合成的I6、YJC-1與HKL-1對人類血癌細胞HL-60抗癌機制之探討; Studies on the anticancer mechanisms of synthetic I6, YJC-1 and HKL-1 in human leukemia HL-60 cells

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題名:	合成的I6、YJC-1與HKL-1對人類血癌細胞HL-60抗癌機制之探討;Studies on the anticancer mechanisms of synthetic I6, YJC-1 and HKL-1 in human leukemia HL-60 cells
作者:	徐美華;Mei-Hua Hsu
貢獻者:	中國醫藥大學：藥物化學研究所博士班
關鍵詞:	抗癌機制;人類血癌細胞株HL-60;細胞週期;細胞凋亡;有絲分裂;anti-cancer mechanism;human leukemia HL-60 cells;cell cycle;cell apoptosis;mitosis
日期:	2007-07-24
上傳時間:	2009-08-13 09:37:15 (UTC+8)
摘要:	本研究目的在於尋求有效之抗癌藥物並探討其抗癌機制。本論文分成三大部分，分別以I6、YJC-1和HKL-1為標的化合物，探討對於HL-60血癌細胞株之抗癌機制。第一部分為I6之抗血癌機制探討。1-(3,4-Dimethoxyphenyl)-3,5- dodecenedione (I6)是本研究團隊過去所合成的gingerdione類衍生物，於本實驗中被發現具有不錯抗血癌活性。本研究中發現，I6對HL-60細胞株所造成的生長抑制作用具有時間與濃度依存性，並以一系列實驗闡明I6對血癌HL-60細胞株之抗癌機制。流式細胞儀分析DNA變化發現，I6造成細胞週期停滯於G0/G1期並伴隨細胞凋亡產生。反轉錄酵素-聚合酵素連鎖反應(RT-PCR)檢測相關細胞週期調控因子變化發現，p15與p27的表現量增加而cyclin D2、cyclin E與cdc25A的表現量下降。型態觀察與DNA瓊膠電泳分析證實I6具有誘導細胞凋亡作用，並發現I6會引起caspase-3和Bax表現量增加與Bcl-2表現量下降。綜合以上結果得知，I6經由活化CDK inhibitor (p15和p27)與抑制cdc25A，使cyclin D2和 cyclin E表現量下降，進而誘導細胞週期停滯於G1期。爾後，I6還會藉由改變粒線體通透性(Bax/Bcl-2比例增加)而活化caspase-3誘導細胞凋亡。由此可推論，細胞週期停滯於G1期並誘導細胞凋亡，可能是I6抗癌作用之重要機制。本論文之第二部分為YJC-1之抗癌機制探討。2-(3-Fluorophenyl)- 6-methoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (YJC-1)是本所研究團隊過去所合成之2-phenyl-4-quinolone (2-PQ)類緣衍生物。這類衍生物的主要作用為抗有絲分裂。本論文中發現，YJC-1對於HL-60、K562、A549、CH27、NCI-H226、HA22T、Hep 3B、J5、Colo 205和HeLa等不同癌細胞均造成有絲分裂停滯及細胞凋亡，其中有絲分裂停滯情形以A549肺癌細胞最明顯，細胞凋亡情形以HL-60血癌細胞最明顯。於A549細胞模式中得知，YJC-1會造成紡錘體移動方向混亂但不影響微管聚合，因而使得細胞週期停滯於有絲分裂之前期(prophase)。同時，YJC-1使A549與HL-60細胞之細胞週期停滯於M 期，均是經由抑制cdc25C與活化p21而改變CDK1與cyclin B1的表現。同時於HL-60細胞模式中得知，p21活化也會增加Bax活性，進而活化caspase-9與caspase-3，最後導致細胞凋亡。由此可推論，細胞週期停滯於prophase而使有絲分裂停滯不前進而導致細胞凋亡，可能是YJC-1抗癌作用的重要機制。本論文的第三部分為HKL-1之抗癌機制探討。2-(3-methoxy- phenyl)-5-methyl-1,8-naphthyridin-4-one (HKL-1)是本所研究團隊過去所合成之2-phenyl-1,8-naphthyridine-4-one (2-PN)類緣衍生物。這類衍生物的主要作用亦為抗有絲分裂。本論文中發現，HKL-1對於HL-60、 A549、CH27、NCI-H226、MCF-7、HA22T、Colo 205和HeLa等不同癌細胞均具有顯著抗癌活性，其中以HL-60血癌細胞作用最顯著，IC50值為0.044 μM。HKL-1主要經由抑制CENP-E而使紡錘體與中心體間連結斷裂，導致紡錘體失去張力與染色體排列錯亂，而抑制微管聚合，因而細胞週期停滯於有絲分裂中期(metaphase)。另外，HKL-1也會藉由抑制cdc25C與活化p21而改變CDK1與cyclin B1的表現，而使細胞週期停滯於M期。同時p21的活化也會增加Bax的活性，進而活化caspase-9與caspase-3，最後導致細胞凋亡。由此可推論，細胞週期停滯於metaphase而使有絲分裂不完全，進而誘導細胞凋亡的發生，可能是HKL-1抗癌作用的重要機制。 The purpose of this study was to search novel anticancer agents and elucidating their action mechanisms. There were three parts in this thesis in which we elucidated the anticancer mechanisms of I6, YJC-1 and HKL-1 in human leukemia HL-60 cells. In the first part, we showed that the anti-leukemia mechanisms of I6. 1-(3,4-Dimethoxyphenyl)-3,5-dodecenedione (I6), a gingerdione deriva- tive, was synthesized in our laboratory, has been demonstrated to be an effective anti-tumor agent in human leukemia cells. In the present study, we found that I6 could inhibit cell proliferation in the time- and dose- dependent manner in human promyelocytic leukemia HL-60 cells. To investigate the anti-proliferation mechanism of I6, cell cycle analysis was performed. Results showed that I6 induced significant G1 arrest and apoptosis in HL-60 cells. It was proved by the reverse transcriptase- polymerase chain reaction (RT-PCR) analysis of regulatory on G1 arrest that the levels of p15 and p27 increased after treatment and mRNA levels of cyclin D2, cyclin E, and cdc25A were decreased. The I6-induced apoptosis was further confirmed by morphological observation and DNA fragmentation assay. I6-induced apoptosis was accompanied by up- regulation of caspase-3, and down-regulation of Bcl-2. Taken together, these results suggest that up-regulation of CDKI (p15 and p27) and down-regulation of cdc25A to cause suppression of cyclin D2 cyclin E may contribute to I6-mediated cell cycle arrest. Furthermore, the mitochondria permeability changes (the ratio of Bax/Bcl-2 increased) and caspase-3 activation may be the plausible mechanism by which I6 induced apoptosis. These results suggest that I6 is a potent anti-HL-60 drug and possess a significant action on cell cycle before commitment for apoptosis occurrence. In the second part, we showed that the anti-cancer mechanisms of YJC-1. 2-(3-Fluorophenyl)-6-methoxyl-4-oxo-1,4-dihydroquinoline-3- carboxylic acid (YJC-1), a 2-phenyl-4-quinolone (2-PQ) derivative, was synthesized in our laboratory, has been demonstrated to be an effective anti-mitotic agent. In the present study, YJC-1 displayed anti-mitosis and apoptosis effects against variety cancer cells including HL-60, K562, A549, CH27, NCI-H226, HA22T, Hep 3B, J5, Colo 205 and HeLa cells. Among these cancer cell lines, YJC-1 induced most typical mitosis phenotype in A549 lung cancer cells, and most typical apoptosis phenotype in HL-60 leukemia cells. In A549 cells, YJC-1 induced the spindle poles not oriented on opposite side of chromosomes, but did not affect the microtubule polymerization, thus the cell cycle progression stopped at early mitosis (prophase). In the meanwhile, YJC-1 suppressed cdc25C and activated p21 to change the expression levels of CDK1 and cyclin B. In HL-60 cells, YJC-1 also activated p21 to increase Bax expression to induce caspase-9 and caspase-3 cleavage, thus apoptosis occurred. Taken together, it is suggested that YJC-1 induced cell cycle arrest at prophase and apoptosis might be a crucial response to its anticancer effect. In the third part, we showed that the anti-leukemia mechanisms of HKL-1. 2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4-one (HKL- 1), a 2-phenyl-1,8-naphthyridine-4-one (2-PN) derivative, was synthe- sized in our laboratory, has also been demonstrated to be an effective anti- mitotic agent. In the present study, HKL-1 displayed significantly anti-cancer activities against variety cancer cells including HL-60, A549, CH27, NCI-H226, MCF-7, HA22T, Colo 205 and HeLa cells. Among these cancer cell lines, YJC-1 induced most anti-cancer activity in HL-60 leukemia cells, with an IC50 value approximate to 0.0441 μM. HKL-1 inhibited the activity of CENP-E to cause spindle-pole fragmentation, loss of chromosome alignment during congression and reduced spindle tension, to inhibit microtubule polymerization, thus the cell cycle progression stopped at middle mitosis (metaphase). In addition, HKL-1 suppressed cdc25C and activated p21 to down-regulated the expression CDK1 and cyclin B. p21 activated also enhanced the activity of Bax to induce caspase-9 and caspase-3 cleavage, thus apoptosis occurred. Taken together, it is suggested that HKL-1 induced cell cycle arrest at metaphase and apoptosis might be a crucial response to its anticancer effect.
顯示於類別:	[藥物化學研究所] 博碩士論文

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