桑椹花青素調節腫瘤微環境抑制胃癌細胞增生移行之研究

The study of Mulberry anthocyanins regulating the microenvironment to inhibit gastric cancer proliferation and migration

黃惠珮(Hui-Pei Huang)

指導教授 ： 王朝鐘

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2013年)

https://doi.org/10.6834/CSMU.2013.00158

摘要

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氧化活性、抑制心血管病變及防癌等作用。台灣特有植物桑椹(Mulberry fruit)含有豐富之花青素(Mulberry anthocyanins; MACs)，本實驗室以HPLC分析，經定量並計算出乾燥後的桑椹含有約5% MACs。我們利用MTT assay、Flow cytometery、TUNEL assay及Western blot 等方法，探討MACs之抗癌作用。結果發現MACs明顯造成AGS細胞DNA斷裂，導致細胞的凋謝死亡，透過三種抑制劑的使用，如SB203580 (p38 抑制劑)、PD98059 (MEK 抑制劑)、and wortmannin (phosphatidylinositol 3-kinase; PI-3K 抑制劑)，結果推測可能經由p38 pathway抑制癌細胞生長。細胞經由MACs的處理，活化p38/JNK造成c-jun的磷酸化，同時也造成p53磷酸化，而活化下游的FasL/Fas使得Bid變成tBid，使得Bax在粒腺體膜上聚集在一起而形成孔洞，結果cytochrome c釋放出來跑到細胞質活化caspase-3造成細胞的死亡。
近年來之臨床數據與研究顯示，腫瘤相關巨噬細胞(tumor-associated macrophage; TAM)可增加癌細胞增生、侵襲與血管新生能力，並導致病人有較差的癒後。因此，本實驗再進一步釐清MACs之抑癌、減少血管新生及轉移作用，與透過抑制TAM形成之關係及其機轉。結果顯示胃癌細胞可誘導THP1細胞成為M2巨噬細胞，而MACs可抑制此現象。不論是與THP1共同培養或以M2細胞收集之CM (conditioned medium)培養之胃癌細胞，MACs皆可減緩其增生、移行、侵襲與血管新生之能力。利用ELISA分析結果，發現MACs可能透過調節腫瘤微環境之TNF-α、IL-6與CSF-1的分泌，抑制胃癌細胞生長、發炎現象與血管新生作用，同時抑制MMP1分泌去改變細胞黏附作用，並降低MMP2/MMP9之表現，以抑制腫瘤侵襲作用。本實驗結果確認桑椹花青素有抑制胃癌之作用。除了可發展為防癌保健食品，未來期待可進入中草藥臨床試驗成為新的癌症轉移之輔助療法用藥。

關鍵字

桑椹花青素 ； 凋謝死亡 ； 細胞生長停滯 ； 腫瘤相關巨噬細胞 ； 侵襲 ； 血管新生

並列摘要

Plant anthocyanins are important part of diet because of their effects on modulating carcinogenesis and cardiovascular disease. Mulberry fruit has abundant anthocyanins, and our laboratory has been quantified and calculated that dried-Mulberry fruit contains 5% anthocyanins by HPLC experiment. Next, we used MTT assay, flow cytometery, TUNEL assay and Western blot to analyze the antitumor effect of Mulberry anthocyanins (MACs). MACs were isolated from the dried fruit of Mulberry and the effect of MACs was to cause the cancer cell apoptosis. We further used SB203580 (p38 inhibitor), PD98059 (MEK inhibitor), and wortmannin (phosphatidylinositol 3-kinase; PI-3K inhibitor) to evaluate their effect on the MACs-induced AGS death. The data showed that only SB203580 had strong potential in inhibiting AGS cell apoptosis and also revealed increased phosphorylation in p38 and c-Jun, cytochrome c release, and expression of tBid, Fas and FasL in the MACs-treated AGS cells. Therefore, we suggested that MACs mediated AGS apoptosis via the p38-FasL and tBid pathway.
Evidences from clinical and experimental researches have suggested tumor-associated macrophage (TAM) functioning in tumor cell proliferation, tumor cell migration and invasion and tumor angiogenesis. Clinical studies have shown a correlation between an abundance of TAM and poor prognosis. Anthocyanins richly exist in mulberry plants and have been well characterized to have various bioactive properties. The aim of the present study is to determine the effect of the MACs the reducing macrophage contributes to gastric cancer (GC) cell migration, invasion and angiogenesis. Using cell proliferation study and flow cytometry, we find that AGS cell can induce THP1 monocyte differentiation to macrophage, which can be inhibited by treatment of MACs. The MACs inhibit proliferation, migration and metastasis of AGS cell enhanced by macrophage through regulating the secretion of MMP1, IL-6, CSF-1 and interferon-gamma by using ELISA assay. Also, after co-culture of AGS and THP1 cell, MACs can decrease the MMP2/9 levels of AGS cells. Beside, MACs can decrease the formation of tumor angiogenesis inducing by TAM in vitro and in vivo via inhibiting VEGF formation. Comprehensive, this work will provided the research model about the natural food inhibiting the tumor formation, metastasis, and angiogenesis. Besides, we can prove the mulberry anthocyanins as an efficient agent against the cancer.

並列關鍵字

Mulberry anthocyanins (MACs) ； apoptosis ； cell cycle arrest ； tumor-associated macrophage (TAM) ； invasion ； angiogenesis

參考文獻

1. Huang, H. P., Ou, T. T., and Wang, C. J. (2013) Mulberry (桑葚子 Sang Shèn Zǐ) and its bioactive compounds, the chemoprevention effects and molecular mechanisms in vitro and in vivo, J Traditional and Complementary Med. 3, 7-15.

2. Chen, C. C., Liu, L. K., Hsu, J. D., Huang, H. P., Yang, M. Y., and Wang, C. J. (2005) Mulberry extract inhibits the development of atherosclerosis in cholesterol-fed rabbits, Food Chem 91, 601-607.

3. Liu, L. K., Lee, H. J., Shih, Y. W., Chyau, C. C., and Wang, C. J. (2008) Mulberry anthocyanin extracts inhibit LDL oxidation and macrophage-derived foam cell formation induced by oxidative LDL, J Food Sci 73, H113-121.

4. Liu, L. K., Chou, F. P., Chen, Y. C., Chyau, C. C., Ho, H. H., and Wang, C. J. (2009) Effects of mulberry (Morus alba L.) extracts on lipid homeostasis in vitro and in vivo, J Agric Food Chem 57, 7605-7611.

5. Peng, C. H., Liu, L. K., Chuang, C. M., Chyau, C. C., Huang, C. N., and Wang, C. J. (2011) Mulberry water extracts possess an anti-obesity effect and ability to inhibit hepatic lipogenesis and promote lipolysis, J Agric Food Chem 59, 2663-2671.