過敏原Der p 2促進肺癌細胞轉移之機制探討

Investigation of aeroallergen Der p 2 promoted motility of lung cancer cell.

林君香(Chun-Hsiang Lin)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化微生物免疫研究所/博士(2016年)

摘要

利用大腸桿菌表現並以Glutathione Sepharose 4B純化出重組塵螨過敏原蛋白Der p 2(DP2)，再以DP2刺激人類肺癌細胞株A549，來探討 (1)塵蟎主要過敏原DP2是否可以直接引起肺癌細胞株的上皮-間質轉化現象，以及(2)塵蟎主要過敏原DP2是否可以誘發肺癌細胞株轉移的能力，並進一步了解其中的分子作用機轉。在第一部分的研究，利用transwell與wound healing assay 分析發現DP2會促進呼吸道上皮細胞的移動能力。進一步的研究發現DP2會改變細胞黏附蛋白的表現，包括降低E-cadherin和ZO-1但增加vimentin的表現。此外，也發現DP2可促進人類肺癌細胞A549參與上皮間質轉換(Epithelial-to-mesenchymal transition,EMT)的重要轉錄因子Snail與Slug的表現。這些研究結果顯示DP2可以調控細胞黏附分子以及EMT相關轉錄因子的表現，進一步增強肺癌細胞的EMT。在第二部分的研究，我們想更進一步了解DP2對提升肺癌細胞移動性的分子機制。研究結果顯示DP2會促使A549肺癌細胞的Integrin表現增加，並造成其下游FAK/Src訊息路徑的活化，進而提高細胞移動相關的蛋白表現。接下來的研究發現DP2所誘發的Integrin/FAK訊息，是透過類鐸受體(Toll-like receptor, TLR)活化其下游Extracellular signal-regulated kinae (ERK)路徑，進而提高Urokinase-type plasminogen activator (uPA)的表現以及活化其受體uPAR。透過活化的uPAR與integrin結合後進而誘發Integrin/FAK訊息路徑。
綜合以上第一與第二部分的研究結果，證實塵螨過敏原蛋白DP2不僅具有呼吸道調控上皮細胞以及肺癌細胞的細胞黏附分子表現之能力與增強EMT，更可以透過活化類鐸受體TLR2與提高uPA的表現，進而誘發uPAR/integrin以及其下游的FAK/Src等訊息路徑，來增加肺癌細胞的移動能力。因此，我們推測塵螨過敏原蛋白DP2可能會提高肺癌細胞的侵襲與轉移能力。

關鍵字

過敏原 ； 肺癌 ； 轉移

並列摘要

In the present study, we isolated and purified recombinant Der p 2 with GST fusion protein by using E. coli expression system and glutathione Sepharose 4B affinity chromatography. The purified recombinant Der p 2 (DP2) was then used to investigate whether aeroallergen DP2 could (1) trigger epithelial-to-mesenchymal transition (EMT) in human lung carcinoma cell A549, and (2) promoted cell motility of human lung carcinoma cells. For the part (1), our results showed that DP2 promoted migration of airway epithelial cell by using wound healing and transmigration assay. We also found that DP2 was able to regulate expression of cell adhesion molecules (CAMs) including decrease of E-cadherin and ZO-1 and increase of vimentin in A549 cell. In addition, expression of important transcription factors involved in EMT such as Snail and Slug was increased in response to DP2 stimuli. These findings indicate that DP2 can promote EMT in lung cancer cell A549. For the part (2), we aimed to investigate whether DP2 promote motility of lung carcinoma cells and the underlying mechanisms. Our results showed that cell growth and cell cycle distribution of human lung carcinoma cell A549, H1299 and H460 were insignificantly affected by recombinant Der p 2 (DP2). In addition, migration and invasiveness of the three lung carcinoma cells were both enhanced in response to DP2 stimuli. Further investigation revealed that integrin aV level was increased and the downstream signaling effectors including focal adhesion kinase (FAK) and paxillin were activated in A549 exposed to DP2. In parallel, DP2 also induced activation of FAK-associated signaling effectors such as Src, phosphotidyl inositol 3-kinase (PI3K), AKT, p38 mitogen activated protein kinase (P38), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Further investigation showed that DP2 increased level of urokinase type plasminogen-activated kinase (uPA) and uPA receptor (uPAR) and the subsequent interaction of uPAR and integrin aV, attributing to activation of toll-like receptor 2 (TLR2) and ERK1/2. Collectively, our results revealed that DP2 stimuli induced TLR2 activation, elevated uPA and uPAR expression, enhanced interaction of uPAR and integrin aV, and triggered FAK signaling cascades, contributing to the promoted cell motility of lung carcinoma cells. These findings indicate that aeroallergen DP2 can promote migration and invasiveness of lung carcinoma cell via uPAR/integrin aV/FAK signaling, suggesting that DP2 may have potential for exacerbating lung carcinoma metastasis.

並列關鍵字

aeroallergen ； lung cancer cell ； metastasis

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