探討花青素peonidin 3-glucoside與穿山龍抑制癌瘤細胞轉移機制之研究

癌細胞的轉移往往是造成癌症病人病灶復發致死及最難根治的主要原因，同時也是一個複雜的過程，其成因包括細胞骨架的改變、細胞的貼附、細胞外基質的降解、蛋白水解酶的大量表達及細胞轉移及侵襲能力的改變。花青素是一種天然的多酚類的抗氧化物，廣泛的存在於日常食用的蔬菜與水果中，已有許多文獻指出花青素在癌症的化學預防上已有許多顯著的生物活性，促進腫瘤細胞凋亡、抑制癌細胞生長及預防正常細胞癌化等作用，但是對於花青素peonidin 3-glucoside的抗癌細胞轉移的相關研究至今還不清楚。因此，在本篇論文中，我們選用黑糯米所萃取的花青素(Oryze sativa L. Anthocyanins ; OAs)及純化後得花青素peonidin 3-glucoside，利用MTT assays、modified Boyden chamber assay、傷口癒合實驗、gelation zymography與 casein zymography assay探討花青素對多種人類肺癌細胞存活率、侵襲和移動能力及分泌蛋白酶能力的影響。研究結果證實OAs與peonidin 3-glucoside不會影響細胞的存活性，且peonidin 3-glucoside能有效的抑制肺癌細胞(H1299)的侵襲和移動能力與matrix metalloproteinase-2 (MMP-2)與matrix metalloproteinase-9 (MMP-9)及urokinase-type plasminogen activator (u-PA)的活性，並呈現濃度效應關係。我們利用Western blot及electrophoretic mobility shift assay (EMSA)，探討其中可能參與的訊息傳遞路徑，發現peonidin 3-glucoside會抑制H1299細胞中磷酸化態之ERK1/2的表現，利用MEK1 overexpression 證實peonidin 3-glucoside的確是透過抑制p-ERK這條路徑，此外peonidin 3-glucoside也可抑制c-Fos及c-Jun的表現及AP-1與DNA binding的活性。最後在動物實驗中，我們利用BALB/c nu/nu 老鼠皮下異種移植H1299細胞，其結果與控制組作比較，發現有處理peonidin 3-glucoside的組別中，會明顯抑制腫瘤的大小。並以C57BL/6以尾靜脈注射老鼠LLC細胞，發現peonidin 3-glucoside具有抑制肺癌細胞轉移至肺臟的能力。綜合以上結果，推測peonidin 3-glucoside可能是藉由抑制H1299細胞c-Fos及c-Jun的表現及AP-1與DNA結合的能力，使得MMP-2、-9及u-PA的表現降低，進而抑制腫瘤細胞的侵襲及轉移能力。此外，利用有機分層萃取方式將穿山龍作不同有機溶劑的萃取物，分別得到甲醇萃取物(DNE1)、氯仿層(DNE2)、乙酸乙酯層(DNE3)、正丁醇層(DNE4)以及水層(DNE5)，將萃取物分別處理老鼠黑色素瘤細胞(B16F10)與人類黑色素瘤細胞(A2058)，在乙酸乙酯層(DNE3)的部份發現其會有效抑制癌細胞的侵襲、移動、分泌MMPs與u-PA的能力，利用動物實驗尾靜脈注射老鼠黑色素癌細胞，以口餵食穿山龍乙酸乙酯萃取物，與注射腫瘤的對照組(餵vehicle)作比較發現其可有效抑制癌細胞轉移到肺部的能力(抑制效果達99.5%)，以西方墨點法探討其可能的相關機轉，證實穿山龍乙酸乙酯萃取物可減少B16-F10細胞磷酸化Akt的蛋白表現量，同時也會抑制轉錄因子NF-kappa B的核蛋白表現量與同時會增加其內生性抑制劑IkappaB的表現量，以上結果或許能在日後應用在預防癌的轉移或輔助肺癌的治療上。

關鍵字

花青素；轉移；侵襲；癌症

並列摘要

Metastasis, the major cause of cancer death and various treatment strategies have targeted on preventing the occurrence of metastasis, is a multi-step process involving change of cytoskeleton, cell adhesion and proteolytic degradation of the extracellular matrix , the expression of proteases , and upregulation of cell motility. Anthocyanins, present in various vegetables and fruits as a nature colorant, have broad activities including anti-carcinogenesis and anti-mutagenesis, which are generally attributed to their antioxidant activities. However, limited studies have been available concerning the inhibitory effect of peonidin 3-glucoside (P3G) for cancer metastasis. Here, we demonstrated that black rice anthocyanoins (Oryza sativa L. anthocyanins; OAs) and P3G could significantly inhibit the invasion (P<0.001), motility (P< 0.05), migration, secretion of matrix metalloproteinase (MMP)-2, -9, and urokinase-type plasminogen activator (u-PA) of lung cancer cells. To investigate the possible mechanisms involved in these events, we performed western blot analysis to find that P3G inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and overexpression with MAPK kinase 1 (MEK1) blocked P3G-inhibited H1299 cell invasion. A treatment withP3G to H1299 cells also inhibited the activation of AP-1 as shown by Western blot and electrophoretic mobility shift assay (EMSA). Finally, these compounds were evidenced by its inhibition on the metastasis of Lewis lung carcinoma (LLC) cells in vivo (P<0.001). Recent studies have revealed pleiotropic anticancer and antiproliferative capabilities of Dioscorea nipponica Makino, whereas the effect of this plant on metastasis of cancer cells has not been clearly clarified. In the present study, we extracted Dioscorea nipponica Makino with methanol (DNE1), chloroform (DNE2), ethyl acetate (DNE3), n-butanol (DNE4), and water (DNE5). We first demonstrate that DNE3 was found to be effective in reducing the lung metastases formation by about 99.5% as compared to vehicle treated control animals. When a non-toxic concentration of the extract was treated directly to highly metastatic murin melanoma cells (B16F10) and human melanoma cells (A2058) in vitro, it exerted a dose-dependent inhibitory effect on the invasion (P<0.001), motility (P<0.001), secretion of MMPs (P<0.001), and u-PA (P<0.001) of both cell lines. To investigate the possible mechanisms involved in these events, we performed western blot analysis to find that DNE inhibited phosphorylation of Akt. A treatment with DNE3 to B16F10 cells also inhibited the activation of NF-kappa B and increased the expression of IkappaB. Taken together, these findings suggested that P3G and DNE3 could reduce the metastasis of cancer cells, thereby constituting an adjuvant treatment for metastasis control.

並列關鍵字

Anthocyanins ； Metastasis ； Invasion ； Cancer

參考文獻

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探討花青素peonidin 3-glucoside與穿山龍抑制癌瘤細胞轉移機制之研究

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