- 學位論文
洛神花花青素Delphinidin透過microRNA-204-3p調控下游路徑抑制結直腸癌轉移之機制
Delphinidin inhibits human colorectal cancer metastasis through microRNA-204-3p targeting
摘要
Delphinidin屬於類黃酮族裡花青素的一員，與其他植物多酚一樣具有很強的抗氧化力，而先前已經在洛神花花青素裡發現其存在，並推定洛神花花青素裡主要的抗癌物質為Delphinidin。而本篇實驗我們探討的重點是 Delphinidin與轉移性結直腸癌的關係，利用細胞與動物實驗加以驗證其調控轉移機制的作用。首先從細胞毒性試驗與群落形成能力來分析Delphinidin對於人類結直腸癌的效果，發現到Delphinidin濃度於100 μM以內時，皆不會對DLD-1、SW480、SW620產生細胞毒性，並能有效抑制癌細胞聚集成群落，顯示出Delphinidin於適當的劑量內時似乎會減緩結直腸癌細胞的惡化，而並不會對細胞的存活率造成影響。接著透過細胞黏附、爬行、侵襲實驗皆證明Delphinidin確實可以降低人類結直腸癌細胞株轉移的能力。接下來從western blotting的結果我們推測Delphinidin可能會抑制integrin αvβ3/FAK/Src、RhoA GTPase路徑的活化，以及EMT相關轉錄因子Twist、Snail、Slug的表現量，進而調控癌細胞轉移的作用。從以上實驗我們已經初步確認Delphinidin所調控的分子機制，不過我們另外還想看microRNA是否參與其中。藉由microRNA微陣列分析出Delphinidin會促進miR-204-3p的表現上調，接著我們再以處理過Delphinidin的細胞內加入anti-mir以確認其與上述路徑之相關性，從western blotting的結果我們發現與單純只加Delphinidin的組別相比，同除處理anti-mir-204-3p/Delphinidin的細胞其integrin αvβ3 訊號路徑蛋白濃度皆有上升的趨勢，推測Delphinidin會藉由mir-204-3p來調控下游路徑，達到減緩癌症轉移的效果。最後再進行動物實驗確認Delphinidin的抗癌效果，分別注射有無處理Delphinidin的DLD-1 cells至裸鼠的脾臟內以模擬活體腸癌轉移的過程，飼養五週後先利用活體冷光影像觀察裸鼠體內腫瘤的分佈，再犧牲並取下實驗動物的臟器與腫瘤來分析檢測，我們認為Delphinidin確實可以減少結直腸癌肝轉移的機率。而本篇是第一個利用高純度的Delphinidin化合物來檢測其抑制結直腸癌轉移機制的研究，我們確實也發現到Delphinidin於過去未曾探討過的抗癌效果，未來希望能往藥物開發的方向發展，以造福末期結直腸癌病患的生活。
並列摘要
Delphinidin, a flavonoids that belongs to the group of anthocyanidin, and possesses potent antioxidant, anti-inflammatory properties as other polyphenols, has been reported to possess anti-cancer effects that abundant in Hibiscus sabdariffa Linnaeus in present studies. In our study, we aimed to investigate the anti-cancer action of delphinidin on metastatic colorectal cancer in vitro and in vivo. First we tested the toxicities and colony formation activities of Delphinidin on colorectal cancer cell lines, treatment of colorectal cancer (CRC) cell lines DLD-1、SW480、SW620 with Delphinidin (< 100 μM) resulted in inhibition of anchorage-independent cell growth without having significant toxic effects, that meant Delphinidin (< 100 μM) might trend to influence tumor malignancy & metastasis. Using these cells, we next determined the effects of delphinidin (< 100 μM) on cell adhesion、migration、invasion abilities, Delphinidin treatment of CRC cells inhibited cancer metastasis. Additionally, treatment of CRC cells with Delphinidin (< 100 μM) resulted in downregulation of focal adhesion protein integrin αvβ3/FAK/Src/paxillin、Rho GTPase RhoA/Rac-1/Cdc42, and inhibiting VEGF-induced angiogenesis by decreasing expression of PI3K/Akt. Furthermore, we also analyzed the expression of microRNA in CRC cells treated Delphinidin (100 μM) using microRNA microarray, upregulation of mir-204-3p was confirmed in treatment of CRC cells with Delphinidin. Then we determined the relation between mir-204-3p and integrin αvβ3/FAK/Akt pathway, and western blotting analysis indicates that higher protein levels after transfection of anti-mir-204-3p and Delphinidin treatment than Delphinidin treatment alone. Finally, we elucidated the role of Delphinidin in cancer metastasis from CRC cells in vivo, using bioluminescence imaging and in vivo metastasis assay confirmed that in nude mice intrasplenic injected with DLD-1 cells with or without Delphinidin, Delphinidin treatment caused decreasing in liver metastasis of CRC. Taken altogether, our findings reveal that delphinidin compound has a potentially new role for anti-metastasis effect by mir-204-3p/integrin αvβ3/FAK/Akt axis, we suggest that delphinidin can be used for treatment of metastatic colorectal cancer.
參考文獻
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