臺灣博碩士論文加值系統

Azatyrosine﹝L-b-(5-hydroxy-2-pyridyl)-alanine﹞，一種由Streptomyces chibanensis 中所分離而得的抗生素，能夠抑制已活化之人類 c-Ha-ras、c-rad，c-ErbB-2 致癌基因所引發的轉型NIH3T3細胞(transformed NIH3T3 cells)的生長。然而，azatyrosine的主要缺點是必須在高劑量下才有抗癌作用，因此台大及長庚大學的王惠珀教授將其結構經過修飾並合成一系列azatyrosine醯胺類衍生物(HPW 98系列)，以增加進入細胞內的能力。本實驗室先前結果已證明，azatyrosine類似物98-1與98-2的抗癌活性遠比azatyrosine強(IC50較低)。也發現HPW 98-1與HPW 98-2的作用機制為抑制DNA與蛋白質的合成，而在蛋白質合成上，HPW 98-1與HPW 98-2不會與tyrosine競爭結合至蛋白質上。利用NIH3T3-wt或NIH3T3-ras或SW480細胞來篩選azatyrosine衍生物，其IC50約為10-500 mM之間，比起azatyrosine要低了很多。另外，利用細胞毒殺試驗及SW480、colon #3或colon #8細胞，用HPW 98-13與5-FdUR合併處理發現藥效有相加的作用，但無相成的效果。T24或Paca-2細胞以HPW 98-13與doxorubicine合併處理，發現藥效有些相成的作用。在遺傳毒理(genotoxicity assay)方面，利用ouabain來篩選，發現代表藥物HPW 98-13的突變率都與自然突變率相差不多，因此推測HPW 98-13很可能不是一致突變劑。經由mitotic index得知HPW 98-13會使M時期的細胞增加。在Balb/c小鼠的動物模式研究方面，Balb/c小鼠皮下(s.c.)打入NIH3T3/7-4 (ras over- expression) (5x106/mouse)，兩天後開始每天腹內(i.p.)施打HPW 98-1，發現HPW 98-1雖不能完全抑制，但卻可以部分抑制腫瘤的生長。另一種動物模式，亦即Balb/c小鼠以i.p.投與NIH3T3/7-4細胞，小鼠於14天內因內臟腐爛導致死亡，因此放棄此一模式。我們曾嘗試用一個新的方法─藥物-西方法(Drug- Western)─來尋求細胞內用來結合HPW98衍生物的特殊蛋白質─標的分子(target molecule)。但可能因為使用的cDNA基因庫不表現HPW 98的標的分子或表現過於微弱，以致於偵測不出來。

Azatyrosine [L-b-(5-hydroxypyrid-2-yl)-alanine], a chemical isolated from Streptomyces chilanesis, has been reported to inhibit the growth of c-Ha-ras-, c-raf- and c-erb-2-transformed NIH3T3 cells. The chemical also inhibits 7,12-dimentyl-benz [a]- anthracene-induced carcinogenesis in mice. A series of azatyrsoine analogues, modified to be more lipophilic, were tested for their anticancer activities. The analogues, termed the HPW 98 series, exhibit IC50 values lower than that of azatyrosine in magnitudes of 104 to 105, affecting cell cycle and inducing apoptosis in a few cancer cell lines tested. HPW 98-1 and HPW 98-2 inhibit DNA and protein synthesis, but do not compete with tyrosine for incorporation into protein. The IC50 of azatyrosine analogues were between 10-500 mM and were lower than that of azatyrosine in NIH3T3-wt or NIH3T3-ras or SW480 cells. In drug combination studies where SW480, colon #3 and colon #8 cells were treated with the concurrent presence of HPW 98-13 and 5-FdUR, the drugs showed additive effects, but not synergistic effects on all cell lines treated. However, a weak synergistic effect was observed when T24 and Paca-2 cells were treated with HPW 98-13 and doxorubicine. In a genotoxicity assay, using V79 cells and ouabain as a mutation marker, the mutation frequency induced by HPW 98-13 was found to be similar to the spontaneous mutation frequency. HPW 98-13 therefore is very probably not a mutagen. HPW 98-13 increases the mitotic index of V79 cells, indicating that HPW 98-13 may arrest NIH3T3 cells at the G2/M phase. We also tried to establish an animal model for testing the antitumor activity of HW 98-1. The best result was found when Balb/c mice were subcutaneously injected with NIH3T3/7-4 cells (5x106cells/mouse) and two days later intrapertonealy administered by HPW 98-1 everyday. The drug inhibited the growth of the tumor cells. We have also tried to identify the molecular target(s) of the analogues by a method entitled Drug-Western that was reported to ensure quick identification of cDNAs of the drug targets. But we failed, due probably to the nonexpression or too weak of the expression of target protein(s) by the cDNA library used.

第一章 研究背景………………………………………………………..1
第二章 研究目的………………………………………………………8
第三章 實驗材料………………………………………………………10
第一節 細胞株……………………………………………………10
第二節 試劑……………………………………………………….10
第四章 實驗方法………………………………………………………13
第一節 細胞培養…………………………………………………13
第二節 離體細胞毒性……………………………………………15
第三節 遺傳毒性測試……………………………………………18
第四節 合併治療…………………………………………………24
第五節 細胞有絲分裂指標………………………………………27
第六節 西方藥物法………………………………………………29
第七節 動物實驗…………………………………………………35
第五章 實驗結果………………………………………………………37
第一節 離體細胞毒性的分析……………………………………37
第二節 遺傳毒性的分析………………………………………...37
第三節 離體細胞合併使用藥物的研究…………………………38
第四節 細胞絲狀分裂的指標…………………………………….39
第五節 以西方藥物法尋找HPW 98-13之標的蛋白質……….…40
第六節 動物實驗………………………………………………….40
第六章 討論……………………………………………………………42
參考文獻…………………………………………………………………..46

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黃秋芬著，Azatyrosine類似物抗癌活性機制之研究，國立成功大學藥理學研究所，碩士論文 (1999)。