研究α1交感神經受體阻斷劑對於抑制血管新生的效果及機轉

Abstract

本研究的目的是希望比較不同的α1交感神經受體阻斷劑對血管新生抑制的效果，並選出最具有潛力的一種，進一步去研究其作用機轉，希望藉由瞭解這類藥物抑制血管新生的作用機轉，奠定其在臨床上應用的基礎。研究方法主要是進行人類血管內皮細胞的體外研究，所使用的藥物為目前臨床上常使用的α1交感神經受體阻斷劑，包含doxazosin、terazosin、prazosin及tamsulosin。我們先利用Sulforhodamine B (SRB) assays來定量細胞的生長作用，測定以及比較不同α1交感神經受體阻斷劑抑制細胞生長的影響程度。結果顯示prazosin的IC50 (6.53 µM)以及doxazosin的IC 50 (7.6 µM)明顯較terazosin (24.62 µM)及tamsulosin (25 µM)佳，對人類臍靜脈內皮細胞的生長抑制有明顯較強之效果。們進一步選擇prazosin當我們進一步研究的對象，去探討其抑制血管新生的機轉。我們利用人類臍靜脈內皮細胞來針對其細胞生長(growth)、附著(adhesion)、以及管狀生成(tube formation)三部份來評估，結果發現prazosin對三個部份都有明顯的抑制效果，prazosin在12.5 µM的濃度下就能抑制80%的細胞生長，然而在抑制附著的部份，需到30 µM才有明顯抑制效果，至於tube formation部份，在2.5 µM濃度就有明顯抑制的效果。於prazosin抑制人類臍靜脈內皮細胞生長的機轉研究，流式細胞儀結果顯示prazosin在濃度20 µM作用6小時就有明顯subG1 phase增加的情形，而且subG1 phase增加的情形並且會隨著時間和濃度的增加而增加，但細胞週期分析並無明顯的G0/G1 arrest或G2/M arrest。我們也利用Comet assay來評估單一細胞DNA受損的方法，即使用到40 µM的prazosin仍無法觀察到DNA damage的情形。至於粒線體膜電位，在20 µM的prazosin作用下，12小時就有膜電位變化 (loss of mitochondrial membrane potential)的情形。方點墨法實驗發現在20 µM的prazosin作用下，抑制細胞凋亡的Mcl-1在6小時就有很明顯的減少，而Bad及Bcl-xL也有些許增加。而藉由粒線體及細胞質蛋白萃取，12小時細胞質中的cytochrome C就有增加的情形。再加上caspase-9、caspase-3和caspase-7有明顯裂解的情形，而caspase-8無變化。因此我們認為przaosin會造成人類臍靜脈內皮細胞細胞凋亡，而其主要路徑是和內生性細胞凋亡有相關。們結論是認為，在目前臨床上常使用的α1交感神經受體阻斷劑包含doxazosin、terazosin、prazosin及tamsulosin中，prazosin對人類臍靜脈內皮細胞的生長抑制有明顯較強之效果，而其主要路徑是和內生性細胞凋亡相關。此外，prazosin對人類臍靜脈內皮細胞的附著及管狀生成也都有抑制的效果。因此我們認為prazosin用來抑制血管新生是具有潛力的，雖說距離臨床上正式使用來治療癌症還有很長的一段路要走，但目前在臨床上合併其他抗癌藥物或放射治療的使用，以及評估減少攝護腺癌發生率的效果是目前值得進行的方向。

The purpose of this study is comparing the anti-angiogenetic effect of different α1-adrenoceptor antagonists and investigating the possible mechanism of one with greatest potential. e performed in vitro study using primary culture of human umbilical vein endothelial cell (HUVEC), and evaluated the effect of most common used α1-adrenoceptor antagonists, including doxazosin、terazosin、prazosin, and tamsulosin, on HUVECs. Sulforhodamine B (SRB) assays were used to define cell proliferation, and compared the effect of different α1-adrenoceptor antagonists on HUVEC cell proliferation. The concentration of 50% inhibition on cell growth (IC50) was obtained. Our data showed that prazosin (IC50: 6.53 µM) and doxazosin (IC50: 7.6 µM) have greater growth inhibition than terazosin (IC50: 24.62 µM) and tamsulosin (IC50: 25 µM).e further chose prazosin to inveatigate its anti-angiogenetic mechanism. We found that prazosin has significant inhibition on HUVEC cell growth, cell adhesion, and tube formation. In a concentration of 12.5 µM prazosin, growth inhibition was found in 80% of HUVECs. Significant inhibition of cell adhesion and tube formation was found in a concentration of 30 µM and 2.5 µM, respectively.low cytometric analysis showed that prazosin induced an increase of subG1 population in a concentration of 20 µM for 6 hours. Quantified data showed that the increase is time-dependent and concentration-dependent. However, there is no significant G0/G1 or G2/M arrest. Comet assay was used to evaluate single cell DNA damage, but no DNA damage was observed even 40 µM prazosin was used. Loss of mitochondrial membrane potential was found in a concentration of 20 µM prazosin. estern blotting analysis showed a significant decrease of Mcl-1 in a concentration of 20 µM prazosin for 6 hours. Increase of Bad and Bcl-xL was also found. Cytochrome C in cytosol increases in a concentration of 20 µM prazosin for 12 hours. Expression of cleavage of caspase-9、caspase-3, and caspase-7 were also detected by Western immunoblotting.n conclusion, prazosin has greater growth inhibition of HUVEC than other common α1-adrenoceptor antagonists. The pathway of growth inhibition is suggested to be association with intrinsic apoptotic pathway. In addition, adhesion and tube formation of HUVEC are also inhibited by prazosin. We think prazosin may have a potential role in anti-angiogenesis. Although there is still a long distance of prazosin to be used clinically as an anti-angiogenetic medication, using in combination with chemotherapeutic medication or radiation may be a considered way. To evaluate the preventive effect for prostate cancer is another possible consideration.