黑糯米花青素抑制肝癌細胞侵襲轉移之作用機制探討

Study of the inhibitory effects and mechanisms of black rice anthocyanins on liver cancer cell invasion and migration

林新萍(Hsin-Ping Lin)

指導教授 ： 江蕙玲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00017

摘要

癌細胞的侵襲及轉移往往是癌症病患致死且導致預後不良的主要原因之一，而其侵入則與細胞間質的分解、細胞的貼附能力及移動性的改變有關。花青素是一種天然的多酚類，廣泛的存在於日常食用的蔬果中，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，具有促使腫瘤細胞凋亡及預防正常細胞癌化等作用。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)、peonidin 3-glucoside (P3G) 與 cyanidin 3-glucoside (C3G)），利用MTT assays、modified Boyden chamber assay、gelation zymography與 casein zymography assay探討對SKHep-1人類肝癌細胞存活率、侵襲和移動能力及分泌proteinases能力的影響。研究結果證實OAs不會影響細胞的存活性，且peonidin 3-glucoside及cyanidin 3-glucoside能有效的抑制SKHep-1細胞的侵襲和移動能力與matrix metalloproteinase-9 (MMP-9)及urokinase-type plasminogen activator (u-PA)的活性，並呈現濃度效應關係，此外cyanidin 3-glucoside也可促進SKHep-1細胞tissue inhibitor matrix metalloproteinase-2 (TIMP-2)之蛋白表現。我們利用Western blot及EMSA，探討其中可能參與的訊息傳遞路徑，發現peonidin 3-glucoside及cyanidin 3-glucoside會抑制SKHep-1細胞c-Fos及c-Jun的表現及AP-1與DNA binding的活性。另外，我們也進一步探討peonidin 3-glucoside 與 cyanidin 3-glucoside對於各種癌細胞的影響，結果顯示兩者皆能抑制SCC-4、Huh-7、HeLa細胞的侵襲力及u-PA的表現。最後在動物實驗中，我們利用BALB/c nu/nu 老鼠皮下異種移植SKHep-1細胞，其結果與控制組作比較，發現有處理OAs的組別中，會明顯抑制腫瘤的大小。綜合以上結果，推測OAs、peonidin 3-glucoside 及 cyanidin 3-glucoside可能是藉由抑制SKHep-1細胞c-Fos及c-Jun的蛋白表現及AP-1與DNA結合的能力，使得MMP-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，以上結果或許能在日後應用在預防肝癌的轉移或輔助肝癌的治療上。

關鍵字

轉移 ； 花青素 ； 侵襲 ； 基質金屬蛋白酶

並列摘要

Metastasis is one of the major reason which causes death and after-effects of cancer. The invasion of metastasis is connected with proteolytic degradation of the extracellular matrix (ECM) and cell adhesion, essential to achieving cell motility. Anthocyanins, a large group of natrual polyphenols existing in a wide range of daily fruits and vegetables, have been widely recognized to possess several potential as cancer chemopreventive agents. Recent studies have also revealed pleiotropic anticancer and antiproliferative capabilities of anthocyanins. This study first demonstrates that effects of black rice anthocyanoins （Oryza sativa L. anthocyanins (OAs), peonidin 3-glucoside (P3G), and cyanidin 3-glucoside (C3G)）in SKHep-1 cells. We assayed the cell viability, cell invasion/motility, matrix metalloproteinases (MMPs) activity, and urokinase-plasminogen activator (u-PA) activity of SKHep-1 cells with flavonoid treatments by MTT assays, cell-matrix adhesion assays,invasion/motility assays, gelatin zymography, and casein zymography. We found that peonidin 3-glucoside and cyanidin 3-glucoside significantly inhibited the cell invasion/motility capacities and the activities of MMP-9 and u-PA in a concentration-dependent manner, while OAs would not affect cell viability . Following a treatment of cyanidin 3-glucoside coud significanty increase the tissue inhibitor matrix metalloproteinase-2 (TIMP-2) expression in SKHep-1 cells. To investigate the possible mechanisms involved in these events, we performed Western blot analysis and EMSA to find that peonidin 3-glucoside and cyanidin 3-glucoside suppressed the nuclear levels of c-Jun and c-Fos and the DNA binding activity of activator protein-1(AP-1) in SKHep-1 cells. Furthermore，peonidin 3-glucoside and cyanidin 3-glucoside also exerted an inhibitory effect of cell invasion and u-PA expression on various cancer cells(SCC-4、Huh-7、HeLa). Finally, an in vivo anti-tumor study using nude mice (BALB/c nu/nu) xenograft model by a subcutaneous inoculation of SKHep-1 cells was performed. The average tumor volume of treatment groups was statistically lower than that of control group. Together, these results suggest that cyanidin 3-glucoside, peonidin3-glucoside, or OAs may act to decrease the MMP-9 and u-PA expression of SKHep-1 cells via suppressing the nuclear levels of c-Jun and c-Fos and the DNA binding activity of AP-1 and therefore, reduce the invasion and metastasis of tumor cells. In conclusion, anthocyanins may be a powerful candidate for a preventive agent against liver cancer development and metastasis.

並列關鍵字

metastasis ； anthocyanins ； invasion ； MMP

參考文獻

Wang, Q., Han, P., Zhang, M., Xia, M., Zhu, H., Ma, J., Hou, M., Tang, Z., and Ling, W. (2007) Supplementation of black rice pigment fraction improves antioxidant and anti-inflammatory status in patients with coronary heart disease. Asia Pacific Journal of Clinical Nutrition 16 Suppl 1, 295-301.

Liabakk, N. B., Talbot, I., Smith, R. A., Wilkinson, K., and Balkwill, F. (1996) Matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Research 56, 190-6.

Alvarez, O. A., Carmichael, D. F., and DeClerck, Y. A. (1990) Inhibition of collagenolytic activity and metastasis of tumor cells by a recombinant human tissue inhibitor of metalloproteinases. Journal of the National Cancer Institute 82, 589-95.

Bao, J., Cai, Y., Sun, M., Wang, G., and Corke, H. (2005) Anthocyanins, flavonols, and free radical scavenging activity of Chinese bayberry (Myrica rubra) extracts and their color properties and stability. Journal of Agricultural and Food Chemisty 53, 2327-32.

Boone, T. C., Johnson, M. J., De Clerck, Y. A., and Langley, K. E. (1990) cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proceedings of the National Academy of Sciences of the United States of America 87, 2800-4.