蜂膠萃取物咖啡酸酚酯對血癌細胞氧化自由基與細胞凋亡的雙刃效應

Abstract

抗氧化功能被廣泛認為是蜂膠的眾多藥理作用之一，在世界各地及台灣的各類蜂膠萃取物同樣也展現抗氧化的能力。例如咖啡酸酚酯 (caffeic acid phenethyl ester，簡稱CAPE) 無論在細胞培養或活體實驗中，其均可降低細胞因自由基過載所導致的氧化傷害。不過我們卻發現CAPE本身除了抗氧化功能外，亦會促使自由基的產生。分析結果顯示CAPE在早期具有抗氧化的功能；在晚期則會反轉為促氧化的角色，這樣的現象跟處理時間有密切的關聯，而無關於劑量。進一步釐清其中的機制，我們發現CAPE會誘發抗氧化蛋白heme-oxygenase 1 (HO-1) 的表現，此可解釋CAPE抗氧化的現象，但在晚期，CAPE所引起HO-1的表現則呈現過量狀態，而促使細胞鐵離子濃度失衡，過多的游離鐵離子易與過氧化氫作用而生成具有高度破壞性的氫氧自由基，進而造成氧化壓力與細胞凋亡。當HO-1的活性被專一擷抗劑抑制後，我們不僅發現CAPE在晚期所造成細胞毒性與細胞凋亡減低，其同時也遮蔽了CAPE抗氧化及促氧化的雙重效應，由此可見HO-1確實在CAPE對於細胞自由基的載覆性扮演重要的角色。雖然CAPE晚期促氧化的現象可能具有潛在風險，但是此促氧化的特質可誘使NB4血癌細胞由粒線體的途徑進行細胞凋亡，其機制涉及抗細胞凋亡Bcl-2家族蛋白Bcl-2及Mcl-1的抑制；以及提升同家族蛋白促細胞凋亡的Bmf、Noxa和Bax之表現，進而導致粒線體膜電位差的喪失及Cytochrome c的溢漏，相繼造成Caspase-9與Caspase-3的活化造成細胞凋亡現象，最後而達到抑制癌細胞生長的目的。本論文評估CAPE具有癌症臨床醫療的可能性，而利用蜂膠或CAPE於保健上的應用亦需謹慎。

Caffeic acid phenethyl ester (CAPE), an active component from bee propolis, has been recognized as a strong antioxidant agent. However, several previous documents report the pro-oxidant effect of CAPE. In this investigation to specify the role of CAPE on intracellular intracellular reactive oxygen species (ROS), we report CAPE acts either as a protective antioxidant at early phase of treatment or a deleterious pro-oxidant during late time period to human promyelocytic NB4 cells. This double-edged effect of CAPE depended on the treatment time phase rather than the dose. Short-term CAPE treatment was protective due to the induction of heme-oxygenase-1 (HO-1) at an appropriate level which catalyzed anti-oxidative heme degradation. Long-term CAPE treatment was deleterious because overexpression of HO-1 led to inhibition of ferritin and thereby the transient existence of ferrous iron, which in turn reacted with hydrogen peroxide through the Fenton reaction to produce more-harmful hydroxyl radicals. The pro-oxidative nature also is involved in CAPE-induced typical mitochondrion-dependent apoptosis. The molecular mechanism of CAPE-driven apoptosis is associated with multiple Bcl-2 family proteins including inhibition of Bcl-2 and Mcl-1 and up-regulation of Bmf, Noxa, and Bax. The results of interaction of those Bcl-2 family proteins cause loss of mitochondrial membrane potential and leaking of Cytochrome c, and subsequently activate Caspase-9 and Caspase -3 to complete the intrinsic apoptotic signal finally. The conclusion of this dissertation suggests that CAPE (or propolis containing CAPE) could be a potential candidate for cancer therapy in the clinic and should be applied carefully.