膽酸與雙氫青蒿素混成化合物於肝細胞癌細胞株之抗癌活性評估

Evaluation of anticancer activity of bile acid-dihydroartemisinin hybrids in hepatocellular carcinoma cells

黃子恩(Tzu-En Huang)

指導教授 ： 許麗卿

國立臺灣大學/醫學院/藥學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901639

摘要

肝細胞癌 (Hepatocellular carcinoma, HCC) 是在成人中最常見的原發性肝臟惡性腫瘤，也佔有原發性肝癌的 90%。根據國際癌症研究署 (International Agency for Research on Cancer, IARC) 在 2018 年所估計的資料，肝癌是造成全球癌症死亡的第四大主要原因。雙氫青蒿素 (Dihydroartemisinin, DHA) 是青蒿素衍生物主要的活性代謝物，也是用於治療瘧疾的著名藥物。在之前的文獻研究中，DHA 已被證明對多種人類癌症具有抗腫瘤作用。為了要增加對癌細胞的細胞毒性作用，合成了一系列的膽酸與雙氫青蒿素混成化合物 (bile acid-dihydroartemisinin, BA-DHA)。其中，UDC-DHA (#12) 是對 HCC 細胞最有效的化合物，其對 HepG2 細胞的 IC50 值為 1.75 μM 以及對 Huh-7 細胞的 IC50 值為2.16 μM。此外，UDC-DHA (#12) 相較於 DHA (#1) 及 UDCA (#2) 以 1:1 莫爾比率的合併使用更有效。在本篇的研究中，也探討了 UDC-DHA (#12) 的作用機轉。藉由流式細胞儀分析經過 propidium iodide (PI) 染色的細胞，可以觀察到 UDC-DHA (#12) 引起細胞停滯於 G0/G1 期以及增加代表細胞凋亡的 subG1 細胞群。UDC-DHA (#12) 在 HepG2 細胞中顯著地引起細胞凋亡指標，包括 caspase-9 與 caspase-3 的活化以及使 PARP 失去活性。有趣的是，透過 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay 可以觀察到 DHA (#1) 與 UDC DHA (#12) 隨著時間顯著地提升活性含氧物 (reactive oxygen species, ROS)，而 ROS 分別在 12 小時與 24 小時達到最高峰。我們的結果更進一步顯示 ROS 的誘發及粒線體膜電位 (mitochondrial membrane potential, MMP) 去極化皆可被抗氧化劑 N-acetylcysteine (NAC) 抑制，因此說明此兩現象促成 UDC-DHA (#12) 的抗癌作用。綜合以上所述，UDC-DHA (#12) 可做為對抗 HCC 的候選藥物，因此還需要更深入地探討詳細的藥物機轉。

關鍵字

肝細胞癌 ； 膽酸與雙氫青蒿素混成化合物 ； G0/G1 期停滯 ； 細胞凋亡 ； 活性含氧物 ； 粒線體膜電位去極化

並列摘要

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for up to 90% of all primary liver cancer. Based on the data estimated by the International Agency for Research on Cancer (IARC) in 2018, liver cancer is the fourth common cause of cancer death overall in the world. Dihydroartemisinin (DHA), a main active metabolite of artemisinin derivatives, is a well-known drug used in the treatment against malaria. In previous studies, DHA has been demonstrated to exhibit antitumor effects toward a wide range of human cancers. In order to improve the cytotoxic effect against cancer cells, a series of bile acid-dihydroartemisinin (BA-DHA) hybrids were synthesized. Among them, UDC-DHA (#12) is the most potent anticancer compound against HCC cells with IC50 values of 1.75 μM (HepG2 cells) and 2.16 μM (Huh-7 cells). Moreover, UDC-DHA (#12) is more potent than the combination of DHA (#1) and UDCA (#2) at 1:1 molar ratio. In this study, the mechanism of action of UDC DHA (#12) was also investigated. The propidium iodide (PI) staining and flow cytometric analysis demonstrated that UDC DHA (#12) significantly induced G0/G1 arrest and further induced subG1 populations, which represent the apoptotic cells. Apoptotic markers including cleaved caspase-9, caspase-3, and cleaved PARP were significantly induced by UDC DHA (#12) in HepG2 cells. Interestingly, the 2′,7′ Dichlorofluorescin diacetate (DCFH DA) assay demonstrated that DHA (#1) and UDC DHA (#12) significantly elevated cellular reactive oxygen species (ROS) levels over time and the ROS levels peaked at 12 h and 24 h, respectively. Furthermore, our results indicated that both induction of ROS and depolarization of mitochondrial membrane potential (MMP), which could be reversed by an antioxidant N acetylcysteine (NAC), contributed to the anticancer effect of UDC DHA (#12). In conclusion, UDC-DHA (#12) could be a drug candidate against HCC and further investigation of the detailed mechanism is warranted.

並列關鍵字

Hepatocellular carcinoma ； bile acid-dihydroartemisinin hybrids ； G0/G1 arrest ； apoptosis ； ROS ； depolarization of MMP

參考文獻

1. American Cancer Society. What Is Liver Cancer.

https://www.cancer.org/cancer/liver-cancer/about/what-is-liver-cancer.html.

2. Ozakyol, A. Global epidemiology of hepatocellular carcinoma (HCC epidemiology). J Gastrointest Cancer 2017, 48, 238-240.

3. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A. and Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018, 68, 394-424.

4. Ministry of Health and Welfare. Statistics of Causes of Death.