黃安正老師的歷程檔案

專題研究計畫名稱	Ethyl-2-anilino-4-oxo-4,5-dihydrofuran-3-carboxylate(HAJ02)誘導人類骨髓血癌細胞（HL-60）之細胞凋亡研究
資料日期	2012-02-22

[摘要] :
2010年執行本校獎助教師專題研究：氮-取代芐基-不同位置雙取代基-2,3,4,9-四氫氟呋喃并[2,3-b]喹啉-3,4-雙酮類化合物之細胞毒性生理活性評估，發現Ethyl-2-anilino-4-oxo-4,5-dihydro- furan -3-carboxylate(以下代號HAJ02)有很明顯的抗癌活性，故探討HAJ02作用於人類癌細胞（Human Premyelocytic Leukemia Cells; HL-60）之抗癌活性及可能機轉。結果：HAJ02對HL-60具有抗細胞增殖活性，其IC50為27 μM。HAJ02在25μM即可以明顯活化caspase- 3、增加細胞內鈣離子濃度，進而導致活性氧物質(ROS)之產生及細胞膜電位之喪失，最終造成HL-60進行細胞凋亡進行。由實驗結果推測：HAJ02以劑量依賴型方式造成HL-60細胞凋亡。因此，HAJ02可以當作一種有潛力的前驅化合物(Lead compound)，用於開發新的抗白血病的藥物。

結論：
這是一個 furoquinoline中間合成衍生的具有抗白血病作用的報告。我們證實HAJ02通過細胞內鈣離子濃度的增加，伴隨ROS的產生和線粒體膜電位的損失，誘導HL - 60細胞進行 caspase依賴的細胞凋亡。 HAJ02誘導細胞凋亡機制類似先前 furoquinoline衍生物誘導細胞凋亡的報告15,16（Lin et al., 2007; (Huang et al., 2007）。 Furoquinoline衍生物ethyl 2- [N-p-chlorobenzyl- (2'-methyl)] anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01007)在人類子宮頸癌細胞，透過線粒體依賴路徑，造成細胞質鈣離子濃度增加，引起細胞凋亡(Lin et al., 2007)。此外，ethyl 2- [N-m-chlorobenzyl- (2'-methyl)] anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01006) 在人類子宮頸癌細胞，透過caspase依賴路徑，導致ROS的過度生產，引起細胞凋亡(Huang et al., 2007)。除了抗癌活性，furoquinoline及其衍生物，已知有一些不同的藥理作用，例如拮抗5 - HT2受體活性(1 Cheng JT 1994)、經由抑制鈣離子內流而舒張血管(2 Yu SM 1992)、抗過敏作用(3 Huang AC 1995)和阻斷外向鉀電流和Na +通道(4 Su MJ 1997)。最近，furoqunoline衍生物對 A2780人類卵巢癌細胞株發現有中度抗增殖活性(5 Cao S)。因此，furoquinolone衍生物的生理功能機制可能是多樣性。
總之，實驗結果指出HAJ02是一種很有前途的化合物，可用於開發新的抗白血病藥物的前導化合物。我們的研究結果強調了抗白血病作用的生理學機制，有助於闡明 furoquinoline及其衍生物的生物活性。

[英文摘要] :
According to previous studies “Assessment of physiological cytotoxicity of N-substituted- benzyl-various bisubstituted 2,3,4,9-tetrahydrofuro[2,3-b] quinolin -3,4-dione”. F ind Ethyl-2-anilino-4-oxo-4,5-dihydro- furan -3-carboxylate (abbr. HAJ02) has clear anti-cancer activity. In this study, we invenstigated anti-leukemia activity of HAJ02 in HL-60 cells. HAJ02 exhibited the anti-proliferative activity, showing a 27 μM of IC50 for HL-60 cells. HAJ02 treatment significantly activated caspase-3 and increased intracellular Ca2+ concentration in 25 μM. Next, the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential were also involved in caspase-dependent apoptosis of HL-60 cells by HAJ02. These results suggest that apoptosis of HL-60 cells by HAJ02 is induced dose-dependent, correlating with initialing apoptosis upstream pathways. HAJ02 might be a promising compound used as lead compounds for the development of new anti-leukemia agents.

This is the first report of a furoquinoline derivative which has anti-leukemic effect. We demonstrate that HAJ02 induced caspase-dependent apoptosis via the increase of intracellular calcium concentration, accompanying with the ROS production and the loss of mitochondrial membrane potential in HL-60 cells. The mechanism of HAJ02-induced apoptosis was similar to previous reports of apoptosis induced by furoquinoline derivatives (Lin et al., 2007; Huang et al., 2007). Furoquinoline derivative ethyl 2- [N-p-chlorobenzyl- (2'-methyl)] anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01007) through via a mitochondria-dependent pathway closely related to the level of cytoplasmic Ca2+ in that in apoptosis of human cervical cancer Ca Ski cells (Lin et al., 2007). In addition, ethyl 2- [N-m-chlorobenzyl- (2'-methyl)] anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (JOT01006) induced ROS overproduction which was responsible for caspase-dependent apoptosis of human cervical cancer cells (Huang et al., 2007).
In addition to anti-cancer activity, furoquinoline and its derivatives are known to have some diverse pharmacological effects, such as antagonizing 5-HT2 receptor activity (1 Cheng JT 1994), vasorelaxation by suppressing the calcium influx flow (2 Yu SM 1992), antiallergic effect (3 Huang AC 1995) and blocking outward K+ current and Na+ channels (4 Su MJ 1997). Recently, furoqunoline derivative was found to have moderate antiproliferative activity against the A2780 human ovarian cancer cell lines (5 Cao S). Therefore, mechanisms of biological function of furoquinolone derivatives could be diversity.
In summary, the results indicate that HAJ02 furoquinolone derivative is a promising compound that could be used as lead compounds for the development of new anti-leukemia agents. Our results emphasize biological mechanism of anti-leukemia action, being helpful for elucidating the biological activities of furoquinoline and its derivatives.