洛神花花青素誘發人類血癌細胞週期停滯之分子機制研究

洛神花，其名稱是由英名 Roselle 音譯而來，學名為Hibiscus sabdariffa Linnaeus，由於栽種方便且適應力強，台灣各地均有大量栽培，而其主要產地分布在台灣東部沿海一帶。洛神花營養價值極高，其內含有豐富的類黃酮、花青素、原兒茶酸等天然抗氧化成分外，在過去許多的研究中，結果證實洛神花中的花青素成分具有抗氧化活性、護肝及抗癌的功效。因此，在本研究中我們主要探討洛神花花青素（HAs）對人類血癌細胞（HL-60）之作用機轉。首先，以MTT分析HAs對HL-60細胞之生長作用，發現在處理24小時下，其IC50為2.008 mg/ml。接著，利用流式細胞儀分析HAs（0 ~ 0.7 mg/ml）處理HL-60細胞之細胞週期，發現細胞週期會停滯在G2/M期，並且隨著劑量與時間增加而愈加顯著。進一步我們擬研究其機制，藉由西方點墨法指出是透過CDC2Tyr15、p21和p27的調控，繼續深入觀察相關上游蛋白，我們發現在 0.7 mg/ml HAs處理下會增加phospho-CDC25C(Ser216)的表現，而磷酸化後的CDC25C提供了一個可與14-3-3蛋白結合的位置，致使從核中被釋出到細胞質。另外，我們亦觀察到HAs使ATM活化，下游的Chk1/2也隨之活化並導致CDC25C磷酸化。綜合以上的結果，HAs會調控ATM–Chk1/2–CDC25C的路徑，並磷酸化CDC2而讓血癌細胞週期停滯在G2/M期。根據本篇實驗結果，我們認為HAs可作為一種化學預防物質，增進人類健康之福祉。

關鍵字

血癌；花青素；週期停滯

並列摘要

Hibiscus sabdariffa Linne (Malvaceae) is widely cultivated in Eastern Taiwan. It contains highly valuable nutrients, such as protocatechuic acid, ﬂavonoids, and anthocyanins. Some previous reports demonstrated that H. sabdariffa anthocyanins (HAs) were antioxidant, liver-protective and anti-tunorigenic. In the present study, we explored the effect and molecular mechanisms of HAs -induced cell cycle arrest in human leukemia cell line HL-60. MTT was used to analyze the growth- inhibiting effect of HAs. The IC50 was 2.008 mg/ml after 24-hour-treatment. Flow cytometry analysis showed that HAs (0–0.7 mg/ml) markedly induced G2/M arrest of HL-60 cells in a dose- and time-dependent manner. The results of Western blot revealed that HAs induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and the induction of Cdk inhibitors p27 and p21 (WAF1/CIP1) with a p53-independent manner. Observing the further upstream signals, we found that HAs induced phosphorylation of cell division cycle 25C (CDC25C) tyrosine phosphatase at Ser-216, which led to its translocation from nucleus to cytoplasm, increasing the bind with 14-3-3. HAs-induced phosphorylation of CDC25C could be activated by ataxia telangiectasia-mutated (ATM) checkpoint kinases, Chk1 and Chk2. We first time confirmed that ATM–Chk1/2–CDC25C pathway as a critical mechanism for DNA damage and G2/M arrest in HAs-induced leukemia cell cycle arrest. According to these results, HAs could be developed as a chemopreventive agents.

並列關鍵字

leukemia ； anthocyanins ； G2/M arrest

參考文獻

1. Lin, H. H., Huang, H. P., Huang, C. C., Chen, J. H., & Wang, C. J., Hibiscus polyphenol-rich extract induces apoptosis in human gastric carcinoma cells via p53 phosphorylation and p38 MAPK/FasL cascade pathway. Mol Carcinog, 2005. 43(2): p. 86-99.

2. Lin, H. H., Chen, J. H., Kuo, W. H., & Wang, C. J., Chemopreventive properties of Hibiscus sabdariffa L. on human gastric carcinoma cells through apoptosis induction and JNK/p38 MAPK signaling activation. Chem Biol Interact, 2007. 165(1): p. 59-75.

3. Tseng, T. H., Hsu, J. D., Lo, M. H., Chu, C. Y., Chou, F. P., Huang, C. L., & Wang, C. J., Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin. Cancer Lett, 1998. 126(2): p. 199-207.

4. Chang, Y. C., Huang, K. X., Huang, A. C., Ho, Y. C., & Wang, C. J., Hibiscus anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages apoptosis. Food Chem Toxicol, 2006. 44(7): p. 1015-23.

5. Shih, P.H., C.T. Yeh, and G.C. Yen, Effects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells. Food Chem Toxicol, 2005. 43(10): p. 1557-66.

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洛神花花青素誘發人類血癌細胞週期停滯之分子機制研究

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