| 摘要: | 之前的研究報告顯示HMJ-38是一種具有抑制細胞微管蛋白聚合作用的2-phenyl-4-quinazolinones 結構衍生物，發現其能有效抑制許多人類腫瘤細胞株之生長。 本論文主要之研究目的，在探討HMJ-38對HL-60細胞株之毒殺作用與抗癌機制，並探討HMJ-38對WEHI-3血癌細胞所誘發之BALB/c血癌病變小鼠之抗癌活性。 我們首先研究HMJ-38 對血癌細胞株 (HL-60，U937與K562)及人類正常週邊單核球(PBMC) 之細胞存活率、細胞週期與細胞凋亡的影響。 HL-60，U937與K562細胞經HMJ-38處理24小時後，細胞存活率呈現劑量與時間依存性的抑制，其50 % 抑制生長濃度 (IC50) 分別為 4.48、5.06與8.87??M。 HMJ-38 對人類正常週邊單核球於24小時或48小時細胞毒殺作用顯著地低於HL-60細胞。 細胞免疫螢光染色分析發現 HMJ-38與秋水仙素一樣具有影響細胞微管蛋白質聚合作用。 細胞週期分析顯示HMJ-38明顯地誘導HL-60細胞停滯於G2/M 期後，進而誘導細胞凋亡。 細胞處理24小時後，HMJ-38會影響CDK1/cyclin B蛋白質活性，並促使Chk1、Wee1、 p21等蛋白質量增加，及Cdc25C 蛋白質量降低。 我們進一步藉由細胞型態觀察與DNA電泳分析證實HMJ-38具有誘導細胞凋亡之作用。 HMJ-38誘導細胞凋亡主要是藉由粒線體Cytochrome c 的釋放，Bcl-2與Survivin 蛋白質量的降低，Bax與Bak 蛋白質量的提升，Caspase-9、-3及Bid 蛋白質的活化，與PARP 的裂解。 由於Caspase專一性抑制劑能抑制 Caspase 的活性及細胞凋亡，顯示HMJ-38誘導細胞凋亡主要是經由活化Caspase-9及 Caspase-3路徑。 HMJ-38能明顯地誘導HL-60之ERK活化。細胞經 ERK的專一性抑制劑 U0126與PD98059 前處理後，不僅會減低HMJ-38活化 ERK，也會降低細胞凋亡百分比，但是對G2/M 期的停滯現象則不影響。 在進行HMJ-38的活體抗癌測試前，我們先確認HMJ-38對WEHI-3小鼠血癌細胞的毒殺作用，其IC50為5.00 ?M。 並且藉由細胞週期分析、細胞外型觀察與DNA膠體電泳分析的結果，證實HMJ-38也會誘導WEHI-3之G2/M 期停滯，繼而誘導細胞凋亡。 WEHI-3 (1X106細胞) 以尾部靜脈注射組織抗原同源 (MHC syngeneic) 之 BALB/c 小鼠體內，14-28天後成功地誘導小鼠產生血癌。 我們於小鼠注射WEHI-3細胞以誘發血癌的第14天時，再將小鼠分組，分別以靜脈注射HMJ-38 (2.5 mg/kg/Q2d) 連續7次給藥，或以Taxol (5 mg/kg/day) 連續7天給藥。 在一個月的動物實驗觀察中，HMJ-38 與Taxol均能增加致癌小鼠的存活率與體重，且顯著地降低致癌小鼠脾臟、淋巴結和肝臟重量至正常範圍以及抑制癌細胞轉移至肝臟的現象。 以H-E染色的脾臟切片的結果顯示HMJ-38與Taxol均能降低致癌小鼠之脾臟紅漿區侵潤的未成熟血癌細胞數。 以流式細胞儀分析小鼠週邊白血球之細胞族群顯示，HMJ-38與Taxol均能顯著地增加致癌小鼠白血球中CD19+ 的細胞族群，並且降低CD14+和 Mac-3+的細胞族群至與正小鼠相同的程度。 綜合以上結果，HMJ-38具有抑制細胞週期進行與誘導細胞凋亡之作用，並且對WEHI-3/BALB/c 血癌小鼠具顯著抗癌作用，因此我們認為HMJ-38 是一個深具潛力之抗癌藥物。; We previously reported that HMJ-38 was the most potent 2-phenyl-4-quinazolinone derivative in inhibiting tubulin polymerization and showed significant cytotoxicity against several human tumor cell lines. In this study, we studied its cytotoxic effect on HL-60 leukemia cells and underlying mechanisms and investigated the anticancer activity of HMJ-38 in syngeneic BALB/c mice bearing murine WEHI-3 leukemia cells. We first investigated the effects of HMJ-38 on viability, cell cycle and induction of apoptosis in leukemia cell lines (HL-60, U937 and K562) and normal human peripheral blood mononuclear cells. After 24 hours of treatment with HMJ-38, concentration- and time-dependent decreases in the viability of HL-60, U937 and K562 cells were observed and the IC50 value were estimated to be 4.48, 5.06 and 8.87 ?M. The cytotoxic effect of HMJ-38 on PBMC was less significant than that on HL-60 cells after 24 or 48 hours of treatment. Immunocytochemistry staining of ?-tubulin demonstrated that, which was also observed in colchicine-treated cells, HMJ-38 inhibited the polymerization of microtubules. Cell cycle analysis showed that HMJ-38 induced significant G2/M arrest and apoptosis in HL-60 cells. The HMJ-38 induced-G2/M arrest occurred before the onset of apoptosis. Within 24 hours of treatment, HMJ-38 affected the CDK/cyclin B activity by increasing Chk1, Wee1 and p21, and decreasing Cdc25c protein levels. The HMJ-38-induced apoptosis was further confirmed by morphological assessment and DNA fragmentation assay. Induction of apoptosis in HMJ-38-treated HL-60 cells was accompanied by an apparent increase of cytosolic cytochrome c, down-regulation of Bcl-2 and survivin, up-regulation of Bax and Bak, and cleavage of Bid, pro-caspase-9, -3, and poly(ADP)ribosylpolymerase (PARP). Results of the significant reduction of caspase activities and apoptosis by caspase inhibitors indicated that the HMJ-38-induced apoptosis was mainly mediated by activation of caspases-9 and -3. HMJ-38 also activated ERK in HL-60 cells. Pre-incubating cells with ERK inhibitors (U0126 and PD98059) attenuated the HMJ-38 induced ERK activation and apoptosis. Nevertheless, cells remained arrested in G2/M. Before performing animal studies, we confirmed that HMJ-38 concentration-dependently decreased viability of WEHI-3 cells with an IC50 of 5.0 ?M. HMJ-38-induced G2/M arrest companioned |
