對於鋰鹽在治療惡性腦瘤以及使用GDNF基因和CGS26303藥物可減緩因胸腹主動脈手術中缺血再灌流傷害引起的脊髓損傷之藥物實證試驗研究

Experimental trials of Lithium on the treatment of brain cancer, GDNF gene and CGS 26303 drug on the reduction of spinal cord ischemia- reperfusion injury induced by thracoabdominal aortic surgery

周安國(An-Kuo Chou)

指導教授 ： 洪純隆

共同指導教授 ： 關皚麗(Aij-Lie Kwan)

高雄醫學大學/醫學院/醫學研究所/博士(2012年)

https://doi.org/10.6832/KMU.2012.00043

摘要

I.鋰鹽與腦瘤
已知的鋰鹽的神經保護作用已被證實乃經由相當多個的交叉機制，並且可在用於治療急性的大腦損傷，例如：大腦缺血，以及慢性的神經退化性疾病，例如：阿茲海默症、帕金森氏症，亨丁頓病和肌萎縮性側索硬化(ALS)等等的疾病。
在治療濃度下，鋰鹽可能的治療機轉是鋰鹽抑制了肝醣合成酶激酶glycogen synthase kinase 3 (GSK3)。GSK3是一種絲胺酸-蘇胺酸激酶 (serine-threonine kinase)，在正常細胞況狀下其活性即相當活躍但亦有相當多的訊息傳遞路徑可將它去活性化。肝醣合成酶激酶(GSK3)有α與?狳潃胺soforms。在細胞中，有相當多的受質可為肝醣合成酶激酶其標的，視其在不同訊息途徑中扮演著不同的作用。BCL2L12，一個BCL2家族中新近被鑑出的成員，曾經被報導與神經膠質母細胞瘤的後粒腺體凋亡事件有關，但是對於BCL2L12A而言，雖然其為BCL2L12的接合變體之一，但在神經膠質母細胞瘤中所扮演的角色及其在凋亡反應中之作用卻尚不清楚。在本項研究中，我們發現在臨床的神經膠質母細胞瘤（GBM）的檢體中，不論是BCL2L12和BCL2L12A均呈現著過度的表現。在大規模酵母菌雙雜合篩選實驗中，我們亦發現BCL2L12是GSK3?猁漱@個重要的結合伙伴，而且我們也證實GSK3?狳瓣˙PBCL2L12A發生交互作用，原因可能跟BCL2L12A其C -端缺乏可與GSK3?猁熊畢X區域有關。並且我們發現，BCL2L12與GSK3β的結合區域乃是位於C -端BH2模體外的153- 191的片段負責，相比之下，因為BCL2L12A沒有上述的區域所以和GSK3β之間並沒有產生相互作用。除此之外，在活體外激酶實驗和LAMDA磷酸酶實驗顯示，GSK3β主要是對於BCL2L12在S156的位置上產生磷酸化，而BCL2L12A並沒有這個位置。我們也發現BCL2L12的153- 191的片段對於GSK3β調控Tau的磷酸化呈現劑量依賴模式的抑制關係。此外，當我們由外源送入融合綠色螢光蛋白及BCL2L12或BCL2L12A基因進入U87MG的細胞株中，此腫瘤細胞株對於staurosporin (STS)的毒殺細胞反應減緩並導致細胞凋亡標記的減少，這些結果顯示，表明BCL2L12和BCL2L12A的確在神經膠質母細胞瘤中均扮演著抗凋亡的角色。相反的，若是在BCL2L12的156的位置上產生突變（S156A）則此變異後的BCL2L12(S156A)將不具有抗凋亡的能力，並且若將鋰鹽合併STS處理有表現BCL2L12的U87MG細胞株，此細胞株將進行凋亡。簡言之，在本研究中我們建立了一個模型，並證明了GSK3β乃藉由和BCL2L12的交互作用及將其磷酸化，並且亦能影響 BCL2L12A在神經膠質母細胞瘤的凋亡信號調控，並基於以上這些結果，我們認為LiCl對於治療神經膠質母細胞瘤將可能成為一個有潛力的治療藥物。

II.神經膠質細胞神經營養因子GDNF基因和內皮素轉換酶抑制劑CGS26303對於缺血再灌流引起的脊髓損傷
神經膠質細胞神經營養因子(glial cell line-derived neurotrophic factor; GDNF) 屬於轉型生長因子-?? superfamily的一份子，其能促進多巴胺神經元及運動神經元的神經突的存活與生長，對於周邊的感覺神經和交感神經亦有相同的作用。根據過去的文獻，在活體及離體實驗中，GDNF對於不同類型的神經傷害期間具有對神經元的保護作用。然而，因其短半衰期的影響，且無法通過腦血流屏障等等的阻礙了其臨床上的應用。半身癱瘓是胸主動脈手術中可能引起的一個災難性的併發症。在我們的研究中，以帶有GDNF基因的重組腺病毒以及帶有綠色螢光蛋白質的腺病毒作為此基因傳送的對照實驗，我們評估了GDNF基因對於防止因脊髓缺血引起的病理變化的預防效果。另外，在以氯化鈷處理脊髓神經細胞的實驗中，氯化鈷會引起依劑量依賴模式產生神經細胞的bcl-2 和synaptophysin downregulation，而此作用能被GDNF的基因傳送所抑制。因為在Intrathecal注射帶有GDNF基因的重組腺病毒能在2-7天內在脊髓內測得最大的GDNF表現，因此，我們選在intrathecal注射帶有GDNF基因的重組腺病毒3天之後，將SD大鼠施以暫時性的主動脈阻塞來引起脊髓缺血並觀察後續的行為損傷。在實驗中，被注射帶有GDNF基因的重組腺病毒的SD大鼠顯現出半身癱瘓的比率只有約40%，而對照組則呈現相當高的比率約75-85% (P<0.01)。另外，在運動功能的評估上，被注射帶有GDNF基因的重組腺病毒的SD大鼠亦展現出與對照組有明顯的差異(P<0.001)。此外在組織染色的分析中，顯示因GDNF基因傳送而減少了在脊髓缺血後白血球的浸潤及脊髓腹角 VI-VIII區神經細胞的死亡及神經突觸素synaptophysin的減少。總結而言，我們相信GDNF應是藉由對於神經細胞元和神經突觸的保護作用機制，才得以減輕因脊髓缺血引起的半身癱瘓。CGS26303，是一種內皮素轉換酶的抑制劑，過去的研究顯示CGS26303對於蜘蛛膜下腔出血及短暫性中大腦動脈阻塞有正面的效果。在我們的研究中，我們使用CGS26303並在SD大鼠脊髓缺血模式中觀察其運動功能以及在脊髓中第一型血色素氧化酶(HO-1)的表現。研究結果顯示，相較於生理食鹽水前處理之對照組而言，給予CGS26303前處理的這組在經過脊髓缺血再灌流傷害之後的第1及第3天，其半身癱瘓的比率較低而其運動功能的評估結果亦明顯較佳。分析SD大鼠其脊髓內的HO-1結果顯示給予CGS26303的組別表現出明顯較高的HO-1 mRNA。我們的研究結果顯示，CGS26303應是藉由調控HO-1在脊髓神經中的表現達到對於神經細胞的保護作用，亦得以減輕因脊髓缺血再灌流傷害所引起的神經損傷。基於這些結果，我們認為CGS26303對於脊髓缺血再灌流傷害具有神經保護的作用並可能成為一個有潛力的治療藥物。

關鍵字

鋰鹽 ； 惡性腦瘤 ； 肝醣合成酶激酶3β ； BCL2L12 ； BCL2L12A ； 磷酸化 ； staurosporine ； 凋亡 ； 脊髓損傷 ； 神經膠質細胞神經營養因子 ； 氯化鈷 ； 神經突觸素 ； CGS26303 ； 內皮素 ； 缺血再灌流傷害

並列摘要

I. Lithium v.s. Brain cancer
Lithium neuroprotection is provided through multiple intersecting mechanisms and can be used in the treatment of acute brain injuries such as ischemia and chronic neurodegenerative diseases as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS).
The possible mechanism is lithium at therapeutic concentrations inhibits glycogen synthase kinase 3 (GSK-3). GSK-3 is a serine–threonine kinase that is normally highly active in cells. It is deactivated by signals from numerous signaling pathways. This enzyme has two isoforms: α and β. Cellular targets of GSK-3 are numerous and often depend on the signalling pathway that is acting upon it.
BCL2L12 , a newly identified member of BCL2 family, has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3β binding partner in a testis cDNA library. Our data demonstrated that GSK3β interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12153–191 fragment located outside of the C-terminal BH2 motif is responsible for GSK3β binding. In contrast, no interaction was detected between BCL2L12A and GSK3β. In vitro kinase and l-phosphatase assays showed that GSK3β phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12153–191 fragment directly interrupted GSK3β mediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3β interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.

II. Glial cell line-derived neurotrophic factor (GDNF) and Endothelin-converting enzyme inhibitor, CGS 26303 v.s. Ischemia-Reperfusion spinal cord injury
GDNF belongs to transforming growth factor-?? superfamily and promotes survival and neurite outgrowth of dopaminergic and motor neurons, as well as peripheral sensory and sympathetic neurons. It has been reported that GDNF conferred protection to neurons during various types of injuries to nervous systems in vitro and in vivo. However, continuous application of therapeutic proteins into central nerve system (CNS) is hampered by the short half-life of protein, concomitant injection trauma, and the difficulty to across blood–brain barrier to CNS including spinal cord.
Paraplegia is a disastrous complication of thoracic aortic surgery. In our study, we evaluated the effect of GDNF gene delivery on prevention of the pathological changes due to spinal ischemia. Recombinant adenovirus vectors encoding GDNF (Ad-GDNF) and green fluorescent protein (Ad-GFP) were used for gene transfer studies. Treatment with cobalt chloride induced dose-dependent bcl-2 and synaptophysin downregulation in spinal neuronal cells, which could be effectively reversed by GDNF gene transfer. Intrathecal injection of Ad-GDNF led to maximal GDNF expression in spinal cord within 2–7 days. Thus, after intrathecal administration of adenovirus vectors for 3 days, Sprague–Dawley rats received transient aortic occlusion to induce spinal ischemia and were monitored for behavior deficits. The Ad-GDNF-treated rats showed significantly lower paraplegia rate (40%) than that of Ad-GFP- or saline-treated groups (75–85%; P＜0.01). In addition, the Ad-GDNF-treated rats exhibited significantly improved locomotor function comparing with rats of control groups ( P ＜ 0.001). Histological analysis revealed that GDNF gene delivery profoundly attenuated the infiltration of leukocytes in spinal cord after ischemic insults. Furthermore, GDNF gene delivery prominently attenuated the ischemia-induced neuronal loss in ventral horn lamina VI–VIII and reduction in synaptophysin expression in spinal cords. In conclusion, GDNF gene transfer confers protection to the neuronal cells and synapses networks, thereby alleviated the paraplegia due to spinal ischemia.
CGS 26303, an endothelin-converting enzyme inhibitor, possessed beneficial effects for the treatment of SAH and transient middle cerebral artery occlusion. In this study, we investigated the neuroprotective effect of CGS 26303 on the locomotor function and mRNA expression of heme-oxygenase-1 (HO-1) in rats subjected to a 15-min spinal cord ischemia-reperfusion injury. The results showed that pretreatment with CGS 26303 significantly preserved the locomotor function and decreased the paraplegia rate at Days 1 and 3 as compared with a saline-treated group. Furthermore, rats pretreated with CGS 26303 had a significant increase in the levels of HO-1 mRNA expression at Day 3 when compared with animals pretreated with saline after spinal cord ischemia and the sham operation group. These results suggest that CGS 26303 may have a promising neuroprotective effect in the spinal cord after ischemia-reperfusion injury, and beneficial result may be due to an adaptive mechanism involved by HO-1 overexpression.

並列關鍵字

Lithium ； brain cancer ； GSK3β ； BCL2L12 ； BCL2L12A ； phosphorylation ； staurosporine ； apoptosis ； spinal cord injury ； GDNF ； cobalt chloride ； synaptophysin ； CGS26303 ； endothelin ； ischemia‐reperfusion injury

參考文獻

PART I:

參考文獻

Abraha, A., N. Ghoshal, et al. (2000). "C-terminal inhibition of tau assembly in vitro and in Alzheimer's disease." J Cell Sci 113 Pt 21: 3737-3745.

Chernova, O. and J. K. Cowell (1998). "Molecular definition of chromosome translocations involving 10q24 and 19q13 in human malignant glioma cells." Cancer Genet Cytogenet 105(1): 60-68.

Cohen, G. B., R. Ren, et al. (1995). "Modular binding domains in signal transduction proteins." Cell 80(2): 237-248.