Quinazoline類α1-腎上腺素受體拮抗劑在培養心肌細胞毒性作用機制之探討

Quinazoline類α1-腎上腺素受體拮抗劑最初是開發用於高血壓治療的藥物，然而許多的臨床試驗結果顯示，這類藥物如Prazosin及Doxazosin，用於心臟衰竭病人時會增加突發性心血管疾病及死亡的風險。因此，Quinazoline類α1-腎上腺素受體拮抗劑目前已不再被視為第一線的心血管疾病用藥，但這類藥物仍被廣泛的運用在良性攝護腺肥大（benign prostate hyperplasia, BPH）的治療上。此外，這類藥物所特有的與α1-腎上腺素受體拮抗無關之細胞毒性作用已有許多相關的研究及報告。在本研究中，我們以H9C2小鼠心肌母細胞株作為實驗模型並分析Quinazoline類α1-腎上腺素受體拮抗劑之中參與細胞毒性的分子機制。研究結果顯示，在我們所測試的四種Quinazoline類α1-腎上腺素受體拮抗劑（Prazosin, Doxazosin, Terazosin及Bunazosin）中，只有Peazosin及Doxazosin對H9C2具有明顯的細胞毒性。在後續針對Prazosin及Doxazosin的研究，我們發現了兩種全然不同的分子機制分別參與了Prazosin及Doxazosin所引起的細胞死亡過程。在Doxazosin引起細胞死亡研究中，我們發現transforming growth factor-β（TGF-β）第一型受體（TβRI/ALK5）參與了Doxazosin所引發的H9C2細胞凋亡。經由測定細胞存活率、凋亡之細胞核及caspase-3的活性，我們發現Doxazosin可以引起濃度及時間相關性的H9C2細胞凋亡，且在10 ng/ml的TGF-β1存在下，30 μM Doxazosin所引發的H9C2細胞凋亡現象被明顯的加強。H9C2細胞以Doxazosin處理時，其p38 MAPK的蛋白質磷酸化被顯著的提升，若以TGF-β1處理時，則Smad3的蛋白質磷酸化被顯著的提升。然而，同時以Doxazosin及TGF-β1處理H9C2細胞時，TGF-β1所引起的Smad3的蛋白質磷酸化被顯著的減少，而Doxazosin所引起的p38 MAPK蛋白質磷酸化被顯著的增加。此外，TβRI/ALK5的抑制劑（SB431542）、p38 MAPK的抑制劑（SB202190）及TβRI/ALK5的knockdown都可以明顯的減少Doxazosin在H9C2細胞所引起的細胞凋亡現象。我們於是歸納出TβRI/ALK5-p38 MAPK蛋白質磷酸化的細胞信息傳遞路徑參與了Doxazosin在H9C2細胞所引起的細胞凋亡現象，此路徑可經由TGF-β1相關的Smad3蛋白質磷酸化降低而進一步被加強。在Prazosin引起細胞死亡的研究中，我們發現Prazosin會在H9C2細胞引發自我吞噬（Autophagy）的現象，包括細胞質中出現空泡（vacuoles）、細胞微小管蛋白質輕鏈（microtubule-associated protein 1 light chain 3, LC3）發生結構改變及細胞內酸性空泡狀胞器（acidic vesicular organelle, AVO）的大量堆積等。經由西方點墨法分析蛋白質磷酸化發現，在prazosin的處理下，p53與AMPK的蛋白質磷酸化程度呈現明顯的增加，相反的mTOR, Akt與p70S6K的蛋白質磷酸化程度則呈現明顯下降的趨勢。此外，雖然在Autophagy抑制劑3-Methyladenine及p53抑制劑Pifithrin-α的前處理下，Prazosin所引發的AVO大量堆積會被明顯抑制，但是Prazosin所引發的細胞死亡作用並無法被阻斷，且Prazosin所引發的caspase-3活化有被加強的現象。基於這些發現，我們推測Prazosin可經由p53的媒介作用引發自我吞噬性細胞死亡（autophagic cell death），而當這個機制被阻斷時，Prazosin仍可轉而加強細胞凋亡路徑中的caspase-3活性，引發細胞死亡。

關鍵字

自我吞噬；細胞凋亡

並列摘要

Quinazoline-based α1-adrenoceptor antagonists are drugs initially developed for the treatment of hypertension. However, results from trials reveal that some of them, especially prazosin and doxazosin, are associated with greater mortality and risks of cardiovascular accidents among heart failure patients. Therefore, quinazoline-based α1-adrenoceptor antagonists has no longer been considered as the first-line drug in the treatment of hypertension, but introduced primarily in the treatment of benign prostate hyperplasia (BPH). The α-adrenoceptor blockade-independent cytotoxic effects of these drugs also attract great attentions of researchers. In present study, we used H9C2 mouse ventricular myoblast cell line as experiment model and attempted to dissect the molecular mechanisms lying in the cytotoxicity of quinazoline-based α1-adrenoceptor antagonists. Our results demonstrated that in the four kinds of quinazoline-based α1-adrenoceptor antagonists we tested (prazosin, doxazosin, treazosin and bunazosin), only prazosin and doxazosin showed significant cytotoxicity to H9C2 cells. In the subsequent analysis focused on prazosin and doxazosin, we identified two distinct molecular mechanisms participated in prazosin- and doxazosin-induced cell death. In doxazosin-induced cell death, we proposed that the transforming growth factor-β type I receptor (TβRI/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell viability, apoptotic nuclei and caspase-3 activity, we found that doxazosin induced concentration- and time-dependent apoptosis in H9C2 cells. The cell apoptosis induced by 30 μM doxazosin was exacerbated by the addition of 10 ng/ml transforming growth factor-β1 (TGF-β1). Doxazosin or TGF-β1 alone respectively elevated p38 MAPK and Smad3 protein phosphorylation in H9C2 cells. However, the co-treatment of doxazosin and TGF-β1 attenuated the TGF-β1-induced Smad3 protein phosphorylation and increased doxazosin-induced p38 MAPK protein phosphorylation. Furthermore, inhibitors of TβRI/ALK5 (SB431542) and p38 MAPK (SB202190) or TβRI/ALK5 knockdown all dramatically reduced the doxazosin-induced apoptosis in H9C2 cells. We concluded that TβRI/ALK5-p38 MAPK phosphorylation signaling pathway could contribute to doxazosin-induced cell apoptosis, which could be further enhanced by TGF-β1 in association with attenuating Smad3 phosphorylation in H9C2 cells. In prazosin-induced cell death, we found that prazosin could induce patterns of autophagy in H9C2 cells, including formation of intracellular vacuoles, conversion of microtubule-associated protein 1 light chain (LC3) and augmentation of acidic vesicular organelles (AVO). Analysis of phosphorylated proteins by Western blotting revealed that, under the exposure of prazosin, the level of phospho-p53 and phospho-AMPK was increased, where in contrast, phospho-mTOR, phospho-Akt and phospho-p70S6K were dramatically decreased. Furthermore, pretreatment of pharmacological autophagy inhibitor 3-methyladenine and p53 inhibitor pifithrin-α although both suppressed prazosin-induced AVO formation, it did not reverse prazosin-induced decline in cell viability but enhanced prazosin-induced caspase-3 activation. Based on these results we suggest that prazosin induced autophagic cell death via p53-mediated mechanism. When autophagy pathway was blocked, prazosin could still induce programmed cell death, at least in part, through the enhancement of apoptotic caspase-3 cascade.

並列關鍵字

quinazoline ； prazosin ； doxazosin ； TGF-β ； TβRI ； ALK5 ； p38 MAPK ； autophagy ； apoptosis ； p53

參考文獻

Adams JM, Cory S (2007) Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 19:488-96

Akduman B, Crawford ED (2001) Terazosin, doxazosin, and prazosin: current clinical experience. Urology 58:49-54

ALLHAT Collaborative Research Group (2000) Major cardiovascular events in hypertensive patients randomized to doxazosin vs chlorthalidone: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT). Jama 283:1967-75

Amann T, Hellerbrand C (2009) GLUT1 as a therapeutic target in hepatocellular carcinoma. Expert Opin Ther Targets 13:1411-27

Anglin IE, Glassman DT, Kyprianou N (2002) Induction of prostate apoptosis by alpha1-adrenoceptor antagonists: mechanistic significance of the quinazoline component. Prostate Cancer Prostatic Dis 5:88-95

國際替代計量

Quinazoline類α1-腎上腺素受體拮抗劑在培養心肌細胞毒性作用機制之探討

全文下載

主題瀏覽