柑橘類水果中的成分5-demethyltangeretin對於Hep3B肝癌细胞之抗增殖活性

Anti-proliferative activity of citrus fruit constituent 5-demethyltangeretin in Hep3B hepatocellular carcinoma cells

歐珍珠(Suvd-Erdene Oyunbileg)

指導教授 ： 陳與國

國立屏東科技大學/國際學院/食品科學國際碩士學位學程/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00158

摘要

多甲氧基類黃酮 (PMFs) 已被發現存在於柑橘類果皮中，且已被證實具有多種生物活性，包括抗癌、消炎及抗腫瘤活性之功效。最近，科學家們指出5-hydroxylated PMFs具有對抗不同型態癌症之潛力。許多數據表明，5-demethyltangeretin (5DT，5-hydroxy-6、7、8、4ʼ-tetramethoxyflavone) 為在柑橘類水果中發現之5-hydroxylated PMFs的一種，並對於三種不同類型的人類肺癌和結腸癌細胞進行抗增殖活性之評估。此項研究著重於 5DT 對於人類肝癌細胞 Hep3B之細胞生長、細胞週期和細胞凋亡之研究分析。在許多國家中，包括台灣及蒙古，hepatocellular carcinoma (HCC) 為肝癌最常見之型態，具有高死亡率。目前肝癌的治療方法已包括外科手術，化學療法和標靶治療，但目前還沒有完美的治療方式，所以另尋新方法是必要的。因此藉由飲食來預防癌症是一實用、經濟及有效之方法，以減少此疾病的風險。在這項研究中，例如細胞型態之改變，包括起泡、皺縮及細胞由培養皿上分離，經由5DT處理後均能經由倒置相位差顯微鏡觀察到。經由細胞型態觀察後，細胞存活率測定(MTT分析)結果顯示，5DT能抑制Hep3B細胞之細胞生長且呈現劑量效應，其IC50經由5DT處理24、48及72小時後分別為7.7 ± 1.2、2.98 ± 0.9 和 4 ± 1.5 µM。此外，經由流式細胞儀分析顯示5DT可以使細胞停滯在細胞週期之G2/M期並誘導Hep3B細胞之細胞凋亡。caspase-3、-8 and -9的活化在細胞凋亡中扮演重要之角色，且經由流式細胞儀分析結果顯示亦具有劑量效應。DNA片段化和粒線體膜電位分析亦能測定5DT誘導細胞凋亡的能力。DNA萃取和破碎分離經由0.2%瓊脂糖凝膠電泳。在粒線體膜電位實驗中，扣除5DT最高濃度處理組。 凋亡調節蛋白BAX的表達及DNA損傷信號蛋白PARP經由西方轉漬法結果顯示有上升之趨勢，並能減少細胞週期相關蛋白Cyclin B1及Cdc-2之表現。此外抗凋亡蛋白Bcl-xL經由24小時處理後相較於控制組，3、6及12小時能降低其蛋白表現量。另外5DT亦能增加自嗜作用相關蛋白Atg7及Beclin-1之表現量。此發現給了我們一個新的研究方向，對於5DT能誘導細胞產生自嗜作用之能力，但仍需更多的研究及評估。總之，我們已證實5DT能誘導Hep3B細胞產生細胞凋亡和使其細胞停滯於G2/M期顯示5DT具有抗肝癌之能力。

關鍵字

5-demethyltangeretin ； Hep3B肝癌細胞 ； 細胞生長抑制 ； 細胞凋亡 ； 細胞週期停滯

並列摘要

Polymethoxyflavones (PMFs) exist almost exclusively in citrus peels and have been demonstrated to have various biological activities including anti-carcinogenic, anti-inflammatory and anti-tumor activities. Recently, scientists have demonstrated anti-carcinogenic potential of 5-hydroxylated PMFs against different types of cancers. Accumulating data showed that anti-proliferative activity of 5-demethyltangeretin (5DT, 5-hydroxy-6,7,8,4ʼ-tetramethoxyflavone), one of the 5-hydroxylated PMFs found in citrus fruits, has evaluated on three different types of human lung and colon cancer cells. This study was focused on the effects of 5DT on cell growth, cell cycle distribution and apoptosis induction in Hep3B hepatocellular carcinoma (HCC) cells. HCC is the most common type of liver cancer which is prevalent cancer with high mortality in many countries including Taiwan and Mongolia. The therapies of liver cancer have included surgery, chemical therapy and target therapy, but there is no perfect treatment. An alternative therapy for liver cancer is certainly needed. Therefore, cancer prevention by dietary components may be a practical, economical and effective approach to reduce the risk of this disease. In this study, cell morphological changes such as blebbing, shrinking and detaching from cell dishes observed after 5DT treatment under phase-contrast inverted microscope. After morphological observation, cell viability assay (MTT assay) demonstrated that 5DT inhibited Hep3B cell growth in a dose-dependent manner. IC50 concentrations of 5DT were 7.7 ± 1.2, 2.98 ± 0.9 and 4 ± 1.5 µM for 24, 48 and 72 h of incubation. Furthermore, flow cytometric analysis indicated that 5DT could arrest cells in G2/M phase of cell cycle and induced apoptosis in Hep3B cells. Activation of caspase-3, -8 and -9 that play essential role in apoptosis, increased in dose-dependent fashion according to flow cytometric analysis. DNA fragmentation and mitochondrial membrane potential (Δψm) assays were evaluated apoptosis inducing ability of 5DT. DNA extracted and fragmentations separated by 0.2% agarose gel electrophoresis. Δψm deducted by 5DT treatment most at the highest concentration treated group. Overexpression of apoptosis regulator protein BAX and DNA damage signaling protein PARP, and decreased expression of cell cycle associate proteins, Cyclin B1 and Cdc-2, indicated by western blotting for further elucidation of anti-HCC mechanism of 5DT activity. Also, expression of anti-apoptotic protein Bcl-xL decreased after 24 h than the expression of control, 3, 6 and 12 h incubated groups. Furthermore, the expression of Atg7 and Beclin-1 which are autophagy, another type of cell death, inducement related proteins increased. This finding gave us a new suspension about autophagy inducing ability of 5DT. It need to be studied more for clearer understanding and evaluation. In conclusion, we have confirmed that 5DT has anti-HCC ability via inducing apoptosis and arresting cells in G2/M phase in Hep3B cells.

並列關鍵字

5-demethyltangeretin ； Hep3B HCC cells ； cell growth inhibition ； apoptosis ； cell cycle arrest

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