| 摘要: | 本論文將針對AE對於人類上皮癌細胞株(A431)、人類頭頸部鱗狀癌細胞株(SCC25)、人類黑色素瘤細胞株(A375)、人類皮膚角質株化細胞株(HaCaT)和人類纖維母細胞細胞株(Hs68)進行細胞毒殺(cytotoxicity)、調控細胞週期(cell cycle)和細胞凋亡(apoptosis)作用機制之研究。在細胞毒殺性試驗中，結果發現經AE作用之癌細胞呈現劑量與時間依存性之關係，由光學顯微鏡觀察發現AE對於A431與SCC25具有抑制細胞增生，且細胞型態出現不規則狀，包括：空泡狀(membrane blebbing)、細胞皺縮等凋亡特徵。在流式細胞儀分析DNA含量的試驗中，結果發現在A431和SCC25細胞的細胞週期分佈中G0/G1期下降， sub-G1和S-G2/M期比例增加，由此可推斷AE使A431與SCC25細胞停滯在S-G2/M期，伴隨著sub-G1增加而引起細胞凋亡。在免疫螢光染色試驗中，發現凋亡小體和DNA斷裂等特徵，特定蛋白質(TNF-ㄐBTNF-R1、TNF-R2、FasL、Fas、TRADD、FADD、p53、cytochem c、bax、caspase-3、caspase-8和caspase-9)的表現量皆有增加，但Bcl-2蛋白質表現量則下降而且在流式細胞儀中的免疫螢光表現量分析得到相似的結果，包括p53上調節，細胞內活性氧(reactive oxygen species, ROSs)的濃度增加，相對使榖胱甘肽(intracellular-reduced glutathione, GSH)濃度降低。
將5-氟尿嘧啶(5-fluorouracil, 5-Fu)與AE合併使用，評估其是否具加乘效果。在細胞毒殺性試驗中，發現5-Fu與AE合併使用對於A431與SCC25對抑制細胞增生具協同作用，在經由combination index (CI)計算，數值小於1，證實是具有協同作用。在免疫螢光表現量試驗中，caspase-3、caspase-8和caspase-9特定蛋白質的螢光表現量皆比單一藥物之表現量高，因此推斷5-Fu與AE可能是經由調控A431與SCC25細胞中caspase-3、caspase-8和caspase-9特地蛋白質的表現來達到協同毒殺作用。5-Fu與AE合併對於抑制皮膚癌細胞株的增生效果，比單獨使用5-Fu或AE效果來的好。因此，AE具有取代皮膚癌藥物的潛力。進一步以微脂粒包覆AE，測試其細胞毒殺效果與穿透皮膚能力。選擇無毒性的微脂粒與低濃度的AE(IC20和IC50)合併作用於A431和SCC25細胞株，結果證實合併作用比單獨使用AE更可加速細胞在短時間(24和48小時)死亡，這些實驗證明微脂粒合併AE具協同作用，推測可能是因為微脂粒主要是由磷脂質所構成的，此結構體與動物及人的細胞膜非常近似，因此比較容易進入皮膚。另外，經皮穿透力的實驗證實，將(1)帶電空包微脂粒與AE混合、(2) AE分散水溶液以及(3)利用微脂粒包覆AE，進行經皮穿透分析，分別於1、2、3、4、5、6和7小時，以經微脂粒包覆的AE比未經包覆的具較強的經皮穿透力。因此微脂粒-AE的組合療法對臨床是有幫助的。
In this study, the cytotoxicity results show that AE expressed less cytotoxicity to human skin normal cells (HaCaT and Hs68 cells) than the cancer cells (A431 and SCC25 cells), suggesting that AE is selective for targeting highly proliferating cancer cells. Additionally, AE-treated skin cancer cells displayed several features of apoptosis, including morphological changes of chromatin condensation, DNA fragmentation and arrest of cells in the S-G2/M phase along with increase in the sub-G1 population. AE revealed dose-dependent upregulation the death receptor-mediated pathway proteins expressions by upregulation of tumor necrosis factor-?(TNF-? and FasL and their cognate receptors (TNFRs and Fas) and downstream adaptors TNF-R1-associated death domain (TRADD) and Fas-associated death domain (FADD), and activation of executing caspase-8. Moreover, AE-displayed apoptosis is associated with mitochondria-mediated pathway, including upregulation of p53, increase the intracellular reactive oxygen species (ROSs) levels, deplete the intracellular-reduced glutathione (GSH), upregulation of cytochrome c and Bax, downexpression of Bcl-2, and activation of executing caspase-9 and -3 in A431 and SCC25 cells. The combinatory use of clinical drug of 5-fluorouracil (5-Fu) and AE accelerated greater apoptotic cell death than each drug did alone in A431 and SCC25 cells, these synergistic effects may through significantly enhanced caspase-8, -9 and caspase-3 expressions after combined treatment of 5-Fu and AE in both cancer cells. The AE (5 mol %) is the first time incorporated into a liposomal formulation for in vitro cytotoxicity and skin penetration tests in this study. The combinatory use of non-toxic liposome with the low concentrations of AE (IC20 and IC50) accelerated greater cell death than AE did alone for short times (24 and 48 h) in A431 and SCC25 cells. These data demonstrate positive cooperation of liposome and AE and emphasize the potential clinical usefulness of liposome-AE combination therapy. Furthermore, results of skin permeation profile suggest that the liposomal formulation could enhance the transdermal delivery of AE, which may be useful to increase the efficiency of AE delivery. |
