- 學位論文
探討肉桂萃取物抑制人類肺腺癌A549和H1299細胞上皮間質轉換之影響
Effect of Cinnamomum cassia extracts on inhibition of Epithelial to Mesenchymal Transition in human lung adenocarcinoma A549 and H1299 cells
摘要
肺癌是國人癌症前五大的死亡原因之一，過去有許多的研究希望能提高肺癌患者存活率，但其預後仍然很差，主因是肺癌細胞具有高侵襲及轉移能力所致，目前研究趨勢是以抑制肺癌轉移來當作治療肺癌的策略。肉桂過去被報導具有抗氧化、抗癌的效果，但是肉桂對癌症侵襲及轉移的作用仍然不清楚。本研究利用肉桂對兩株人類肺癌細胞進行研究 (A549 細胞和 H1299 細胞)，研究肉桂萃取物 (Cinnamomum cassia extract；CCE) 對肺癌是否有抗轉移、抗腫瘤以及回復 EMT 的作用。實驗將肉桂分別處理 A549 與 H1299 細胞，利用 MTT assay 分析肉桂對人類肺癌細胞的毒性評估。利用 Colony formation assay 分析肉桂對肺癌細胞群落形成影響。Adhesion assay 分析肉桂對人類肺癌細胞黏附力影響。進一步研究肉桂在人類肺癌細胞侵襲和移動的抑制作用，利用侵襲實驗 Boyden chamber assay 和傷口癒合實驗 Wound healing assay 進行分析以及癌細胞分泌蛋白酶試驗。利用培養過細胞的培養液分別進行基質金屬蛋白酶(MMPs)以及尿激酶型纖溶酶原激活物(u-PA)酵素活性分析。進一步使用 Western blot 分析肉桂對抑制肺癌細胞轉移和上皮間質轉化(EMT)的作用路徑。總結：1.隨著濃度與時間的增加，肉桂可以抑制人類肺癌細胞的細胞存活率。2.在 Colony formation assay 中發現肉桂可以抑制肺癌細胞的群落生成。3.Adhesion assay 分析顯示肉桂能夠降低肺癌細胞的黏附能力。4.透過細胞侵襲及傷口癒合實驗可以發現隨著肉桂濃度增加，有效降低 A549 和 H1299 細胞的侵襲及爬行能力。5.酵素活性分析顯示肉桂可以降低人類肺癌細胞 MMP-9、MMP-2 以及 u-PA 的活性。6.肉桂也可以抑制由 TGF-β 誘導的 EMT。7.在動物實驗中發現肉桂可以抑制腫瘤的生長。本研究證實肉桂可以減少人類肺癌細胞的侵襲及轉移能力，肉桂可抑制訊息傳導路徑中的 FAK、ERK1/2 的磷酸化。結果推論，肉桂可以減少人類肺癌細胞侵襲和轉移同時也可以回復人類肺癌細胞由 TGF-β 所誘導的 EMT作用。
並列摘要
Lung cancer is top five cancer that cause of death, the main reason that lead to death is because malignant tumor metastasis. There is a lot of research hope that can elevate the lung cancer patient’s survival rate, but the prognosis is still not optimistic, the main reason is that lung cancer can highly invasion and migration that cause the metastasis, so we can focus on inhibit the lung cancer metastasis as a strategy of treating lung cancer patient. Cinnamomum cassia is reported to have anti-oxidation and anti-cancer effect, but the treatment to cancer invasion and migration is still inconclusive. In this study, we use two kinds of human lung cancer cell (A549 and H1299) and treat with Cinnamomum cassia for the research, studying the effect of Cinnamomum cassia on lung cancer cell’s metastasis, anti-tumor and reverse EMT. The experiment that treat A549 and H1299 with Cinnamomum cassia, and analyze cancer cell proliferation with MTT assay. Then we use colony formation assay in order to analyze the effect of Cinnamomum cassia on cell colony formation. Utilize of adhesion assay to understand the effect of Cinnamomum cassia on cancer cell adhesion ability, and with the use of Boyden chamber assay and Wound healing assay, we can analyze the inhibition of the human lung cancer cell invasion and migration. In order to analyze the activity of MMPs and u-PA, we use the medium that had cultured with the lung cancer cell. Then we run the Western blot to detect the effect of Cinnamomum cassia on lung cancer cell metastasis and EMT. With the increase of concentration and time, Cinnamomum cassia can inhibit the survival rate of human lung cancer cell. Enzyme activity analyze shows that Cinnamomum cassia can reduce the human lung cancer cell activity of MMP-9, MMP-2 and u-PA. Through the experiment of cell invasion assay, we found that the increase concentration of Cinamomum cassia can reduce A549 and H1299 cell invasion ability effectively. Cinamomum cassia can also inhibit EMT that induced by TGF-β, the result show Cinamomum cassia can reduce the ability of human lung cancer cell invasion. We found that the phosphorylation of the FAK and ERK1/2 in the tumor progression pathways were also inhibited by the Cinamomum cassia. Above all the experiment, we know that Cinamomim cassia can reduce human lung cancer cell invasion and migration, and also reverse the EMT that is induced by the TGF-β in human lung cancer cell.