膽酸與雙氫青蒿素混成化合物於乳癌細胞株之抗癌活性評估

Abstract

乳癌是全世界女性中最常見的惡性腫瘤，其發生率或致死率都居高不下。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。先前文獻研究，DHA對多種人類癌症具有抗腫瘤作用，但其化學不穩定性以及葡萄醣醛酸化作用可將其迅速消除，導致半衰期短。為了解決穩定性不好的問題，且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)。我們先前研究發現在膽酸與雙氫青蒿素混成化合物中，UDC-DHA、HDC-DHA 和N3UDC-DHA在HepG2及HL-60效用最好。在這篇研究，我們針對乳癌細胞株也可觀察到此結果。DHA對MCF-7 細胞的 IC50 值約為40μM; UDC-DHA、HDC-DHA 和N3UDC-DHA的 IC50 值約為2 μM，為DHA 20倍的功效。針對MDA-MB-231細胞，DHA的 IC50 值約為100 μM; HDC-DHA 和N3UDC-DHA的 IC50 值分別約為20 μM及40 μM，為DHA 2.5-5倍的功效。雙氫青蒿素混成化合物比 DHA和相對應膽酸以 1:1 莫爾比率的合併使用更有效。探討在MCF-7中，DHA、HDC-DHA 和N3UDC-DHA之作用機轉，並使用相等功效的濃度進行實驗。在培養液及細胞中，可以發現UDC-DHA、HDC-DHA 和N3UDC-DHA較 DHA穩定，而DHA相較HDC-DHA 和N3UDC-DHA更快展現細胞毒性。可以觀察到HDC-DHA及N3UDC-DHA引起細胞停滯於 G0/G1 期和增加代表細胞凋亡的 subG1 細胞群，並發現可降低cyclin D、cyclin B、cyclin E的表達及提高p21、p27的表達。DHA、HDC-DHA及N3UDC-DHA在MCF-7細胞中通過內在凋亡途徑在MCF-7細胞中發揮細胞毒性作用。DHA、HDC-DHA及N3UDC-DHA誘導粒線體活性含氧物 (reactive oxygen species, ROS)並誘發細胞質ROS，隨後導致粒線體膜電位去極化。結果更進一步顯示ROS 的誘發及粒線體膜電位去極化皆可被抗氧化劑 N-acetylcysteine (NAC) 抑制，因此說明此ROS促成 DHA、HDC-DHA及N3UDC-DHA的抗癌作用。在處理DHA、HDC-DHA及N3UDC-DHA後可以使Chk2 磷酸化及g-H2AX上升且增加 p53 表現及活化以及Rad51代表的HR修復途徑下降，顯示出DNA 破壞性。在處理DHA、HDC-DHA及N3UDC-DHA後，可提升AMPK/ autophagy途徑。此外，DHA、HDC-DHA及N3UDC-DHA可抑制細胞遷移。Akt/mTOR 訊息傳遞路徑與細胞生長有相關性，此路徑在一般抗癌情況會被抑制，意外地是，在加藥處理後DHA、HDC-DHA及N3UDC-DHA誘導該路徑。因此將Akt抑製劑MK-2206與膽DHA、HDC-DHA及N3UDC-DHA做合併，顯示出協同作用。綜合以上所述，HDC-DHA及N3UDC-DHA可做為對抗乳癌的候選藥物。

Breast cancer is the most common malignancy in women worldwide, in which incidence and the lethal rate remain high. Dihydroartemisinin (DHA) is the main active metabolite of artemisinin derivatives and also a well-known anti-malaria drug. Previous studies have shown that DHA has antitumor effects in a variety of human cancers, but it can be rapidly eliminated by glucuronidation and it is highly chemically fragile, resulting in a short half-life. To solve the problem of poor stability and increase the cytotoxic effect on cancer cells, a series of bile acid-dihydroartemisinin (BA-DHA) compounds were synthesized. Our previous studies have found that UDC-DHA, HDC-DHA and N3UDC-DHA are the most effective compounds in HepG2 and in HL-60 cells. In this study, we also observed anticancer activity against breast cancer cells. The IC50 value of DHA for MCF-7 cells was ~40 μM; and the IC50 value of HDC-DHA and N3UDC-DHA was ~2 μM, which was 20 times more potent than DHA. The IC50 value of DHA for MDA-MB-231 cells was 100 μM; and the IC50 values of HDC-DHA and N3UDC-DHA were 20 μM and 40 μM, respectively, which were 2.5-5 times more potent than DHA. The BA-DHA hybrids were more effective than the combination of DHA with the corresponding bile acid at a 1:1 molar ratio. The mechanism of action of DHA, HDC-DHA, and N3UDC-DHA was investigated using concentrations of equal efficacy in MCF-7 cells. In the medium and cells, UDC-DHA, HDC-DHA and N3UDC-DHA were more stable than DHA, while DHA showed cytotoxicity faster than HDC-DHA and N3UDC-DHA. HDC-DHA and N3UDC-DHA caused G0/G1 arrest and increased subG1 cell population that represents apoptosis., These compounds reduced the expression of cyclin D, cyclin B, cyclin E and increased the p21 and p27 levels. DHA, HDC-DHA and N3UDC-DHA exerted cytotoxicity in MCF-7 cells through the intrinsic apoptotic pathway. DHA, HDC-DHA and N3UDC-DHA induced mitochondrial reactive oxygen species (ROS) and subsequently cellular ROS, finally led to mitochondrial membrane potential loss. The induction of ROS and MMP loss was attenuated by an antioxidant N-acetylcysteine (NAC), suggesting that ROS contributed to the anticancer effects of DHA, HDC-DHA and N3UDC-DHA. After treatment with DHA, HDC-DHA and N3UDC-DHA, p-Chk2, g-H2AX and p53 were upregulated, and Rad51 involved in HR repair was downregulated, indicative of DNA damage response. The AMPK/autophagy pathway was also activated by DHA, HDC-DHA and N3UDC-DHA. Furthermore, DHA, HDC-DHA and N3UDC-DHA inhibited cell migration. The Akt/mTOR signaling pathway is related to cell growth. This pathway is usually inhibited in general anti-cancer treatment. Unexpectedly, DHA, HDC-DHA and N3UDC-DHA induced this pathway. Therefore, an Akt inhibitor MK-2206 was combined with DHA, HDC-DHA and N3UDC-DHA and displayed a synergistic effect. All in all, HDC-DHA and N3UDC-DHA can be used as drug candidates against breast cancer.