碳酸酐異構酶第三型在口腔癌侵襲與轉移所扮演的角色及其機制之探討

碳酸酐異構酶 (carbonic anhydrase, CA) 是一種存在於細胞質中的催化酵素，主要的功能是參與催化可逆性水解反應。近年有多篇文獻證實細胞溶質中 CAs 的 CA III 參與蛋白質的硫化作用同時提供細胞抗氧化的角色，而這些抗氧化的能力可能對腫瘤轉移與侵襲相關。上皮細胞間質化 (epithelial-mesenchymal transition, EMT) 是一種上皮細胞轉化成間皮細胞的過程，是癌症轉移的主要機制之一。然而對 CA III 在口腔癌的發展與轉移的過程中許多機制仍不清楚，本研究為探討 CA III 對口腔癌轉移與 EMT 之間的關係並其詳細機制為何。在臨床部份，我們收集了 248 位來自中部地區的口腔癌患者，採集其癌組織，分析其中 CA III表現量與癌症分期、復發情形以及死亡率之間之相關。從結果中發現在 CA III 表現量較低的患者顯著的具有其較高的復發率以及較高的死亡率；在口腔癌細胞株方面，在比較過各細胞內 CA III 的表現量後我們選擇 SAS與 SCC-9這兩株低表現內源性 CA III 之細胞株為目標細胞株，並嘗試建立大量表現 CA III 之 SAS 及 SCC-9 並利用此細胞株做後續的研究探討。首先透過 MTT assay發現在大量表現 CA III 的環境下對細胞生長率存活率沒有顯著的影響。再利用細胞侵襲與轉移分析實驗發現大量表現　CA III　對細胞移動、侵襲、貼附和轉移能力的影響都有明顯促進的情形。同時用西方墨點法技術偵測細胞內蛋白在大量表現　CA III　的環境下觀察到與　EMT　相關的 E-cadherin 蛋白表現下降， vimentin 以及 fibronectin 蛋白表現量上升以及轉錄因子 TWIST, SNAI1 及 SNAI2 表現量增加。更進一步分析其中訊息傳遞路徑可能是透過 FAK 與 Src 訊息傳遞路徑做調控。透過以上實驗推論由 CA III 所造成刺激細胞侵襲與轉移可能是透過影響 FAK 與 Src 訊息傳遞路徑影響轉錄因子 TWIST, SNAI1 與 SNAI2 的表現進而影響 E-cadherin 的表現所引起。然而對於其中詳細的機制仍需要透過更多實驗結果及文獻探討證實。

關鍵字

碳酸酐異構酶口腔癌；癌症轉移；上皮細胞間質化

並列摘要

Carbonic anhydrase III (CA III) is a cytoplasmic enzyme that exhibits carbon dioxide hydratase activity and promotes transformation and invasion in cancer metastasis. During the epithelial–mesenchymal transition (EMT), epithelial cells become mesenchymal cells and metastasis is initiated for cancer progression. However, the functions and mechanisms of CA III in oral cancer are mostly unknown. We collected 248 oral cancer patients from the central area of Taiwan and analyzed the relationship between the CA III expression level and the clinical stage, recurrence, and survival rate. Using a CA III overexpression system, the effects on cell proliferation were determined by the MTT assay and on cell migration and invasion by the Boyden chamber assay. To clarify the signaling pathways, we investigated the expression of CA III, EMT-related proteins, and signaling proteins using western blotting and real-time polymerase chain reaction. Based on our results, we suggested that the patients with lower CA III expression had higher recurrence and lower survival rate. The mobility and invasive ability of the CA III-overexpressing oral cancer cells, SAS and SCC-9, were higher than those of the control cell lines. Western blotting identified EMT-related proteins such as E-cadherin, fibronectin, SNAI1, SNAI2 and TWIST as candidate downstream targets of CA III. Moreover, CA III-mediated cell migration and EMT-related protein expression were modulated through the phosphorylation of the signaling molecules FAK and Src. In conclusion, we revealed that CA III may induce cell migration and invasion through EMT induction in the SAS and SCC-9 cells. The activation of FAK and Src signaling may be involved in the CA III-mediated EMT in these oral cancer cells.

並列關鍵字

Carbonic anhydrase III ； oral cancer ； metastasis ； epithelial–mesenchymal transition.

參考文獻

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碳酸酐異構酶第三型在口腔癌侵襲與轉移所扮演的角色及其機制之探討

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