity for SCC25 cells among the other five cell lines. In this study, 5-epi-sinuleptolide induces apoptosis of SCC25 cells and the mechanism was characterized. After treatment 5-epi-Sinuleptolide with SCC25 cells, typical morphological features of apoptotic, such as cell shrinkage, chromatin condensation, and apoptotic bodies were observed. Flow cytometry indicates that 5-epi-Sinuleptolide sensitized SCC25 cells in S-G2/M phases with a concomitant significantly increase of sub-G1 population.
5-epi-Sinuleptolide increases of TNF-α and FasL to the SCC25 cells, and up-regulates TNF-R1, TNF-R2 and Fas expressions and their subsequent TRADD and FADD signal cascades. Additionally, in RT-PCR and western blot analysis, 5-epi-Sinuleptolide triggers mitochondria-mediated pathways, release of Cytochrome c, down-regulation of Bcl-2, up-expression of Bax, increase of Caspase-9 and Caspase-3 activities. These findings suggest that 5-epi-Sinuleptolide induces SCC25 cells apoptosis via death receptor and mitochondria pathways. Furthermore, 5-epi-Sinuleptolide is promising as an important for further evaluation for its application as an anti-skin cancer agent. |
