胰臟癌是西方國家是造成癌症死亡的第四項惡性腫瘤，在臺灣也是十大癌症死因之一。百分之 八十五的胰臟癌在診斷確立時，已有遠處轉移或局部侵襲至臨近器官，此時只有少於百分之十能夠 接受根除性手術治療。胰臟癌患者的平均存活時間只有四至六個月，並且整體的五年存活率小於百 分之五。咖啡酸苯乙酯是蜂膠中所具有生物活性的成份。之前對咖啡酸苯乙酯的研究顯示咖啡酸苯 乙酯是具有抗病毒、抗發炎、抗氧化、放射線治療的致敏劑等作用的性質。本實驗將以上列藥物作 用於人類胰臟癌細胞株，希望能得到一些調控細胞週期及誘導細胞凋亡的相關機轉。對於中度及低 度分化人類胰臟癌細胞株 BxPC-3 與 PANC-1, 我們以 trypan blue dye exclusion test 來偵測咖啡酸苯 乙酯對細胞株生長的抑制作用。我們藉著觀察 morphology ， DNA histogram ， annexin-V/PI 雙染法
， agarose gel electrophoresis 來檢驗咖啡酸苯乙酯造成人類胰臟癌細胞株 BxPC-3 的死亡形式。隨著 細胞株加入 10 μg/mL 濃度的咖啡酸苯乙酯處理， BxPC-3 細胞株在第三天達到 80.4 ± 4.1% 的生長 抑制， PANC-1 細胞株在第三天達到 74.3 ± 2.9% 的生長抑制。 BxPC-3 細胞株在咖啡酸苯乙酯處理 的第二天見到典型的細胞凋亡型態變化但並未見到明顯的 DNA 片斷化。隨著細胞株加入不同濃度 5 μg/mL 至 20 μg/mL 的咖啡酸苯乙酯處理而隨著時間增加 sub-G1 population 的趨勢，第 2 日起有較 高的細胞株的 sub-G1 population 增加。 BxPC-3 以 10 μg/mL 濃度的咖啡酸苯乙酯處理、處理的細 胞株產生粒線體膜電位下降，在二天後呈現下降至未處理細胞的 66.2 ± 11.4% 。 BxPC-3 在經 CAP E 培養之前加入泛 caspase 抑制劑 Z-VAD-fmk 50 μM 處理，呈現部分的生長的抑制作用及 sub-G1 細胞群減少。並且 BxPC-3 細胞加入 CAPE 10 μg/mL 處理，在 2 小時後發現 caspase-3/7 的活性有 2 倍增加。說明 caspase 在咖啡酸苯乙酯誘導人類胰臟癌細胞株細胞凋亡中扮演相關的角色。本實驗 結果顯示咖啡酸苯乙酯有強力的誘導人類胰臟癌細胞株細胞凋亡的活性，細胞死亡的同時也伴隨著 粒腺體膜電位的減少及 caspase 的活化。
咖啡酸苯乙酯誘導人類胰臟癌細胞凋亡之作用及 相關機轉
The Effect of Caffeic Acid Phenethyl Ester on Induc ing Apoptosis of Human Pancreatic Cancer Cells a
nd Related Mechanisms
Pancreatic cancer is the fourth leading cause of cancer death in the western countries and the tenth in Tai wan. There are more than 85% of pancreatic cancers metastasized or extended locally at the time of diagn osis. Finally there were about less than 10% of the pancreatic cancer patients able to undergo curative res ection. The average survival duration for the patients with pancreatic cancer is about 4–6 months, and the overall 5-year survival rate is less than 5%. An effective treatment or novel agent for this devastating dise ase is urgently needed. Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of honeybe e propolis. The previous studies showed several biological functions, including anti-viral, anti-inflammat ory, antioxidant and radiation sensitization effects of CAPE. This study was aimed to investigate the effec t of caffeic acid phenethyl ester on inducing apoptosis in human pancreatic cancer cells. The growth inhib itory effect of the CAPE on BxPC-3 and PANC-1, moderate to poorly differentiated human pancreatic car cinoma cell lines, were estimated by trypan blue dye exclusion test. We examined the type of cell death in BxPC-3 after CAPE treatment by observation of morphology, DNA histogram, annexin-V/PI staining and DNA agarose gel electrophoresis. The CAPE treatment (10 μg/mL) resulted in marked growth inhibition on BxPC-3 cells up to 80.4 ± 4.1% and PANC-1 to 74.3 ± 2.9% at day 3. CAPE also induced characteristi c morphological changes of apoptosis but there were no DNA fragmentation in BxPC-3 cell at day 2. Esti mation of the apoptotic percentage showed a time-dependent increase after CAPE treatment. CAPE (10 μ g/mL) induced a significant decrease in mitochondrial transmembrane potential to 66.2 ± 11.4% of the un treated level after 2 days. The growth inhibition and sub-G1 population was partially blocked by pretreat ment with pan-caspase inhibitor Z-VAD-fmk (50 μM), which indicating a caspase-related mechanism inv olved in CAPE-induced apoptosis. The caspase-3/7 activity were approximately 2 times greater after treat ment by caspases substrate activity assay. These results suggested that CAPE is a potent apoptosis-induci ng agent and its action is accompanied by loss of mitochondrial transmembrane potential and activation o f caspase.
