桑椹花青素經由Ras/Akt路徑抑制小鼠黑色素細胞瘤轉移作用

癌症預防已為一世界趨勢，因此尋找一化學預防物質(chemopreventive agent)為一重要工作。花青素(anthocyanins)廣存於蔬菜、水果等植物中，且有許多報告證明花青素具抗化活性、抑制心血管病變及防癌等作用。桑椹(Mulberry fruit)含有豐富之花青素(MAC)，近年來我們初步發現MAC可以抑制黑色素瘤細胞對小鼠的轉移作用，其詳細機制尚不清楚。我們利用MMP電泳、轉移分析及傷口癒合實驗來觀察花青素是否可以在in vitro抑制B16-F1細胞轉移，結果顯示在MAC處理以後，MMP2及MMP9的表現量減少；此外，在MAC高劑量(3 mg/ml)處理以後，傷口癒合及轉移能力明顯變差。因此，我們進一步以immunoblotting分析其相關蛋白的表達，實驗數據顯示出Ras、PI3K、Akt及NF-κB都相對減少，故推測MAC可能經由Ras與PI3K的路徑去抑制B16-F1的轉移能力。為了確定MAC抑制動物腫瘤轉移的詳細機制，C57BL/6小鼠先於右鼠蹊注射B16-F1細胞，同時餵管MAC 5週後發現，MAC可抑制B16-F1細胞轉移至肝、肺、皮膚等，其中以抑制轉移至肝臟最明顯。從以上結果我們可以提出MAC可以預防及治療癌症轉移，並說明其機轉。由於桑椹易在國內種植，MAC產率高也易分離，因此本研究成果可應用於發展為抗癌物質。

Over the pass decade, advances in understanding carcinogenesis have made possibility the identification of the candidates of chemopreventive agents that are being developed to hit the key molecular targets. Mulberry fruit has abundant anthocyanins (MAC). Recently, Mulberry anthocyanins (MACs) were isolated from the dried fruit of Mulberry to know the effect of MACs caused cancer cell apoptosis. We have proved that MAC inhibited B16-F1 melanoma migration ability, but the mechanism of inhibition was still unclear.The experiments are design to confirm MAC as a metastasis inhibitor evaluated by gelatin zymography, migration assay and wound healing analysis to exhibit a key role in inhibition of metastasis-associated metalloproteinase secretion. As was expected, the expression levels of MMP9 and MMP2 were inhibited when treated with MAC (1, 2 and 3 mg/ml). After treating 3 mg/ml MAC with B16-F1 cells, the ability of wound healing was abolished at Day 3 and migration numbers of cell were decreased too. We further used immunoblotting assay to evaluate their effect on the MAC-inhibited B16-F1 cells metastasis. The data showed that expression of Ras, PI3K, Akt, and NF-κB in the MAC-treated B16-F1 cells were reduced. Therefore, we suggested that MAC mediated B16-F1 metastasis via the Ras and PI3K pathway. To investigate whether MAC could also affect tumor metastasis in vivo, we design an animal model for the inhibition of liver metastasis induced by B16 melanoma cells in mice, we observe that MAC can inhibit the migration of B16-F1 cell. To summarize these results, MAC can inhibit the development and migration stages of the cancer cell. We suggest that MAC may offer the application in clinical medicine.