臺灣博碩士論文加值系統

微小管是抗癌藥物的一個重要作用標的，經由萃取分離台灣扁柏心材，我們得到一個新穎的抗微小管化合物Chamaecypanone C。經由化學合成我們得到一系列4-aroyl-6,7,8-trimethoxyquinolines的抗微小管化合物，並評估它們的抑癌作用。
Chamaecypanone C 經由結合到微小管的秋水仙素結合部位，影響微小管的聚集，導致細胞停留在細胞週期的M期。 Chamaecypanone C在各種癌細胞抑癌IC50 介於nM等級。藉由流式細胞儀的分析，我們比較不同濃度chamaecypanone C對口腔上皮癌細胞KB之影響，發現在引發細胞死亡前，越高濃度的chamaecypanone C，其造成KB細胞停留於G2-M 期的細胞數目也越多。Chamaecypanone C造成細胞週期停留在G2-M 期， 主要跟 cyclin B1的增加， Cdc2的去磷酸化及Cdc25C的磷酸化有相當程度的關聯，由於M期的標記MPM-2之呈現，顯示Chamaecypanone C主要是讓細胞停留在M 期。值得注意的是Chamaecypanone C造成的細胞凋亡主要是經由caspase8-Fas/FasL 這個途徑，而不是mitochondria/caspase 9 這個途徑。此外，我們也發現Chamaecypanone C在過度表現P-gp170/MDR 和 MRP的多重抗藥性細胞亦具相同的抑癌作用。
以combretastatin A-4的化學結構為基礎，我們合成一系列2-aroyl, 2-aryl-5,6,7-trimethoxyquinolines和 4-aroyl-6,7,8-trimethoxyquinolines的化合物，並評估其抗癌作用，其中先導藥物 4-aroylquinoline (化合物11)，在各種不同人類的癌細胞，口腔上皮癌細胞KB、結腸直腸癌細胞HT29和胃癌細胞MKN45，表現最佳的的抑癌作用，它的抑癌IC50 分別為 217、327和239 nM，另外4-aroylquinoline(化合物11)在產生多重抗藥性的癌症細胞株 KB-vin10、 KB-S15和 KB-7D，亦具同等的抑癌作用，它的抑癌IC50 分別為246、213和252 nM。
綜合我們的研究發現，Chamaecypanone C 和 4-aroylquinoline(化合物11)這兩個抗癌化合物，具有治療各類癌症的潛質，特別是在產生抗藥性的病人族群，亦有良好的抑癌作用，值得做進一步研究發展其成為新穎微小管抑制藥物。

Microtubule is a popular target for anti-cancer drugs. A novel antitubulin compound chamaecypanone C was isolated from the heartwood of Chamaecyparis obtusa var. Formosan. In addition, a series of 4-aroyl- 6,7,8-trimethoxyquinolines antitubulin compounds were synthesized and evaluated for anticancer activity.
Chamaecypanone C induces mitotic arrest through binding to the colchicine-binding site of tubulin, thus preventing tubulin polymerization. Cytotoxic activity of Chamaecypanone C in a variety of human tumor cell lines has been ascertained, with IC50 values in nanomolar ranges. Flow cytometric analysis revealed that chamaecypanone C treated human KB cancer cells were arrested in G2-M phases in a time-dependent manner before cell death occurred. Additional studies indicated that the effect of Chamaecypanone C on cell cycle arrest was associated with an increase in cyclin B1 levels and a mobility shift of Cdc2/Cdc25C. The changes in Cdc2 and Cdc25C coincided with the appearance of phosphoepitopes recognized by a marker of mitosis, MPM-2. Interestingly, this compound induced apoptotic cell death through caspase8-Fas/FasL dependent pathway, instead of mitochondria/caspase9 dependent pathway. Notably, several KB-derived multidrug- resistant cancer cell lines overexpressing P-gp170/MDR and MRP were sensitive to Chamaecypanone C.
A series of 2-aroyl, 2-aryl-5,6,7-trimethoxyquinolines and 4-aroyl- 6,7,8-trimethoxyquinolines based on CA 4 chemical structure were also synthesized to discover novel microtubule inhibitor in this study, and the lead compound 4-aroylquinoline 11 inhibited the growth of the human cancer cells lines KB, HT-29, and MKN45, as well as three human-resistant cancer cell lines of KB-vin10, KB-S15, and KB-7D, with an IC50 of 217, 327, 239, 246, 213, and 252 nM, respectively.
In conclusion, our findings indicate that Chamaecypanone C and 4-aroylquinoline 11 are promising anticancer compounds that have potential for management of various malignancies, particularly for patients with drug resistance. The antitubulin profiles of these two compounds make them interesting compounds for further development.

Abstract…………………………………………………………………………V
Chinese Abstract………………………………………………………………VII
Introduction……………………………………………………………………… 1
Materials and Methods………………………………………………………… 6
Chemistry ……………………………………………………………………… 6
Purification of Chamaecypanone C………………………………………6
Synthesis of 4-aroyl-6,7,8-trimethoxyquinolines.……………………………… 8
Reagents ………………….………………………………………………….… 15
Biological Assays………………………………………………………………16
Cell Cultures………………….…………………………………………………16
Growth Inhibition Assay………………………………………………………17
Cell Cycle Analysis …………………….……………….……….…..…………18
Western Blot Analysis……………………………………………………………18
Microscopic Assessment of Mitotic Index……………………………………… 19
In Vitro Microtubule Assembly Assay………………………………………… 20
In Vivo Microtubule Assembly Assay………………………………………… 20
Immunocytochemistry……………………………………………………..…… 21
Time-lapsed Video Microscopy………………………………………………… 21
Tubulin Competition-Binding Scintillation Proximity Assay………………… 22
Annexin-V/PI Binding Assay…………………………………………….…… 23
Determination of Caspases Activity………………………………….………… 23
Caspase Inhibitory Assay…………………………………………………… 24
FasL Neutralization Assay……………………………………………………… 24
Statistical Analysis……………………………………………………………… 25
Results……………………………………………………………………………26
Chamaecypanone C Induced Growth Inhibition in Human Cancer Cell Lines…26
Chamaecypanone C Induced G2-M Phase Arrest and Subsequent Apoptotic
Cell Death……………………………….………………………………………26
Chamaecypanone C-induced Changes in the Expression and Phosphorylation
of G2-M Regulators………..………..……………………….............………… 27
Chamaecypanone C Binds to the Colchicine-binding Site of Tubulin and Inhibits Microtubule assembly…………………………………………………………..28
Chamaecypanone C Induces Apoptotic Cell Death through Caspase8-Fas/FasL Dependent Pathway……….…….…………………………,………….….…… 31
Chamaecypanone C is Cytotoxic toward Multi-drug resistant Cancer Cell Line...33
Synthesis of 6,7,8-trimethoxyquinolines and 5,6,7-trimethoxyquinolines………34
Growth Inhibition of various trimethoxy-substituted heterocycles on the Cancer Cells…………….…………………………………………………….……..36
Compound 11 is Cytotoxic toward Multi-drug resistant Cancer Cell Line……… 37
Compound 11 slightly Binds to the Colchicine-binding Site of Tubulin….….… 37
Discussion and conclusion……………………………………............60
Reference……………………………………………..……..……………………68

Tables

Table 1. Growth inhibition of chamaecypanone C against various human cancer cell lines…………………………………………………………………..…38
Table 2.Growth inhibition of chamaecypanone C against drug-resistant cell lines……………………………………………………….……………39
Table 3. Growth inhibition of compounds 7-13 against various human cancer cell lines…..…………………………...……………………………...….....40
Table 4. Growth inhibition of compounds 11 against drug-resistant cell lines…...41
Table 5. Inhibition of tubulin polymerization and colchicine binding by compound 11……………………………………………………………….…...…42

Figures

Figure A. Natural tubulin polymerization inhibitors…………………..… ……...43
Figure B. Synthetic 4-aroyl-6,7,8-trimethoxyquinolines, 2-aroyl- and
2-aroyl-5,6,7-trimethoxyquinolines ……………...……………...……44
Figure 1. Chemical structure of chamaecypanone C …….…………….……..…..45
Figure 2. Chamaecypanone C induces G2/M phase arrest before apoptotic cell death occurs………………………………………………………………. ....46
Figure 3. Chamaecypanone C-induced change in G2-M regulators coincides with mitotic arrest occurs in KB cells.…………………………....…………49
Figure 4. Chamaecypanone C inhibits microtubule assembly through binds to the colchicine- binding site of tubulin…………………………...…………51
Figure 5. Chamaecypanone C induced apoptotic cell death mainly through the caspase8/Fas-FasL cascade……………..…………………..……….....56

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