建立一個利用斑馬魚胚胎篩選影響 EMT 藥物的活體平台

Abstract

癌症領域中尋找治癒癌症的藥物至今仍然是一大挑戰，目前研究指出癌症導致死亡有 90% 是因為癌症轉移，而上皮細胞-間質細胞轉變 (EMT) 是癌細胞得到侵襲能力的關鍵。所以，建立一個新穎的活體平台以快速尋找可能抑制癌細胞轉移的藥物及機轉是必要的。本實驗欲利用斑馬魚胚胎做為快速篩選抑制癌細胞轉移藥物的活體平台。首先，先確認斑馬魚的心臟瓣膜形成的 invagination 與癌細胞 EMT 的訊息傳遞路徑是否相同。為釐清此問題，本實驗利用已知會抑制斑馬魚心內皮細胞進行 invagination 的藥物 Amiodarone，探討其可否以相同訊息傳遞路徑抑制癌細胞的 EMT。我們利用傷口癒合法與 Transwell 侵襲實驗觀察，發現 Amiodarone 會造成癌細胞 (B16/OVA、JC、4T-1) 移動與侵襲能力的下降。接著，利用西方浸漬法分析，結果顯示 Amiodarone 會增加癌細胞中 Versican V2 蛋白質的表現量。進一步地發現 Amiodarone 也會降低癌細胞中 Snail 與 Twist 蛋白質表現，而使得 E-cadherin 蛋白質表現量上升但 N-cadherin 蛋白質表現量下降。當利用 siRNA 降低 Versican V2 蛋白表現量後，小鼠癌細胞侵襲能力會提升，且 Amiodarone 抑制 Snail 表現的能力會下降。而本實驗同時也發現癌細胞處理 Amiodarone 後，癌細胞中 epidermal growth factor receptor (EGFR) 磷酸化程度會下降，且AKT、ERK 以及 GSK3β 磷酸化程度都下降。當利用 epithelial growth factor 或 BIO 干擾 Amiodarone 對 EGFR 或 GSK3β 的影響時，Amiodarone 促使 E-cadherin 表現的能力皆受阻。但當利用 myr-AKT 與 MEK1 質體轉染，干擾 Amiodarone 抑制 AKT 與 ERK 磷酸化程度，卻發現只有轉染 MEK1 會降低 Amiodarone 抑制 Snail 表現的能力。因此，證明了 Amiodarone 會增加 Versican V2表現量，並抑制癌細胞株中 EGFR 訊息傳遞路徑，導致 ERK 訊息傳遞路徑受阻，進而增加 GSK3β 活性，使得 Snail 無法抑制 E-cadherin 的表現量，達到抑制癌細胞移動與侵襲的結果。這表示 Amiodarone 可抑制癌細胞進行 EMT，且其調控機制與抑制斑馬魚胚胎瓣膜形成相同。為了證明此現象並非只適合 Amiodarone，本實驗利用另一個會導致斑馬魚心內皮細胞 versican 異位性過量表現的藥物 4-Amiopyridine，發現其也可抑制 B16/OVA 的移動能力，而其他不影響斑馬魚心內皮細胞 versican 表現的藥物，則不影響 B16/OVA 的移動。因此，我們總結本實驗成功建立了以斑馬魚心臟專一發螢光的基因轉殖品系 Tg(cmlc2：HcRFP) 胚胎，可作為快速篩選會影響 EMT 藥物的活體新式動物平台。

To find a drug that cures cancer is still a challenge. It has been reported that approximately 90% reasons caused death by cancer are metastasis, and the key step of cancer cells that acquires invasion ability is epithelial-mesenchymal transition (EMT). Therefore, it is necessary and helpful if we could establish a novel in vivo platform for rapid screening drugs and understanding the mechanism of inhibiting cancer metastasis. To accomplish this goal, in this study, we employed zebrafish embryo as an alternative study material. First, we studied whether the pathway involved in the invagination of cardiac valves formation during zebrafish heart development is similar to that of EMT involved in cancer cells. We used Amiodarone, a drug has been reported to inhibit invagination of endocardial cells during zebrafish cardiogenesis, to investigate its effect on cancer cells. Using wound healing assay and matrigel invasion assay, we found that Amiodarone did inhibit migration and invasion of cancer cell lines B16/OVA, JC and 4T-1. Western blot demonstrated that Amiodarone induced Versican V2 and E-cadherin proteins’ level but reduced Snail, Twist and N-cadherin proteins’ level. Knockdown of Versican V2 by specific siRNA induced cancer cell invasion and lost the inhibition Snail expression mediated by Amiodarone. Signaling pathway analyses demonstrated that Amiodarone treatment reduced the phosphorylation levels of EGFR, AKT, ERK and GSK3β in cell lines B16/OVA, JC and 4T-1. However, Amiodarone failed to induce E-cadherin in cells treated with either epithelial growth factor or GSK3β inhibitor, BIO. Moreover, Amiodarone failed to repress Snail in the MEK1- transfected cells but not in the myr-AKT- transfected cells. The line of evidences suggested that Amiodarone increases Versican V2, blocks EGFR and ERK pathways, and elevates GSK3β activity by decreasing its phosphorylation, then causes E-cadherin overexpression through inhibiting Snail, which in turn, inhibits the migration and invasion of cell lines B16/OVA, JC and 4T-1. Taken together, we demonstrated that Amiodarone inhibits EMT of cancer cells through the signaling pathways which are similar to those of inhibiting the cardiac valve formation of zebrafish embryos. Additionally, to prove this phenomenon is not particularly specific to Amiodarone, we employed another drug, 4-Amiopyridine, which is known to induce versican overexpression in zebrafish endocardial cells, and found that 4-Amiopyridine could inhibit B16/OVA migration. However, Lidocaine and Nifedipine, which could not affect versican expreesion, could not influence B16/OVA migration. Therefore, we clearly demonstrated that using zebrafish embryo derived from transgenic line Tg(cmlc2：HcRFP) is a highly potential in vivo platform for rapid screening putative drugs that could affect EMT.