靈芝酸GAT的抗癌活性研究

本研究主要探討靈芝三萜類Ganoderic acid T (GAT)的抗癌活性，使用GAT針對不同的癌細胞進行研究，觀察是否能促使細胞凋亡或細胞老化。發現GAT可抑制人類血癌細胞株HL-60、肝癌細胞株Hep-G2和Huh7細胞的生長，其IC50分別為35.2，20.3及75.7，而對人類正常週邊血液單核球細胞沒有影響。在HL-60細胞的研究中，藉由劉氏染色法和TUNEL實驗、粒線體膜電位分析、細胞週期分析、7-AAD染色及流式細胞分析，caspase-8、caspase-9及caspase-3活性分析這一系列和細胞凋亡有關的實驗，在Liu’s stain中發現有apoptotic bodies，在TUNEL實驗中有發現DNA斷裂，在粒線體膜電位分析中發現粒線體膜電位的下降，在7-AAD實驗看到細胞膜的完整性降低導致7-AAD螢光染色程度升高，在caspase-9和caspase-3實驗看到caspases活性的上升，顯示GAT有誘導HL-60細胞凋亡的能力。在Huh7 細胞發現GAT會使細胞的粒腺體膜電位下降、細胞週期停滯在G2期。在HepG2的研究，初步觀察到GAT會使HepG2細胞產生老化的細胞型態。除此之外，將加過GAT的細胞培養液的回收，萃取其中的物質，藉由高效液相層析儀，再配合四級柱質譜儀分析後，發現仍可找到結構沒改變的GAT，可見GAT在細胞培養液中的穩定性。由以上結果顯示GAT有發展為抗癌藥物的可能性。

關鍵字

赤芝；靈芝酸； GAT ；細胞凋亡；細胞週期停滯；細胞老化； HL-60 ； HepG2 ； Huh7

並列摘要

Ganoderic acid T (GAT), is a lanostanoid triterpene isolated from Ganoderma lucidum (Leyss.exFr.) Karst., and the aim of this study was to study the effects of GAT on some cancer cell lines. GAT was found to inhibit the growths of human leukemia cell line, HL-60 and hepatoma cell lines, HepG2 and Huh7, and the IC50 are 35.2, 20.3 and 75.7 μM respectively, while GAT showed no effect on non-proliferating human peripheral blood mononuclear cells. In HL-60 cells, Liu’s stain, TUNEL assay, MMP, cell cycle, 7-AAD, caspase-8, caspase-9 were performed. Apoptotic bodies were shown by Liu’s stain, TUNEL assay positive cells were observed in TUNEL assay, and 7-AAD positive cells were observed. Mitochrondrial membrane potential was decreased, and caspase-9 and 3 activities were increased in GAT treatment. In conclusion, GAT was found to induce HL-60 cell apoptosis. In Huh 7 cells, GAT was found to decrease mitochondrial membrane potential and induce cell cycle arrest in G2 phase. In HepG2 cells, preliminary data showed that GAT seemed to induce premature senescence of HepG2 cell. GAT was recovered from cultured medium, and HPLC and Q-TOF mass spectrometry was used to analyze GAT and the recovered fraction. The result showed that GAT is stable in cell culture. In the future, GAT might be recovered from culture medium. The results above indicate that GAT could be a potential anticancer drug.

並列關鍵字

Ganoderma ； Ganoderic acid ； GAT ； apoptosis ； cell cycle arrest ； senescence ； HL-60 ； HepG2 ； Huh7

參考文獻

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靈芝酸GAT的抗癌活性研究

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