| Abstract: | 第一部份 咖啡酸苯乙酯抑制神經膠質瘤體外與體內之增生及誘發分化之研究
Caffeic acid phenethyl ester (CAPE)是多酚抗氧化化合物，是蜂膠主要成分之一；過去文獻指出其具有抗發炎、抗氧化、抗病毒、抗腫瘤等作用。由先前的實驗，發現 CAPE誘導的訊息傳導機制 p38 MAPK (p38 mitogen-activated protein kinase)，啟動可能由 p53-Bax途徑介導，誘發凋謝死亡；所以，在本實驗中我們針對 C6神經膠質瘤進一步探討其對抑制細胞增生之作用機轉。當處理 CAPE不同劑量與歷經不同的時間，藉此觀測細胞的增生動態，我們發現 CAPE能有效的壓抑惡性腫瘤 C6的增殖；除此，透過流式細胞儀分析細胞週期， CAPE（50μM）處理24小時，發現 85 ﹪的細胞停滯在G0/G1期；利用西方墨點法分析相關細胞週期蛋白，處理6小時後發現， Rb phosphorylation蛋白表現減少，且 CDKIs 蛋白 p16、 p21、 p27表現大增；而 cyclin D1與 cyclin E的交互作用遞減，顯示 CDK2活性的確受到影響，因此 CAPE呈現良好的抑制現象。另外，在裸鼠皮下接種 C6神經膠質瘤細胞，測試 CAPE是否影響在皮下之急性生長；給予腹腔注射 CAPE (1-10 mg/kg)能有效抑制轉殖裸鼠腫瘤之生長，摘除局部腫瘤組織，切片利用免疫化學染色觀察，細胞增殖的情形，結果發現， CAPE 刺激下，能抑制皮下癌化細胞的分裂與 PCNA蛋白質的表達。據文獻指出，誘發細胞週期停滯與特異的蛋白表現是分化之現象，所以我們以 CAPE為藥劑來探索是否造成神經膠質瘤 C6細胞分化。結果顯示，在低劑量 CAPE (10 μM)處理下，能有效誘導 C6細胞分化標的蛋白 GFAP表達，細胞型態呈現紡綞體而不見延伸能力。並發現 PKC/ERK訊息傳導激活化，在 CAPE刺激 C6細胞的分化過程裡，可能扮演重要的調控角色。經由以上結果得知， CAPE的抗癌活性作用，在較高的劑量能抑制神經膠質瘤細胞的增生，而且較低的劑量能誘發癌化 C6細胞分化。
第二部份 咖啡酸苯乙酯促進維他命A酸誘發人類血癌細胞分化之研究
急性前骨髓性白血病 (APL)是由於這些骨髓母細胞異常停滯在不同分化階段，白血球不能正常分化，進而造成不成熟的細胞大量累積成為病灶。目前，治療血癌病患的化學治療以維他命A酸 (ATRA)為主；不過美中不足的是， ATRA可能會引起白血球過高及快速發展的耐藥性，致使治療遇上瓶頸，而無法維持病患長期的緩解。而替代方法是尋求另類藥劑，或是以天然物作為複合配方，以加強治療效用。 Caffeic acid phenethyl ester (CAPE)是蜂膠活性成分之一；過去，我們已經證實，刺激癌化細胞凋亡與抑制增生的機制。根據之前文獻，誘發細胞週期停滯與特異的蛋白表現是分化現象，所以我們以 CAPE為藥劑來探索是否造成血癌 HL-60細胞分化，或是與 ATRA作為複合配方研究分化的分子機制。經由實驗結果發現，利用化學染劑 (Wright-Giemsa stain) 觀察型態， CAPE也會促進低劑量 ATRA (1nM)分化的效用，而此種分化經由 NBT reduction，和透過流式細胞儀分析分化標的膜蛋白 CD11b/CD14及細胞週期測試後，證實細胞週期停滯 G1 phase與分化現象的機制有關；再藉由免疫沉降法測試，更證實細胞週期停滯將影響 CDK2的活性。血癌 HL-60細胞分化與細胞核 RARa蛋白受體訊息路徑，促進相關基因的轉錄活性有關。另外，我們也利用 EMSA分析 RARa蛋白受體與 RARE DR5的親合力，發現低劑量 ATRA誘導 RARa轉錄活化會隨 CAPE的劑量增高而加強，直接促進 ATRA相關基因 RARa、C/EBPe與 p21的表達，進而誘導血癌細胞分化的進行；此外， RARa蛋白受 CAPE的處理而活性增加，這樣的現象似乎是 MEK-ERK活絡訊息傳導路徑有關。在以上實驗結果得知， CAPE不僅會誘導分化，也會促進分化的進行，充分了解分化現象的機制，與細胞核蛋白雙受體訊息路徑的角色。這個研究將在抗血癌的化學預防領域有所突破。
Part-1
The mechanistic study of caffeic acid phenethyl ester induced C6 glioma growth inhibition and differentiation in vitro and in vivo
Caffeic acid phenethyl ester (CAPE), a component of honeybee propolis, has been reported to hold various biochemical responses. More recently, work on the anti-inflammatory, anti-bacteria, anti-fungus, anti-virus and anti-tumor activity of propolis has concentrated on caffeic acid phenethyl ester（CAPE）, which is one of components. As our previous studies, we had found CAPE induced apoptosis of C6 glioma cells and was mediated via signaling pathway of p38 MAPK. In the present study, we found that CAPE inhibited the growth of C6 cells in a dose dependent and time dependent manner as shown by the results of trypan blue dye exclusion assay and cell proliferation assay. In addition, the cell number percentage of the G0/G1 phase increased to 85% after the treatment with 50 μM of CAPE for 24h. After treatment with CAPE (50 μM) for 6 h, it demonstrated that the protein level of hyperphosphorylated pRb decreased, and cyclin dependent kinase inhibitors p21, p27, and p16 were marked up-regulated. The association of CDK2 and cyclin E that affects the CDK2 activity decreased. When C6 cells were grown as xenografts in nude mice, treatment with CAPE (1-10mg/kg; ip) induced a significant dose dependent decrease in tumor growth by evaluating tumor volume and tumor weight. Histochemical and immunohistochemical analysis revealed that CAPE treatment significantly reduced the number of mitotic cells and proliferating cell nuclear antigen (PCNA)-positive cells in C6 glioma. We further evaluated the effects of CAPE (10 μM) on the expression of GFAP and PKC isoenzymes in C6 glioma cells. Our results demonstrated that the ability of CAPE to promote activation of PKC/ERK is thought to be a key event in controlling the astrocytic differentiation of C6 glioma cells. These results suggest that CAPE presents an antitumor potential for glioma by inhibiting the growth of tumor cells and triggering astrocytic differentiation.
Part 2-The role of caffeic acid phenethyl ester enhanced all–trans retinoic acid induced differentiation in human leukemia HL-60 cells
Acute promyeloid leukemia (APL) is characterized by the accumulation of myeloid blast cells that are arrested at various differentiation stages and unable to terminally differentiate. All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (ALP), however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. Our previous studies showed that CAPE apparently affect carcinoma cells of by stimulating cellular apoptosis and anti-proliferative effect both in vitro and in vivo. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest at the G1 phase by decreasing the association of CDK2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear receptors such as RARa binding to the DNA response element and enhanced the expression of target genes including RARa, C/EBPe, and p21 protein resulting in the development of leukemia. CAPE alone elicited the expression of RARα, but a marked up regulation in presence of ATRA, which appeared to be modulating MEK-ERK signal pathway. Taken above together, it is suggested that CAPE provides novel drug targets of the potential to enhance the efficiency of ATRA in the induction differentiation of promyelocytic leukemia. |
