| 摘要: | 近幾年在臭氧層破壞的情況下，皮膚癌的發生率有逐年增加的趨勢。目前用於皮膚癌治療的5-fluorouracil (5-Fu)、Diclofenac及Imiquimod等，雖具治療效果，卻會呈現相當程度之疼痛、潰瘍、疤痕、色素沈澱、脫失以及發炎性反應等副作用。先前的研究指出萃取分離出自PL的成分具有抗菌、降低血壓、抗發炎及細胞毒性的活性。因此本研究目的在於以化合物PL1-4作用於人類的皮膚癌之毒殺機制。
以MTS方法測定細胞毒性，結果顯示四種化合物對人類頭頸部鱗狀上皮癌細胞株(SCC25)和人類上皮癌細胞株(A431)皆具有毒殺作用，尤其是PL1和PL4毒殺作用比PL2和PL3明顯，四種化合物對A431細胞又比SCC25細胞敏感，對人類角質細胞株(HaCaT)和人類纖維母細胞株(Hs68)的毒性相對較小。根據毒殺作用結果，分析推算PL1-4毒殺SCC25細胞50% (IC50)的濃度分別是27.5 ± 1.0、77.7 ± 0.9、121.5 ± 1.6和54.0 ± 2.3 μM；A431細胞IC50的濃度為30.1 ± 0.2、33.2 ± 1.7、127.6 ± 1.5和33.1 ± 0.6μM。由光學顯微鏡觀察經PL1-4作用72小時後二株細胞型態上的變化，可觀察到細胞變圓、皺縮、細胞質濃縮和空泡化等凋亡的特徵。經由流式細胞儀分析細胞週期發現，四種化合物皆會造成SCC25和A431細胞週期中的G0/G1和S-G2/M期下降，而sub-G1的比例明顯增加，顯示四種化合物引發SCC25和A431細胞凋亡。進一步利用RT-PCR分析二株細胞內與細胞凋亡有關的基因表現，結果顯示與細胞凋亡相關基因TNF-αBTNF-R1、TNF-R2、FasL、Fas、TRADD、FADD、caspase-8、p53、cytochrome c、bax、caspase-9、caspase-3的表現量，在四種化合物作用24、48和72小時後有明顯增加，bcl-2基因表現量減少。利用免疫螢光染色和流式細胞儀分析蛋白質變化上，亦顯示與細胞凋亡相關蛋白質之表現量皆明顯增加，而bcl-2蛋白質有減少的趨勢。綜合上述結果， PL1-4四種化合物對人類皮膚癌細胞之毒殺作用是藉由誘導細胞凋亡機制而促使皮膚癌細胞死亡，因此認為其具有發展成為抗皮膚癌用藥之潛力。
Cutaneous squamous cell carcinoma (SCC) is one of the most common human non-melanoma skin malignancies, and accounts for over 90% of all skin cancers. Its incidence varies considerably and is reported to be increasing worldwide. Previously studies have shown that PL had exhibited antimicrobial, hypotensive activity, anti-inflammatory and cytotoxic effects, although the underlying mechanisms are yet to be elucidated. This study evaluated the anti-skin cancer mechanism of compound 1-4 (PL1-4). PL1-4 presented more specifically inhibited cell viability in human epithelial carcinoma (A431) and human head and neck squamous cell carcinoma (SCC25) cells than in human skin immortalized keratinocyte (HaCaT) and human skin fibroblast (Hs68) cells as determined by MTS assay, suggesting that PL1-4 is selective for targeting skin cancer cells. Indeed, PL1-4 displayed a dose- and time-dependent cytotoxic effect on human skin cancer SCC25 and A431 cells. PL1 and PL4 displayed a superior cytotoxicity than PL2 and PL3 in both cells. After treatment with PL1-4, SCC25 and A431 cells indicated typical morphological features of apoptotic cell death, such as cell shrinkage, rounded cell bodies, membrane blebbing and nuclear condensation. Flow cytometry indicates that PL1-4 sensitized both cells in the G0/G1 and S-G2/M phases with a concomitant significantly increased sub-G1 population. This apoptosis process was accompanied by death receptor-operated TNF-? TNF-R1, TNF-R2, FasL, Fas, and downstream adaptors TRADD and FADD expressions. Additionally, PL1-4-mediated both cell apoptosis by mitochondria-operated pathway, including upexpression of cytochrome c, bax, and downexpression of bcl-2. Moreover, exposure of both cancer cells to PL1-4 was associated with activation of executing caspase-8, -9 and -3. Hence, these results herein suggest PL1-4 may cross-link between the pathways of apoptosis via both receptor and mitochondria-mediated caspases signaling pathways, and may be effectively exploited in future human skin cancer clinical trials. |
