- 學位論文
脂氧酶在中樞神經疾病與癌症轉移之研究
Role of lipoxygenase in CNS disease and cancer metastasis
摘要
神經性疾病與癌症在已開發國家是很嚴重的醫療與經濟問題。兩者的致病原因都十分複雜，所以截至目前為止，在這領域中雖有許多研究，但仍然無法窺見全貌。我們從2002年起，在生技製藥國家型科技計畫辦公室支持下以小鼠腦中風模式篩選了多種化合物，來評估對中樞神經保護的作用，一共篩選超過1000種化合物。其中只有由中國醫藥大學侯曼貞教授合成的化合物MJ-39效果最為優良。其在大、小鼠腦中風模式下，皆能有效減少中樞神經損傷，並且能提高中風後老鼠的運動能力，經由計畫辦公室委由汎球藥理公司進行一般藥理實驗，證實其選擇性對5-脂氧酶 (lipoxygense) 與12/15-脂氧酶具有抑制的作用。脂氧酶是一類含鐵的酵素，主要催化花生四烯酸 (arachidonic acid) 產生白三烯 (leukotriene) 或羥花生四烯酸 (hydroxyeicosatetraenoic acid, HETE)。這些代謝物很多參與發炎反應或具有抗發炎的作用，因此脂氧酶也常常被認為與神經疾病或癌症有關。最近有許多研究指出脂氧酶在腦中風、阿茲海默症、巴金森氏症都扮演重要的角色。 我們發現MJ-39與其衍生物也有許多具有神經保護的效果，我們以中腦神經與神經膠細胞來測試這些衍生物對MPP+所造成的神經毒性影響，發現其仍然具有不錯的保護效果；而在動物實驗方面，亦能保護中風老鼠大腦皮質的損傷，並能拮抗MPTP造成多巴胺神經元的損傷。 我們實驗發現，12/15-脂氧酶的下游代謝產物羥花生四烯酸，在小鼠腦中風模式下，加重血腦障壁滲透性增加的情形，此一現象在12/15-脂氧酶基因剔除小鼠上，觀察到顯著的改善。在離體實驗中，羥花生四烯酸亦會增加血腦障壁的滲透性增加，還會破壞其緊密連接蛋白 (tight junction protein)。而這些作用我們認為是經由煙醯胺腺嘌呤二核苷酸磷酸氧化酶 (NADPH oxidase) 訊息傳遞路徑而來。 我們進一步研究發現在小鼠注射MPTP後，星狀神經膠細胞 (astrocytes)的5-脂氧酶表現會增加。另一方面5-脂氧酶活化蛋白的抑制劑MK-886，在中腦細胞培養以及SHSY5Y細胞株，皆可有效改善MPP+造成的多巴胺神經死亡。此外，5-脂氧酶下游產物，白三烯B4不論在活體或離體的巴金森氏症模式下，皆會增加多巴胺神經的損傷。根據這些結果我們認為5-脂氧酶抑制劑，有潛力可以做為治療巴金森氏症的候選藥物，而白三烯B4在巴金森氏症的致病機轉，可能也扮演重要角色。 癌症之所以可怕，不在於腫瘤組織不斷的增殖，而在於它會在原發病灶附近造成破壞之外，更會產生遠端轉移，導致病患的死亡。癌症轉移相當複雜，且牽涉到癌細胞的生長、擴散與血管新生。我們發現到羥花生四烯酸會增加黑色素瘤細胞B16F10黏著能力，增加癌細胞轉移。在分離培養小鼠的肺部表皮細胞中，與野生型小鼠相比發現黑色素瘤細胞較少黏著在12/15-脂氧酶剔除小鼠的組別上。我們進一步羥花生四烯酸增加細胞黏著的作用與磷酸化局部沾黏激酶 (Focal Adhesion kinase)有關，且此一現象會被ERK抑制劑抑制。綜合以上所知，我們認為未被癌細胞入侵的器官，因其12/15-脂氧酶活化，進而產生過多的羥花生四烯酸，使得局部沾黏激酶磷酸化促進黑色素瘤的粘著，進而導致癌細胞轉移；可以推論，抑制12/15-脂氧酶活化可以降低癌細胞轉移。 從上述所有實驗可以知道，脂氧酶抑制劑可以開發成為中樞神經保護劑或抑制癌症細胞轉移之藥物。在這兩大領域雖然各大藥廠皆投入大量心血研發，但市場仍然十分需要更有效之藥物，我們的研究提供了一個未來藥物研發的方向。
並列摘要
Neurological illnesses and cancer are among the most common and most serious health problems in industrialized nations. The causes of them are diverse, complex, and only partially understood. Since 2002, we have used the stroke model of mice to screen over 1000 testing compounds under the support office of National Science and Technology Program for Biotechnology and Pharmaceuticals. Only one potential compound was found among all tested sample, with code number of MJ-39, which is provided by the professor M.J Hour in China Medical University. The biochemical assay results show that MJ-39 is a potent and specific inhibitor for 5-lipoxygenase (5-LOX) and 12/15-lipoxygenase (12/15-LOX). LOXs are a family of lipid-oxidizing enzymes that generate a wide array of pro- and anti-inflammatory mediators. The well-known 5-lipoxygenase and its activating protein have recently been intensely studied because of a genetic linkage to stroke and Alzheimer's disease. In addition, 12/15-LOX is also one of the key mediators in stroke and neurodegenerative disease. We have screened several MJ-39 derivatives and found some more effective compounds. MJ serial compounds also exhibit a good neuprotective efficacy after ischemia/reperfusion (I/R) injury. Here we also found that MJ serial compounds significantly inhibited the cell death of dopaminergic neurons after injection of MPTP in vivo and significantly inhibited MPP+-induced neuronal death in vitro. We further examined the role of LOX in the increase of blood brain barrier (BBB) permeability following stoke. It was found that 12/15-LOX metabolites enhanced brain damage and BBB hyperpermeability after I/R injury and these effects were reduced in 12/15-LOX KO mice. In addition, 12(S)- and 15(S)-HETE caused an increase of BBB endothelial permeability and a destruction of ZO-1 in vitro. NADPH oxidase pathway may be involved in 12/15-LOX metabolites enhanced endothelial barrier disruption. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [3H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP+ treatment. In addition, LTB4, one of 5-LOX’s downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB4 also enhanced MPP+-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB4 may play an important pathological role in Parkinson’s disease. Metastasis is the leading cause of cancer-related death. Metastasis is a very complex cascade of events such as tumor growth, invasion and angiogenesis. Here we found that HETEs released from host organ played a critical role in tumor metastasis. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonized by FAK inhibitor or ERK inhibitor. 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signaling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in host organ may affect the metastatic potential of melanoma. In summary, LOX inhibitors may be developed for the treatment of ischemic stroke and PD. Furthermore, 12/15-LOX is a potential therapeutic target for developing drugs to inhibit the progression of melanoma. LOXs may play a pivotal role in neurological illnesses and cancer metastasis.