酯多醣促進TPA誘導神經膠質瘤細胞C6轉型之機轉探討

Lipopolysaccharide Promotion of TPA-Induced Transformation in Rat Glioma Cells

簡蘭孋(Lan-Li Chen)

指導教授 ： 陳彥州

臺北醫學大學/藥學院/生藥學研究所/碩士(2004年)

摘要

癌細胞轉移 (Migration)及侵犯其他組織 (Invasion)常見於神經膠質腫瘤形成的過程，而且基質金屬蛋白酶 (Metalloproteinases; MMPs) 尤其是基質金屬蛋白酶第二型 (MMP-2)和第九型 (MMP-9)的表現在癌細胞轉移過程中扮演著很重要的角色。發炎反應為一常見之生理與病理反應，一氧化氮 (NO)為發炎反應之重要產物之一，目前關於一氧化氮在神經膠質腫瘤細胞轉移之角色尚未明白。我們的實驗結果發現，神經膠質腫瘤細胞glioma C6單獨以LPS處理時能少量且顯著誘導iNOS蛋白質的表現但在細胞形態上並沒有改變；在單獨以TPA (400 ng/ml)處理的時候可以發現細胞有輕微聚集的情形，卻沒有誘導iNOS蛋白質與MMP-9活性的表現，在以LPS和TPA (LPS/TPA)共同處理下，細胞形態上有顯著之聚集且iNOS蛋白質與MMP-9活性大量的被誘導，同時又伴隨著一氧化氮大量產生。機轉方面，我們證實LPS/TPA處理能大量誘導ERK1/2和JNK （沒有p38）的磷酸化，ERK1/2抑制劑PD98059與JNK抑制劑SP600125（而非p38抑制劑SB203580）能有效分別抑制LPS/TPA所誘導之ERK1/2和JNK的磷酸化，同時降低iNOS蛋白質與MMP-9活性表現。同時LPS/TPA所誘導之細胞轉型也被PD98059與SP600125（沒有p38抑制劑SB203580）所抑制。這些結果證實LPS/TPA誘導細胞轉型是經由活化ERK/JNK路徑。進一步我們證實類黃酮化合物中的kaempferol和wogonin能有效的抑制LPS/TPA所誘導的細胞轉型、iNOS蛋白質表現與NO產生，同時降低MMP-9的活性。Soft agar實驗成果證實LPS/TPA能誘導細胞群落之形成，kaempferol和wogonin能有效抑制LPS/TPA能誘導細胞群落之形成。在體內裸鼠實驗發現LPS/TPA對glioma C6腫瘤細胞成長與對照組、LPS或TPA處理組沒有顯著之差異，但是LPS/TPA有誘導癌細胞轉移之發生 (4/10)，以kaempferol和wogonin處理能有效抑制LPS/TPA誘導glioma C6細胞轉移之發生 ( Kaempferol, 0/8; Wogonin, 0/8 )。本實驗成果提供科學證據證實LPS能促進TPA所誘導之神經膠質瘤細胞癌化與轉型，其過程與活化ERK/JNK、iNOS 蛋白質表現與 MMP-9活性有密切關係。

關鍵字

細胞轉型 ； 腦瘤細胞株C6 ； 轉移 ； 基質金屬蛋白酶

並列摘要

In the present study, we found that lipopolysaccharide (LPS) promoted the transformation in glioma C6 cells in the presence of TPA according with inducing iNOS protein expression, nitric oxide production, and MMP-9 enzyme activity. Activation of extracellular signal-regulated kinases (ERK1/2) and c-Jun-N-terminal kinase (JNKs) but not p38 kinase was identified in LPS/TPA-treated glioma C6 cells. ERKs inhibitor PD98059 and JNKs inhibitor SP600125 (but not p38 inhibitor SB203580) significantly blocked LPS/TPA induced transformation with reducing iNOS protein expression, NO production and MMP-9 enzyme activation. In addition, kaempferol and wogonin show significant suppression on LPS/TPA induced transformation with blocking iNOS protein expression, NO production and MMP-9 enzyme activity. Results of soft agar assay suggested that LPS/TPA but not LPS or TPA alone induce colony formation in glioma C6 cells, and kaempferol and wogonin exhibit preventive effect on LPS/TPA-induced colony formation. In vivo study elucidated that LPS/TPA cotreatment induce tumoral metastasis in s.c. injected glioma C6 cells (4/10), and treatment of kaempferol or wogonin inhibited the metastatic ability of glioma C6 cells induced by LPS/TPA in nude mice. These data provide scientific evidences to indicate that LPS possesses ability to enhance the transformation with TPA; activation of iNOS protein expression and NO production in the presence of elevating MMP-9 enzyme activity, located at down stream of ERK/JNK activation, was firstly identified.

並列關鍵字

transformation ； glioma C6 ； metastasis ； MMPs

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