| Abstract: | 根據衛生署統計，癌症多年蟬聯死亡率之首，其中肺癌死亡率增長的速度最為快速，雖然現今科技日新月異，治療技術不斷進步，但對於肺癌的治癒能力仍然相當的低，在癌症病患當中，癌症的轉移仍是癌症致死的主因。肺癌細胞在轉移的過程中，癌細胞會出現上皮-間質型態相互轉換之情形，增加較為侵襲的表型，提高轉移的能力。在近期的研究中，中草藥已有許多的功能被探討發現，舉例來說，如能抗癌、抗轉移、抗增生、抗血管新生等功能。透過本研究可得知，透過50%的乙醇萃取蛇莓(DIE)，以Boyden chamber assay和 would healing assay檢測A549肺腺癌細胞之移動及侵襲能力，利用Western blotting 去分析蛇?乙醇萃取物對A549肺腺癌細胞的轉移影響，當中透過何種蛋白進行訊息傳遞(如MAPK與PI3K/Akt訊息路徑)。將DIE以不同濃度(0, 25, 50, 75, and 100 μg/mL)處理A549，並透過gelatin zymography 和 casein zymography觀察金屬基質蛋白?和尿激?型血纖維蛋白溶解?原活化因子，是否因加藥濃度升高而蛋白表現量受到抑制。並使用轉型生長因子(TGF-β1)對細胞進行型態上的轉化，研究蛇梅萃取物是否能降低癌症間質的特性。結果發現DIE可有效降低A549的移動及侵襲能力，並有效抑制u-PA 以及 MMP-2 的活性，並在對於細胞無毒性的條件下，能有效抑制A549肺腺癌細胞的攤附能力，進一步發現由TGF-β1所誘導的間質轉化，透過處理DIE能回復成較為上皮細胞的特性，使E-cadherin上皮marker的回復，N-cadherin和vimentin等間質蛋白表現量下降。透過DIE的處理也發現磷酸化Akt和磷酸化ERK等蛋白表現量下降。總結來說，當我們乙50%乙醇提取蛇莓萃取物，能夠有效抑制A549人類肺腺癌細胞的移動及侵襲能力，並使具有高轉移特性的間質型態的肺癌細胞，表現E-cadherin上皮細胞的marker，回復成較為上皮的特性，並且降低N-cadherin間質細胞的marker，以及磷酸化的Akt和磷酸化的ERK。最後，本論文將肺癌細胞A549異種移植到裸鼠上，觀察腫瘤是否受到抑制。結果顯示，每天灌食蛇莓萃取物，證實了蛇莓取物的確有抑制肺癌細胞與腫瘤生長的功能。綜合所有結果與討論可以知道中草藥未來足以當作具有潛在的治療方式。
Cancer has ranked the number-one cause of death entire the world. Particular lung cancer death rate is the fastest growing. Although therapeutic advances, the cure rate for lung cancer still low. In all cancer patients, metastasis is often fatal cancer caused by one of the reasons. The epithelial-to-mesenchymal transition (EMT) in lung cancer cells is considered pre-requisite for acquiring invasive/migratory phenotype, and subsequent metastasis. In the recent study, there were some functions Chinese herb medicine have been reported to have various anti-carcinogenesis properties, such as anti-metastasis, anti-proliferation, and anti-angiogenesis.The purpose of this study is to explore the inhibitory effect of Duchesnea indica extract (DIE), extracted by 50% ethanol, on the invasion, motility and migration of A549 cells by Boyden chamber invasion assay, motility assay, and would healing assay. We examined the effect of DIE on factors (such as MAPKs and PI3K/Akt pathways) of cancer metastasis and EMT by Western Blot. We treated A549 cells with various concentrations of DIE (0, 25, 50, 75, and 100 μM), and then subjected cells to gelatin zymography and casein zymography to investigate the expression of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA).We also induced the cell morphology transition by using TGF-β1, and then to observe whether the phenotype will be upside down from mesenchymal to epithelial using a microscope. In this study, we have demonstrated that DIE could inhibit invasive and migratory potential, reduce the u-PA and MMP-2 activities, and have a negative influence on cell spreading of a highly metastatic lung cancer cell line at non-cytotoxic concentration. Further investigation showed that higer migration ability is due to epithelial-mesenchymal transition (EMT) occurred in A549, when we treated DIE detection that EMT phenomenon might be reverse. DIE induced up-regulation of epithelial marker such as E-cadherin and decreased the mesenchymal markers such as vimentin and N-cadherin. DIE also inhibited p-Akt and p-ERK1/2 protein expression.Taken together, our findings showed a strong anti-migratory and anti-invasive efficacy of DIE against human lung cancer A549 cells, which was in-part through promoting E-cadherin expression and decreasing the level of N-cadherin, p-Akt, p-ERK1/2. Furthermore, oral gavage of Duchesnea indica extracted significantly reduced the tumor growth in A549 lung cancer cells subcutaneous xenograft of nude mice. All the results and discussion chinese medicine herbs might be sufficient to investigate the potential of treatment in the future. |
