(I)艾瑞莎誘導H1650肺癌細胞株進行細胞凋亡及細胞自噬之研究 (II)五味子內酯A之純化及抑制A549肺癌細胞株生長之研究

Abstract

I. 艾瑞莎可以專一性抑制上皮生長因子受器的酪胺酸激酶活性，目前已在多國被核准用於治療非小細胞性肺癌。本研究探討艾瑞莎對肺癌細胞株H1650的影響，艾瑞莎可以有效抑制上皮生長因子的磷酸化，並抑制下游Akt分子的活化，但對ERK的磷酸化則沒有影響；艾瑞莎會抑制H1650細胞生長及造成G0/G1細胞週期停滯；艾瑞莎抑制Bcl-2蛋白質表現，提高Bax蛋白質和p53蛋白質表現，進而誘導H1650細胞進行細胞凋亡。研究發現，艾瑞莎會誘導H1650細胞出現細胞自噬現象，包含大量酸性囊胞的累積、LC3蛋白質的轉化以及細胞自噬溶體的形成；我們並進一步藉由3-methyladenine及Bafilomycin A1來抑制細胞自噬，發現會造成H1650細胞株更容易受到艾瑞莎誘導進行細胞凋亡，證實細胞自噬在這裡是扮演著保護癌細胞對抗艾瑞莎誘導細胞凋亡的功能。 II. 本篇研究從黃芝(Ganoderma colossum)中分離純化出一抗癌化合物，經構造鑑定發現是為五味子內酯A (Schisanlactone A)。五味子內酯A最早是在1983年從五味子中被分離純化出來，至今已超過20年，但生物活性方面的研究卻非常少，目前唯一有被報導的是抑制愛滋病病毒蛋白質酶活性。黃芝俗稱棺菇，因為不易取得所以相關研究十分稀少，過去研究曾從黃芝中純化得到20種天然物，五味子內酯A便是其中之一。；本篇研究從黃芝經由酒精萃取、乙酸乙酯層析、矽膠管柱層析以及高效能液相層析等方式分離純化得到五味子內酯A，並進一步探討其對癌細胞的影響；五味子內酯A會抑制肺癌細胞株A549的生長，並造成細胞死亡，其抑制50%細胞活性的藥物濃度為2.4 μM (72小時)；探討其機轉發現五味子內酯A會造成A549細胞株出現細胞週期G0/G1階段停滯，並提高CDK2的表現，而Cyclin D1的表現則不受影響，五味子內酯A長時間處理也會誘導細胞死亡，進一步研究顯示，五味子內酯A會活化Caspase-3的活性，但是會刺激A549細胞株內Bcl-2蛋白質表現量上升以及p53蛋白質表現量下降。實際的死亡機制還需進一步探討。

I. Gefitinib is a clinical use specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for treatment of non-small cell lung cancer. In this study, mechanism of gefitinib-induced cell death in NSCLC H1650 cancer cell line was studied. When gefitinib was used to inhibit EGFR phosphorylation, partly diminished Akt activation and exerted no effect on ERK phosphorylation. The treatment was found to induces G0/G1 cell cycle arrest and reduce cell proliferation rate. Apoptotic cell death was triggered by decrease in the level of anti-apoptotic protein Bcl-2, increases the level of pro-apoptotic protein Bax and p53. On the other hand, gefitinib was found to induce minor autophagic response in H1650 cancer cells as shown by acidic vesicular organelles accumulation, protein LC3 conversion and autophagosome formation. Bafilomycin A1 or 3-methyladenine was used to inhibit the autophagy response induced by gefitinib. Both autophage inhibitors render H1650 cancer cell prone to gefitinib-induced apoptosis. The results indicate that autophagy plays a protective role in H1650 cells preventing apoptotic cell death induced by gefitinib. II. An anticancer compound was isolated from medicinal mushroom Ganoderma colossum (G. colossum). The chemical structure was identified and characterized to be Schisanlactone A which was first isolated from a Schisandra sp. in 1983. Anti-HIV protease activity of the compound is the only bioactivity had been reported so far. G. colossum is a rare species of Ganodermataceae family mushroom. The bioactivity of G. colossum and the bioactive compounds in G. colossum have rarely been reported. In this study, ethanol extraction, ethyl acetate partition, silica gel column chromatography and high performance liquid chromatography were used to isolate schisanlactone A from G. colossum. Schisanlactone A was found to inhibit growth and induce cell death in A549 lung cancer cells with IC50= 2.4 μM (72-h). In A549 cells, schisanlactone A induces G0/G1 cell cycle arrest, CDK2 was up-regulated and cyclin D1 remained unchanged. Schisanlactone A was found to activate caspase-3 to mediate cell death, but increases in Bcl-2, an antiapoptotic protein, and reduces in p53 protein, an proapoptotic protein were observed. The mechanism of cell death induced by schisanlactone A needs further study.