荔枝花乙醇萃取物誘導肝癌細胞(HepG2)凋亡與自體吞噬之研究

Apoptotic and Autophagic Mechanisms of Ethanol Extract from Litchi (Litchi chinensis Sonn.) Flower in the Induction of Death of Human Hepatocellular Carcinoma Cells (HepG2)

張宇霈(Yu-Pei Chang)

指導教授 ： 楊登傑 ； 林昭田

中山醫學大學/健康管理學院/健康餐飲暨產業管理學系碩士班/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00178

摘要

荔枝（Litchi chinensis Sonn.）為無患子科（Sapindaceae）荔枝屬（Sapindaceae）植物。荔枝花開花時期為每年的3~4月，其一般以廢棄物處理。然而，我們先前的研究發現荔枝花萃取物含有豐富的酚類化合物，且展現優異的抗氧化以及抗發炎效果。有文獻指出酚類化合物具有良好的抗癌功效，然而對於荔枝花抗癌之功效目前尚未有相關報導。因此，本研究進一步探討，荔枝花乙醇萃取物之抗癌功效。利用研究各種癌細胞株，包括A549、KB、MCF-7、 HepG2，先行評估荔枝花乙醇萃取物對此些癌細胞之細胞毒性，發現抑制HepG2細胞生長之功效較佳，因此再以正常肝細胞Clone-9 ( normal rat liver cell line )以及臨床抗癌藥物Fluorouracil(5-FU)、Methotrexate(MTX)確認出最佳試驗濃度為0.3mg/ml。利用此濃度探討其對癌細胞生長(proliferation)、促凋亡(apotosis)和自體吞噬(autophagy)之影響。在細胞週期分析中，荔枝花乙醇萃取物主要引起HepG2細胞G2/M期停滯，屬於晚期凋亡，在Western Blot分析中，荔枝花乙醇萃取物可降低Bcl-2蛋白表現，並增加Bid蛋白表現，使之有利於凋亡機轉進行，同時觀察到細胞凋亡下游蛋白 Pro-Caspase-3、-7、-8、-9蛋白表現量降低與 PARP 蛋白降解增加之趨勢。在PI3K/Akt路徑上荔枝花乙醇萃取物可有效增加PI3K、Akt蛋白表現且抑制磷酸化Akt蛋白表現，使癌細胞走向細胞凋亡，亦藉由p53蛋白的增加，進而抑制下游的mTOR、磷酸化p70S6K、PS6K(D57)以及PS6K(D68)蛋白表現量，使LC3 I/II的蛋白表現量增加，而達到自體吞噬的作用(autophagy)。

關鍵字

荔枝花 ； 細胞凋亡 ； 細胞週期 ； 細胞自體吞噬

並列摘要

Litchi (Litchi chinensis Sonn.) belonging to Sapindaceae family is an important commercial crops in Taiwan, which blooms in late March and fruit matures in late June. The flower is usually considered as disposable agricultural byproducts. Our pervious study found that litchi flower extract had a noticeable phenolic level and also exhibited good antioxidant and anti-inflammatory activities. Reports indicated that phenolic compounds had good anti-carcinogenic effect. There are, however, no related reports regarding anti-carcinogenic effect of litchi flower. In this study, cytotoxic activity of ethanolic extract from litchi flower on A549, KB, MCF-7 and HepG2 cancer cell lines was evaluated first. The litchi flower extract had better anti-proliferative effect on HepG2 cells. Through the comparative experiments with Clone 9 normal rat liver cell line and two drugs (fluorouracil (5-FU) and methotrexate (MTX) used in the treatment of cancer), 0.3 mg/ml is the best concentration used to investigate the apoptotic and autophagic mechanisms for the litchi flower extract-induced death in HepG2 cells. Cell cycle analysis showed that HepG2 cell treated with ethanolic extract from litchi flower was arrested at G2/M phase. Western blot analysis showed that ethanolic extract from litchi flower-induced HepG2 cell apoptosis should attribute decrease of Bcl-2 expression and increase of Bid expression; furthermore, decreased expression of procaspases-3, 7, 8 and 9, and increased PARP degradation could also be observed. For PI3K/Akt pathway, expression of PI3K and Akt was increased, and phosphorelation of Akt was down-regulated through treatment of the litchi flower extract, which led to HepG2 cell apoptosis. Moreover, ethanolic extract from litchi flower-induced HepG2 cell autophagy should attribute increase of p53 expression, which suppressed expression of phosphorylated mTOR, phosphorylated p70S6K, PS6K(D57) and PS6K(D58), and up-regulated LC3 I/II expression.

並列關鍵字

Litchi flower ； HepG2 ； apoptosis ； autophagy

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