細胞移行與能量需求在神經膠細胞瘤的探討

Studies of Tumor Cell Motility and Energy Demand in Glioma

葉威蘭(Wei-Lan Yeh)

指導教授 ： 符文美

國立臺灣大學/醫學院/藥理學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.00235

摘要

神經膠細胞瘤(glioma)是中樞神經系統當中最常見也是最惡性的腫瘤。它最大的病理特色就是極容易從原生處侵犯到周圍的腦部組織，導致不論是手術後或是放射線治療後的預後不良。因此，研究神經膠細胞瘤的能量需求以及細胞轉移能力，或許能提供我們對於治療神經膠細胞瘤更多的了解。
腫瘤細胞相對於一般細胞而有較高的能量需求，包括攝入較多的葡萄糖、較旺盛的糖解作用、及較多的三磷酸腺苷產生，尤其是當腫瘤細胞缺乏足夠的氧氣供應時更為明顯。因為葡萄糖是由血液中轉運到腫瘤細胞內，所以葡萄醣分子勢必會經由血管內皮細胞及腫瘤細胞表面上的葡萄糖轉運蛋白來運送。在本論文中我們發現，當神經膠細胞瘤遭受缺氧刺激後會分泌一些調節因子，影響腦部血管內皮細胞上葡萄糖轉運蛋白的表現。從缺氧的成鼠神經膠細胞瘤細胞株(rat C6 glioma)所產生之條件性培養液(conditioned medium)會調增第一型葡萄糖轉運蛋白(glucose transporter type I)在成鼠腦血管內皮細胞株(adult rat brain endothelial cells)的表現；反之，在正常氧分壓下的成鼠神經膠細胞瘤細胞株所產生的條件性培養液則不會造成顯著影響。我們進一步發現，造成這樣顯著改變的原因來自於成鼠神經膠細胞瘤細胞株在缺氧狀況下所大量分泌的血管內皮生長因子(vascular endothelial growth factor)。當轉殖血管內皮生長因子的小干擾核醣核酸到大鼠神經膠細胞瘤細胞株中抑制其表現時，被轉殖細胞在缺氧刺激下產生的條件性培養液則無法如之前調增第一型葡萄糖轉運蛋白在成鼠腦血管內皮細胞株上的表現；再者，加入血管內皮生長因子的中和性抗體到缺氧下產生的條件性培養液中，也表現了類似的抑制作用。此外，在成鼠腦血管內皮細胞上經由血管內皮生長因子刺激所表現的第一型葡萄糖轉運蛋白，是經由磷脂酰肌醇-3激酶路徑所調控(PI3K/Akt)。這些結果顯示，受到缺氧刺激的神經膠細胞瘤會分泌血管內皮生長因子，進而促進葡萄糖轉運穿透過血腦屏障(blood-brain barrier)而由血流進入腫瘤細胞。
瘦素(Leptin)是一種肥胖基因的產物，它藉由調控代謝、進食、神經內分泌的反應來控制體重的維持。然而，中樞神經系統對於瘦素的基因表現和在週邊的脂肪細胞內不同。此外，瘦素被發現對於許多腫瘤細胞株有促進分裂和血管新生的作用。在此我們發現惡性的神經膠細胞瘤細胞株相較於其他非惡性的星狀神經膠細胞(astrocytes)會表現較多的瘦素及瘦素接受器，並發現當外給瘦素之後可刺激神經膠細胞瘤移行(migration)和侵犯週邊(invasion)的能力。此外我們發現在瘦素的刺激之下會增加第十三型基質金屬蛋白酶 (matrix metalloproteinase-13)在神經膠細胞瘤的表現，但第二型和第九型的表現則不受影響，且因瘦素而增加的移行和侵犯能力也會因為加入第十三型基質金屬蛋白酶的中和性抗體或小干擾核醣核酸而被抑制。此外，第十三型基質金屬蛋白酶的表現增加主要是經由瘦素的刺激後引起絲裂素活化蛋白激酶(p38 MAP kinase)和核轉錄因子NF-kappa B路徑的活化所造成。我們更進一步篩出較易移行的神經膠瘤細胞，並發現當細胞具有較強的移行能力時會表現較多的瘦素以及第十三型基質金屬蛋白酶。綜合以上的結果，我們發現瘦素會促進神經膠細胞瘤的移行和侵犯週邊的能力，而這樣的促進作用是來自於第十三型基質金屬蛋白酶的表現增加。
在腫瘤的微環境中，與發炎相關的調節因子或細胞受體扮演了相當重要的角色。在某些狀況下，致癌相關因子會引發出發炎的微環境且進而促進腫瘤的發展與惡化。在神經膠細胞瘤當中有微膠細胞(microglia)的存在，也表示了有與腫瘤相關的發炎現象、及微膠細胞的活化，且這些發炎反應會進而影響腫瘤生長與轉移的能力。在本論文中我們也發現，由神經膠細胞瘤所產生的胞外基質(extracellular matrix)可活化微膠細胞，並促使其分泌第十八型介白素(interleukin-18)；這是一種屬於第一型介白素家族的細胞發炎激素(inflammatory cytokine)，並可以刺激神經膠細胞瘤的移行能力。此外，藉由塗抹純化的胞外基質組成於培養盤表面而發現，微膠細胞在接觸到纖維粘連蛋白(fibronectin)與玻連蛋白(vitronectin)後會被活化而分泌出第十八型介白素；而第十八型介白素所引起的神經膠細胞瘤移行能力增加及細胞微絲重組(microfilament disassembly)，皆會因為加入誘發型一氧化氮合成酶抑制劑(iNOS inhibitor)、鳥苷酸環化酶抑制劑(guanylate cyclase inhibitor)、G型蛋白質激酶抑制劑(protein kinase G)而被抑制。這些結果顯示，神經膠細胞瘤所產生的胞外基質會活化微膠細胞並促進其分泌第十八型介白素，且經由一氧化氮/環磷酸鳥苷(NO/cGMP)的路徑進而刺激神經膠細胞瘤的移行能力。
黏著分子(integrin)是另一個影響細胞移行和侵犯週邊能力的重要因子，它不只可以把細胞物理性的固定在基質上，並且還可以發送和接收調控這個機制的分子訊息；其中黏著分子alpha v beta 3會表現在血管內皮細胞和神經膠細胞瘤的細胞表面上。利用黏著分子的拮抗劑被認為可以經由數種機制去抑制腫瘤的進程。在本論文中我們發現，黏著分子拮抗劑專一性的阻斷黏著因子alpha v beta 3時，可具有抗血管新生的能力、及抑制神經膠細胞瘤細胞群落形成的能力。此外，因為瘦素被發現也會增加黏著分子alpha v和beta 3在神經膠細胞瘤上的表現，因此該黏著分子拮抗劑也會抑制瘦素所引發的細胞移行。
腦血管內皮細胞組成的血腦屏障藉由細胞間的緊密連接蛋白(tight junction protein)的隔離來維持腦部血管的低滲透性及維持腦內組成的平衡。缺血再灌流(Ischemia/Reperfusion)被認為會傷害血腦屏障並造成滲透性的改變，在此我們利用成鼠腦血管內皮細胞株探討YC-1的保護角色，防止化學性缺氧及缺氧再給氧(Hypoxia/Reoxygenation)所引發的血腦屏障滲透性改變。實驗發現YC-1會顯著的減少氯化鈷(CoCl2)和缺氧再給氧所造成的葡萄聚醣(fluorescein isothiocyanate-dextran)滲透度增加。此外，構成血腦屏障的緊密連接蛋白之一ZO-1(zonular occludens-1)會因為氯化鈷和缺氧再給氧的刺激而被破壞，且此現象會因為加入YC-1而被拮抗。進一步的實驗發現，YC-1的保護作用可能來自於抑制第一型缺氧誘導因子(hypoxia-inducible factor-1)在細胞內的累積，並進而減少其下游的血管內皮生長因子的蛋白表現。血管內皮生長因子單獨作用即可顯著增加葡萄聚醣的滲透度，並且調降成鼠腦血管內皮細胞株上ZO-1的表現。我們進一步用動物實驗來評估，發現在缺血再灌流之後YC-1可有效的防止缺血再灌流對於血腦屏障所造成的傷害。總體而言，YC-1可能藉由抑制第一型缺氧誘導因子在細胞內的累積，進一步減少血管內皮生長因子的表現，並進而保護血腦屏障不受到缺氧所造成的傷害。
神經膠細胞瘤於成人造成很高的死亡率，原因在於它很容易在不知不覺間轉移侵犯到周圍組織、腫瘤邊界不明顯，造成治療效果不彰。本論文從腫瘤細胞能量代謝及發炎的角度來探討並了解其影響癌細胞轉移的能力；此外，更初步評估一個新的發展中藥物黏著分子拮抗劑在神經膠細胞瘤方面的應用，以期能開發藥物治療神經膠細胞瘤的方針。

關鍵字

神經膠細胞瘤 ； 細胞移行 ； 能量需求

並列摘要

Glioblastoma multiform (GBM) is the most frequent and malignant tumor of the central nervous system (CNS). The pathological characteristic of their insidious infiltration of the brain leads to the poor prognosis after surgery and/or radiation therapy. Therefore, elucidation of the energy demand and motility of glioma provides more information to develop potential therapeutic targets.
Increased need for glycolysis and glucose uptake for ATP production is observed in tumor cells, particularly in cells lacking of oxygen supply. Because glucose is transported from blood to tumor, glucose molecules must be delivered across glucose transporters of the vascular endothelium and tumor cells. Here we found that glioma suffered from hypoxic insults can secrete factor(s) to regulate glucose transporter expression in brain endothelium. It was found that conditioned medium from rat C6 glioma cells under hypoxia up-regulated glucose transporter type 1 (GLUT1) expression in rat brain endothelial cells, whereas conditioned medium from C6 cells under normoxia caused no significant effect. We further investigated whether the observed potentiating effect was caused by vascular endothelial growth factor (VEGF) production from C6 cells, because secreted VEGF was markedly increased under hypoxic condition. By transfection of C6 cells with VEGF small interfering RNA, it was found that conditioned medium from transfected cells under hypoxia no longer up-regulated GLUT1 expression of endothelial cells. Moreover, the addition of VEGF-neutralizing antibody to the hypoxic conditioned medium could also exert similar inhibitory effects. Furthermore, it was found that the VEGF-induced increase of GLUT1 expression in endothelial cells was mediated by the phosphoinositide-3 kinase/Akt pathway. Our results indicate that hypoxic brain glioma may secrete VEGF to increase glucose transport across blood-brain barrier (BBB).
Leptin, the product of the obese gene, plays an important role in the regulation of body weight by coordinating metabolism, feeding behavior, energy balance, and neuroendocrine responses. However, regulation of leptin gene expression in the CNS is different from that in the adipocytes. In addition, leptin has been found in many tumor cell lines and has been shown to have mitogenic and angiogenic activity in a number of cell types. Here we found that malignant C6 glioma cells expressed more leptin and leptin receptors than nonmalignant astrocytes. Furthermore, it was found that exogenous application of leptin enhanced the migration and invasion of C6 glioma cells. In addition, we found that the expression of matrix metalloproteinase-13 (MMP-13) but not of MMP-2 and MMP-9 was increased in response to leptin stimulation. The leptin-induced increase of cell migration and invasion was antagonized by MMP-13 neutralizing antibody or silencing MMP-13. The up-regulation of MMP-13 induced by leptin was mainly through p38 MAP kinase and NF-kappa B pathway. In addition, migration-prone sublines demonstrate that cells with increasing migration ability had more expression of MMP-13 and leptin. Taken together, these results indicate that leptin enhanced migration and invasion of C6 glioma cells through the increase of MMP-13 production.
The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor-related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here we found that C6 glioma- but not primary astrocyte-derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV-2 cell line, and activated microglia secreted interleukin (IL)-18, a potent inflammatory cytokine of the IL-1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL-18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL-18-induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase (GC) inhibitor and protein kinase G (PKG) inhibitor. Taken together, these results indicate that IL-18 secreted by microglia, which was activated by C6 glioma-derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway.
Integrins play another crucial role for cell invasion and migration, not only for physically tethering cells to the matrix, but also for sending and receiving molecular signals that regulate these processes. Pharmacological blockade of integrins using integrin antagonists are considered to inhibit tumor progression by several mechanisms. Here we found an alpha v beta 3-selective integrin antagonist exhibited an anti-angiogenic effect in vivo and inhibits colony formation of C6 glioma cells. Moreover, since leptin also up-regulated integrin alpha v and beta 3 expression in C6 glioma, this integrin antagonist also antagonized leptin-induced C6 migration.
Cerebral microvascular endothelial cells form the anatomical basis of the BBB, and the tight junctions of the BBB are critical for maintaining brain homeostasis and low permeability. Ischemia/reperfusion is known to damage the tight junctions of BBB and lead to permeability changes. Here we investigated the protective role of 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole (YC-1), against chemical hypoxia and hypoxia/reoxygenation (H/R)-induced BBB hyperpermeability using adult rat brain endothelial cell culture (ARBEC). YC-1 significantly decreased CoCl2- and H/R-induced hyperpermeability of fluorescein isothiocyanate (FITC)-dextran in cell culture inserts. It was found that the decrease and disorganization of tight junction protein zonular occludens-1 (ZO-1) in response to CoCl2, and H/R was antagonized by YC-1. The protection of YC-1 may result from the inhibition of HIF-1 alpha accumulation and production of its downstream target VEGF. VEGF alone significantly increased FITC-dextran permeability and down-regulated mRNA and protein levels of ZO-1 in ARBECs. We further used animal model to examine the effect of YC-1 on BBB permeability after cerebral ischemia/reperfusion. It was found that YC-1 significantly protected the BBB against ischemia/reperfusion-induced injury. Taken together, these results indicate that YC-1 may inhibit HIF-1 alpha accumulation and VEGF production, which in turn protect BBB from injury caused by hypoxia.
Glioblastoma multiforme is the most common malignant glioma in adults. GBM constitutes a major problem in clinical management due to its high infiltrative ability, resistance to the current cancer therapy that account for its bad prognosis and high mortality. In our studies, we investigated tumor cell motility from the aspects of energy demand and tumor-related inflammation. Moreover, we also evaluated the anti-angiogenic and anti-tumor effects of a developing integrin antagonist for becoming a good candidate to treat glioblastoma.

並列關鍵字

glioma ； migration ； energy

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