| 摘要: | 台灣每年約有一萬三千人死於慢性肝炎、肝硬化及肝癌，其中慢性肝炎及肝硬化為全國主要死因的第九名，肝癌則為全國主要癌症死因的第二名，由此可知研究如何預防及治療肝癌仍是台灣一大重要課題。早期肝癌的症狀不明顯，自我察覺不易，因此在臨床上被診斷為肝癌的患者有85%為中、晚期肝癌患者，而臨床上多使用標靶藥物治療晚期肝癌患者。近年有臨床研究發現肝癌患者合併使用cyproheptadine與標靶藥物thalidomide後，肝臟腫瘤與轉移至肺臟的腫瘤皆消失，經體外試驗證實cyproheptadine可以透過活化p38 MAPK以阻斷細胞週期，進而抑制肝癌細胞的生長。先前研究透過細胞存活率分析cyproheptadine的結構類似物，發現第一代抗組織胺藥物deptropine 抑制肝癌細胞HepG2與Hep3B生長的能力最強，並且利用西方墨點法發現deptropine會促進細胞自噬作用的指標蛋白——LC3B表現量上升。基於此結果，本論文研究進一步確認deptropine在細胞自噬作用所扮演的角色，並持續探討此藥物對於抑制肝癌細胞生長的機制。實驗結果顯示，deptropine不會造成HepG2細胞與Hep3B細胞內細胞凋亡相關蛋白Caspase 3、8、9或PARP的切割。而經過deptropine處理的HepG2細胞與Hep3B細胞，LC3B蛋白表現量上升，但SQSTM1蛋白 (p62蛋白) 表現量沒有隨藥物濃度提升而下降。deptropine亦會抑制cathepsin L蛋白的成熟。透過免疫螢光染色LC3發現經過deptropine處理的HepG2細胞與Hep3B細胞，自噬體形成量增加，但deptropine會干擾自噬體與溶酶體的融合。細胞存活率分析結果顯示合併使用deptropine與doxorubicin對HepG2細胞與Hep3B細胞的毒殺效果有協同作用。這些結果顯示deptropine透過阻擋自噬體與溶酶體的融合以抑制自噬作用的進行並導致細胞死亡。另外，合併使用deptropine與doxorubicin有潛力作為新的肝癌治療方式。
In Taiwan, over 13,000 deaths result from chronic hepatitis, cirrhosis and hepatoma per year, with hepatoma being the second leading cause of cancer-related death. Hepatoma is frequently asymptomatic in its early stages. Thus, about 85% of patients diagnosed with hepatoma are in intermediate or advanced stages. For these patients, targeted therapy is the most frequent treatment. Recently, the combination of cyproheptadine, an anti-histamine drug, and thalidomide was reported to result in the disappearance of liver tumors and lung metastasis. Further studies confirmed that cyproheptadine inhibited proliferation of hepatoma by activating p38 MAP kinase and blocking cell cycle. Our previous studies found that deptropine, an analog of cyproheptadine, as a more effective compound to inhibit proliferation of human hepatoma cells. It induced the expression of LC3B, a marker of autophagy. In this study, we investigated the role of deptropine in autophagy and its mechanism of inhibiting proliferation of human hepatoma cells. Results showed deptropine did not cause cleavage of caspase 3, 8, 9 or PARP in HepG2 and Hep3B cells. Deptropine increased the expression of LC3B-II in HepG2 and Hep3B cells, but the amount of SQSTM1 (p62) did not decrease. Deptropine also inhibited the processing of cathepsin L from its precursor form to its mature form. Immunofluorescence microscopy showed an increase of autophagosomes in deptropine-treated HepG2 and Hep3B cells, but deptropine interfered fusion between autophagosomes and lysosomes. Cell viability assay showed a synergistic effect when combining deptropine and doxorubicin. These data suggest that deptropine inhibits autophagy by blocking autophagosome-lysosome fusion and results in cell death. Moreover, the combination of deptropine and doxorubicin might be a potential treatment for human hepatoma. |
