Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Osthole (OST), a dominant active constituent of Chinese herb Cnidium monnieri, exhibits anti-cancer, anti-inflammatory, anti-osteoporotic, anti-bacterial, and anti-allergic effects. It has been reported that OST could inhibit cancer metastasis in vitro and in vivo while the mechanisms were still unclear. Here, we reported the anti-metastatic effect of OST in TGF-β1-induced EMT model in A549 cells and its potential mechanism. The cytotoxic effect of OST was determined by MTT assay. A549 cells were treated with TGF-β1 with or without OST co-treatment, the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by both Western blotting and qRT-PCR. The expression of NF-κB p65, IκBα, p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. Results revealed that low concentration of OST (<40 µM) showed no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration of cells. The upregulation of N-cadherin, Vimentin, NF-κB and the downregulation of E-cadherin after TGF-β1 treatment were detected. The fluorescence changes of E-cadherin and N-cadherin in the plasma membrane were observed by immunofluorescence. The adhesion, migration, and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 increased Snail mRNA expression and induced protein expression of Snail in a time-dependent manner. Furthermore, Western blotting and immunofluorescence showed that TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations induced by TGF-β1were dramatically inhibited by OST co-treatment. In addition, both the NF-κB inhibitor PDTC and the Snail siRNA could inhibit TGF-β1- induced alterations in A549 cells. In conclusion, these results showed that OST inhibited TGF-β1-induced EMT in A549 cells possibly mediated by the inactivation of the NF-κB-Snail signaling pathway. This study provides novel molecular mechanism of OST in the anti-metastatic area.

上皮細胞間充質轉化(Epithelial-mesenchymal transition, EMT)在腫瘤的侵襲和轉 移過程中發揮重要作用，為的腫瘤治療提供了新的思路和策略。蛇床子素 （Osthole, OST）是中藥蛇床子的主要活性成分，具有抗腫瘤、抗炎、抗骨質 疏鬆、抗菌及抗過敏等生物活性。有研究報道，OST 在體內外均有抑制腫瘤轉 移作用，然其具體機制尚不明確。本課題以轉化生長因子（Transforming Growth Factor-β1，TGF-β1)誘導的肺癌 A549 細胞 EMT 為模型，研究了 OST 對 EMT 的抑制效應及其可能機制。體外培養肺癌 A549 細胞，MTT 法檢測 OST 對細胞的毒性作用。TGF-β1 單獨處理或與 OST 共同處理細胞，顯微鏡觀察細 胞的形態學變化。用 Western blotting 及免疫熒光技術分別檢測 E-cadherin，Ncadherin 及 NF-κB p65 的蛋白表達及熒光定位變化。Matrigel 膠粘附實驗、劃痕 實驗及 Transwell 實驗分別檢測了 OST 對 TGF-β1 誘導的 A549 細胞的粘附、遷 移及侵襲能力的變化。用 Western blotting 和實時定量聚合酶鏈反應（qRT-PCR） 測定了 Snail 蛋白及 mRNA 的表達。提取胞漿和核蛋白，Western blotting 分別 檢測 NF-κB p65，IκBα，p-IκBα 在細胞核及胞漿內的表達。實驗結果顯示，濃 度低於 40 µM 的 OST 對 A549 細胞無明顯毒性。TGF-β1 處理后，細胞形態由橢 圓形向梭狀改變。細胞內 N-cadherin, Vimentin，NF-κB 蛋白表達明顯升高，Ecadherin 蛋白表達顯著降低。細胞內 N-cadherin 熒光亮度隨著 TGF-β1 刺激后增 強，而 E-cadherin 熒光亮度減弱。TGF-β1 處理也顯著增強細胞的粘附、遷移及 侵襲能力。同時，TGF-β1 誘導 Snail mRNA 表達增高且時間依賴性增加 Snail 蛋 白表達。Western blotting 和免疫熒光結果顯示，TGF-β1 處理后胞漿內 NF-κB p65 表達明顯下降，而核內明顯增多。加入 OST 和 TGF-β1 共同處理細胞后， OST 可以顯著逆轉 TGF-β1 誘導的以上所有變化。另外，NF-κB 的抑制劑 PDTC 和 Snail siRNA 也可顯著抑制 TGF-β1 誘導的 EMT 相關變化。綜上所述，本研 究顯示，OST 顯著抑制 TGF-β1 誘導 A549 細胞 EMT 作用，其機制可能是通過 抑制 TGF-β1 誘導的 NF-κB-Snail 信號通路激活。本研究為 OST 抑制腫瘤轉移 提供了新的分子機制。