7-Hydroxydehydronuciferine 經由細胞凋亡及細胞自噬雙重途徑誘導人類黑色素瘤A375.S2細胞的死亡

7-Hydroxydehydronuciferine induces human melanoma A375.S2 cell death via apoptosis and autophagy

吳佩芳(Pei-Fang Wu)

指導教授 ： 王惠民

高雄醫學大學/藥學院/香粧品學系碩士班/碩士(2012年)

摘要

人類惡性黑色素瘤為世界各地發病率和死亡率快速增加的癌症之一，且為皮膚癌中最致命的形式。我們證實，從牡丹蓮分離出的化合物7-hydroxydehydronuciferine (7-HDNF)對人類黑色素瘤細胞株A375.S2為一個有效的生物活性抑制劑。在正常人類皮膚細胞的增殖實驗結果顯示，7-HDNF對角質細胞、黑色素細胞及真皮層的纖維母細胞僅具有輕微的細胞毒性；同時7-HDNF對於A375.S2細胞株表現出具有效濃度依賴抗癌作用。另外，藉由Annexin V-FITC和PI雙染可改變細胞膜的對稱性，並利用二維流式細胞儀來測定因7-HDNF而產生的細胞凋亡率；一維的流式細胞儀分析也顯示，7-HDNF使細胞發生DNA損傷，細胞週期中的G2/M期會有停滯現象。在吖啶橙染色中，我們發現7-HDNF會誘導細胞自噬並增強空泡及酸性胞器的形成；接著，透過西方點墨法分別探討細胞凋亡和細胞自噬中其相關蛋白質的表現及反應路徑。此外，於傷口癒合實驗中觀察到7-HDNF對A375.S2細胞具有抗移行的能力。最後，在這項研究中，我們證實7-HDNF會誘導A375.S2細胞株觸發細胞凋亡及細胞自噬雙重的死亡機制，將可作為一個有效的皮膚癌化學治療劑。

關鍵字

牡丹蓮萃取物(7-hydroxydehydronuciferine) ； 皮膚細胞 ； 黑色素瘤 ； 細胞凋亡 ； 細胞自噬

並列摘要

Melanoma, a malignant tumor of melanocytes, has one of the worldwide fastest growing incidences among cancers and is the deadliest form of skin cancer. We identified that 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv.rosa-plena to be a bio-active agent against human melanoma A375.S2 cells. Cell proliferation assay displayed minor cytotoxicities on normal human skin cells including, epidermal keratinocytes and melanocytes and dermal fibroblasts. It also demonstrated the strongest anticancer effect of 7-HDNF on A375.S2 cells to exhibit a dose-dependent behavior. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining to confirm the cellular membrane asymmetry losing. One-dimensional flow cytometric analysis showed that 7-HDNF increased cellular DNA population in cell cycle at G2/M phase. With acridine orange (AO) staining, we found that 7-HDNF induced patterns of autophagy unexpectedly, such as the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). Through western blot, protein expressions were discovered to verify apoptosis and autophagy response mechanisms sharing some common associated pathways. In addition to, 7-HDNF presented the high-quality anti-migratory activity in wound healing assay. Within this study, we confirmed that there were dual cell death machineries via apoptotic and autophagic inductions in 7-HDNF-treated A375.S2 cells to be a potential effective chemotherapeutic agent.

並列關鍵字

7-hydroxydehydronuciferine ； skin cell ； melanoma ； apoptosis ； autophagy

參考文獻

Amaravadi RK, Thompson CB. The Roles of Therapy-Induced Autophagy and Necrosis in Cancer Treatment. Clin Cancer Res. 2007, 13(24), 7271-7279.

An WW, Wang MW, Tashiro S, Onodera S, Ikejima T. Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways. J Korean Med Sci. 2004, 19, 560-566.

Bressenot A, Marchal S, Bezdetnaya L, Garrier J, Guillemin F, Plénat F. Assessment of Apoptosis by Immunohistochemistry to Active Caspase-3, Active Caspase-7, or Cleaved PARP in Monolayer Cells and Spheroid and Subcutaneous Xenografts of Human Carcinoma. J Histochem Cytochem. 2009, 57(4), 289-300.

Chen BH, Chang HW, Huang HM, Chong IW, Chen JS, Chen CY, Wang HM. (-)-Anonaine induces DNA damage and inhibits growth and migration of human lung carcinoma H1299 cells. J Agric Food Chem. 2011, 59(6), 2284-2290.

Chen CY, Cheng KC, Chang AY, Lin YT, Hseu YC, Wang HM. 10-Shogaol, an Antioxidant from Zingiber officinale for Skin Cell Proliferation and Migration Enhancer. Int J Mol Sci. 2012, 13(2), 1762-1777.