欖仁樹葉萃取物與丹寧酸誘發急性骨髓性白血病癌細胞凋亡及增強三氧化二砷療效之相關研究

Study of the effects of Terminalia catappa L. extracts and tannin acid on induction of apoptosis and enhancement of sensitivity to arsenic trioxide in human acute myeloid leukemia

陳國勳(Kuo-Shuen Chen)

指導教授 ： 周明智 ； 謝易修

中山醫學大學/醫學院/醫學研究所/博士(2009年)

https://doi.org/10.6834/CSMU.2009.00052

摘要

欖仁樹 (Terminalia catappa L.)在植物分類上為使君子科，分布於亞熱帶地區，冬天為欖仁樹落葉季節，在台灣、印度與菲律賓民眾會拾取欖仁落葉，用泡茶般的沖泡方式作為保肝茶飲。近年許多研究指出，欖仁樹葉具有預防及治療肝炎、抗發炎、清除自由基、抗氧化等活性。葉部之成份研究報告指出，其中最主要的成分為鞣質(或稱單寧; tannins)。單寧酸(tannic acid; TA)是由多個沒食子酸(gallic acid)聚合而成的糖苷體化合物，且已被證實具有抑制酵素活性、抗菌、抗腫瘤及止血收斂的作用。但是到目前為止，欖仁樹葉是否具有抑制血癌細胞的效果並不清楚。在本研究中，我們發現急性骨髓性白血病(acute myeloid leukemia; AML)細胞株 HL-60以不同濃度欖仁樹葉萃取物(Terminalia catappa L. extracts; TCE) 處理，會造成細胞存活率明顯的降低，且下降趨勢具有濃度及時間關係，此外以TCE處理HL-60細胞，在流式細胞儀的分析中，發現sub-G1 phase的細胞群會隨TCE的濃度提高而增多，以DAPI stain，證實TCE會造成HL-60細胞染色質濃染(chromosome condensation)，顯示TCE具有誘導HL-60細胞走向凋謝死亡。進一步利用單寧酸去處理HL-60細胞，發現HL-60細胞株在單寧酸的處理下，會造成細胞存活率與細胞數顯著降低，並呈現濃度及時間效應。在流式細胞儀的分析實驗中，發現sub-G1的細胞群會隨單寧酸的濃度提高而增加，顯示單寧酸具有誘導HL-60細胞凋亡的效果。以DAPI stain以及DNA電泳的分析，證實單寧酸會造成HL-60細胞核濃染以及DNA斷裂(DNA fragmentation)的效果。並進一步探討其分子機制，結果顯示單寧所誘導的HL-60細胞凋亡，主要是透過破壞粒腺體膜電位(mitochondrial membrane potential)進而釋放出cytochrome C，並且活化caspase 8、caspase 9 以及caspase 3，而造成poly (ADP-ribose) polymerase (PARP)的切割。此現象主要是透過降低細胞內超氧岐化酵素的活性，進而增加細胞內超氧離子的濃度，並進一步促進細胞凋亡內粒線體的訊息傳遞路徑活化。最後，我們發現低濃度單寧酸可以促進臨床上治療急性骨髓性白血病之抗癌藥物(arsenic trioxide)的活性，並具有相當不錯的輔助效果。綜合以上結果，欖仁樹葉萃取物與其所富含單寧可以有效的抑制急性骨髓性白血病細胞HL-60的生長，或許有助於日後對於白血病的治療。

關鍵字

丹寧酸 ； 血病癌 ； 細胞凋亡 ； 三氧化二砷

並列摘要

Terminalia catappa L.is a Combretaceous plant distributed on tropical and subtropical beaches. The leaves of this plant have been claimed to be a folk medicine to treat dermatitis and hepatitis in Taiwan, India, Philippines, Malaysia and Indonesia. The fallen leaves, after drying and shredding, have been used as a folk medicine and also as a drink after infusion with hot water. Previous studies showed that the leaves of Terminalia catappa L. extracts (TCE) exerted hepatoprotective, antiinflammatory, antioxidative, and radical scavenging activities. Tannins are the most abundant components of TCE and have the greatest antioxidative activity. Tannins (tannic acid; TA), a glucoside of gallic acid polymer, has been shown to possess anti-enzymatic, anti-bacterial, anti-tumor and astringent properties. However, the anti-cancer activity of TCE in leukemia is still obscure. In this study, we showed TCE-induced apoptotic death in acute myeloid leukemia (AML) HL-60 cells via dose- and time-dependent manner. In addition, TCE also induced cell death by increasing of sub-G1 fraction and chromosome condensation. The pure compound, tannic acid, was further examined. The result showed that TA-induced apoptotic death in HL-60 cells via dose- and time-dependent manner as well as increase of sub-G1 fraction, chromosome condensation, and DNA fragmentation. Further analysis demonstrated the involvement of activation of caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C, in TA-induced apoptosis. These effects were probably associated with the increase of intracellular superoxide in mitochondrial signaling pathway which attributed to the down-regulation of superoxide dismutase (SOD). Notably, a low dose of TA is sufficient to aggravate As2O3-induced cytotoxicity in HL-60 cells. Altogether, this study suggested the effects of TCE and TA to induce apoptosis in HL-60 and therapeutic potential in acute myeloid leukemia by being an adjunct to As2O3.

並列關鍵字

tannin acid ； leukemia ； apoptosis ； arsenic trioxide

參考文獻

Adhami, V.M., and Mukhtar, H. (2007). Anti-oxidants from green tea and pomegranate for chemoprevention of prostate cancer. Mol Biotechnol 37, 52-57.

Arber, N. (2008). Cyclooxygenase-2 inhibitors in colorectal cancer prevention: point. Cancer Epidemiol Biomarkers Prev 17, 1852-1857.

Bhat, R., and Hadi, S.M. (1994a). DNA breakage by tannic acid and Cu(II): generation of active oxygen species and biological activity of the reaction. Mutat Res 313, 49-55.

Bhat, R., and Hadi, S.M. (1994b). DNA breakage by tannic acid and Cu(II): sequence specificity of the reaction and involvement of active oxygen species. Mutat Res 313, 39-48.

Brenner, C., and Kroemer, G. (2000). Apoptosis. Mitochondria--the death signal integrators. Science 289, 1150-1151.