| Abstract: | 人類流行病學的研究顯示，N-乙醯轉移（N-acetyltransferase ，NAT）的活性和膀胱癌及大腸直腸癌的發生率有相關性。本篇研究是利用paclitaxel在人類骨癌細胞株（U-2 OS）中，檢測是否抑制NAT的活性、基因的表現和AF-DNA鍵結衍生物的形成。NAT的活性是以高效液相層析儀（high performance liquid chromatography, HPLC）來檢測乙醯化的AF（2-aminofluorene）和PABA（p-aminobenzoic acid）以及未乙醯化的AF和PABA所得。人類骨癌細胞（U-2 OS）株的細胞液和完整細胞，被用來檢測NAT的活性、基因的表現和AF-DNA鍵結衍生物的形成。結果顯示在人類骨癌細胞株（U-2 OS）中的NAT的活性、檢測細胞中NAT的百分比、基因的表現和AF-DNA鍵結衍生物的形成，有被paclitaxel所抑制而降低，而且呈現跟劑量相關。同時也顯示paclitaxel，明顯降低人類骨癌細胞株（U-2 OS）中的Km和Vmax值，是故paclitaxel對NAT是一種無競爭性的抑制者。
N-乙醯化被認為是芳香胺類（arylamine）代謝的第一步，而這是被一種所謂的芳香胺類NAT所完成。NAT是利用乙醯輔A，來當乙醯基的供應者。而paclitaxel曾經被研究顯示具有抗腫瘤和抗癌的活性。本篇研究是用paclitaxel在人類血癌細胞株（HL-60）中，檢測是否抑制NAT的活性、基因的表現和AF-DNA鍵結衍生物的形成。結果顯示paclitaxel（0.01-1μM）確實降低NAT mRNA的的表現，而且呈現跟劑量相關。paclitaxel在人類血癌細胞株（HL-60）中，也抑制NAT的活性和AF-DNA鍵結衍生物的形成，而且呈現跟劑量相關。同時，paclitaxel明顯降低人類血癌細胞株（HL-60）中的Km和Vmax值。所以，paclitaxel對NAT可能是一種無競爭性的抑制者。本篇報告是第一個描述paclitaxel影響人類血癌細胞株（HL-60）中NAT的活性、基因的表現和AF-DNA鍵結衍生物的形成。
Paclitaxel是一種從太平洋紫杉樹皮分離出來的成分，被發現在人類骨癌細胞株（U-2 OS）具有抑制NAT活性和基因表現及鍵結衍生物的合成。目前為止，只有一篇文獻告有關paclitaxel對人類骨癌細胞株（U-2 OS）具有抑制細胞生長和細胞週期停止在G2/M期。我們知道細胞凋亡的起始，牽涉很多重要的調節蛋白。為了更進一步了解paclitaxel對人類骨癌細胞株（U-2 OS）之細胞週期的影響及細胞凋亡，我們利用流式細胞計數儀來分析。我們發現：（1）paclitaxel引起人類骨癌細胞株（U-2 OS）的細胞週期停止在G2/M期；（2）paclitaxel引起人類骨癌細胞株（U-2 OS）的細胞凋亡，且和時間與劑量呈現相關：（3）加入caspase-3的抑制劑z-VAD-FMK，paclitaxel引起人類骨癌細胞株（U-2 OS）的細胞凋亡和caspase-3的活性，比不加入的顯著降低。由此可知，paclitaxel會引起人類骨癌細胞株（U-2 OS）的細胞週期停止在G2/M期以及細胞凋亡，而這其中經過caspase-3的活化。
Paclitaxel被發現在人類血癌細胞株（HL-60）具有抑制細胞生長和細胞週期停止在G2/M期。我們知道細胞凋亡的起始，牽涉很多重要的調節蛋白。為了更進一步了解paclitaxel對人類血癌細胞株（HL-60）之細胞週期的影響及細胞凋亡，我們利用流式細胞計數儀來分析。我們發現：（1）paclitaxel引起人類血癌細胞株（HL-60）的細胞週期停止在G2/M期；（2）paclitaxel引起人類血癌細胞株（HL-60）的細胞凋亡，且和時間與劑量呈現相關：（3）加入caspase-3的抑制劑z-VAD-FMK，paclitaxel引起人類血癌細胞株（HL-60）的細胞凋亡和caspase-3的活性，比不加入的顯著降低。由此可知，paclitaxel會引起人類血癌細胞株（HL-60）的細胞週期停止在G2/M期以及細胞凋亡，而這其中經過caspase-3的活化。
研究探討paclitaxel在SD大白鼠體內AF的N-乙醯化情形和AF-DNA鍵結衍生物的分佈狀況。體內實驗開始，SD大白鼠先給于paclitaxel（50 mg/kg）處理48小時後，再給于AF（50 mg/kg），發現在尿及大便中分別減少28%和43%的AAF復原，也就是減少22%從AF到AAF的代謝清除率。paclitaxel不會影響NAT在血液，肝臟，肺臟，大腸和膀胱活性的Michaelis-Menten參數值。同樣的，AF在各個測試組織中的Km值不被paclitaxel所影響。然而，肝臟之NAT活性，在被paclitaxel處理過後的Vmax值有明顯降低。接受AF的SD大白鼠，不管有沒有paclitaxel之前處理，檢測標的組織中（如：肝臟、大腸和膀胱），以及非標的組織中（如：肝臟和循環中的白血球）之AF-DNA鍵結衍生物，發現被paclitaxel前處理過的，在各個組織中的AF-DNA鍵結衍生物都被降低。這是我們首先發現paclitaxel在SD大白鼠，會影響AF的分佈和N-乙醯化以及DNA鍵結衍生物的體內試驗。
在SD大白鼠體內檢測paclitaxel是否會影響化學致癌物AF的分佈和代謝。以高效液相層析儀偵測AF、乙醯化的AF和AF代謝物。只給于AF（對照組）、AF和paclitaxel同時給于、paclitaxel前處理24小時後再給于與AF三組，經過24小時，尿、大便和組織，如：肝臟、腎臟、胃、大腸、膀胱和血液，被收集來分析AF和它的代謝物。比較對照組，paclitaxel增加尿和大便中AF代謝物的排泄。而尿和大便中最主要的排泄代謝物是9-OH-AAF。肝臟是最主要的代謝中心，而在肝臟中最主要的殘留代謝物也是9-OH-AAF。
Human epidemiological studies suggest an association between the N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assaying for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. U-2 OS cell cytosols and intact cells were used for examining the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that the NAT activity, percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in U-2 OS cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact U-2 OS cells. Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.
N-acetylation is recognized as the first step in arylamine metabolism. The enzyme responsible for N-acetylation is called arylamine NAT which uses acetyl coenzyme A as the acetyl group donor. Paclitaxel has been shown to exhibit antineoplastic and anticancer activity. In this study, paclitaxel was selected to determine the inhibition of arylamine NAT activity, gene expression (NAT mRNA) and DNA-AF adduct formation in human leukemia HL-60 cell line. Paclitaxel (0.01-l μM) did decrease the level of NAT mRNA in a dose dependent manner. The results demonstrated that paclitaxel inhibited NAT activity and DNA-AF adduct formation in HL-60 cells in a dose-dependent manner. Using standard steady-state kinetic analysis, it was demonstrated that paclitaxel was a possible uncompetitive inhibitor to NAT activity in cytosols based on the decrease in apparent values of Km and Vmax. This report is the first demonstration which showed paclitaxel affected HL-60 cells NAT activity and DNA-AF adduct formation.
Paclitaxel, an isolated component from the bark of the Pacific yew Taxus brevifolia, was found to inhibit NAT activity, gene expression and AF-DNA adduct formation in U-2 OS cells. There was only one report regarding the inhibition of cell growth and cell cycle progression mainly by inducing arrest of the G2/M phase in U-2 OS cells. Several key regulatory proteins involved in the initiation of apoptosis. To obtain information regarding cell cycle arrest and apoptosis induced by paclitaxel, we examined its effect on the regulating factors of cell cycle arrest and apoptosis in U-2 OS cells treated with paclitaxel by flow cytometry and Western blotting. Our observations were: (1) paclitaxel induced cell cycle arrest of the G2/M phase in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) z-VAD-FMK (caspase-3 inhibitor) inhibited U-2 OS cells apoptosis and caspase-3 activation that were induced by paclitaxel. These results suggest that paclitaxel can induce cell cycle arrest of the G2/M phase and apoptosis via caspase-3 activity in U-2 OS cells.
Paclitaxel was found to inhibit cell growth and cell cycle progression mainly by inducing arrest of the G2/M phase in HL-60 cells. Several key regulatory proteins involved in the initiation of apoptosis. To obtain information regarding cell cycle arrest and apoptosis induced by paclitaxel, we examined its effect on the regulating factors of G2/M phase and apoptosis in HL-60 cells treated with paclitaxel by flow cytometry and Western blotting. Our observations were: (1) paclitaxel induced cell cycle arrest of the G2/M phase in HL-60 cells; (2) time and dose dependent apoptosis of HL-60 cells was induced by paclitaxel; (3) z-VAD-FMK (caspase-3 inhibitor) inhibited HL-60 cells apoptosis and caspase-3 activation that were induced by paclitaxel. These results suggest that paclitaxel can induce cell cycle arrest of the G2/M phase and apoptosis via caspase-3 activity in HL-60 cells.
Paclitaxel was selected for investigating the effects of the in vivo distribution and the levels of N-acetylation of AF and AF-DNA adducts in Sprague-Dawley rats. For in vivo examination, pretreatment with paclitaxel (50 mg/kg) 48 hr prior to the administration of AF (50 mg/kg) resulted in a 28% and 43%, decrease respectively in the urinary and fecal recovery of N-acetyl-2-aminofluorene (AAF), and a 22% decrease in the metabolic clearance of AF to AAF. Paclitaxel did not affect Michaelis-Menten parameters for NAT activity in blood, liver, lung, colon, and bladder. Similarly, the Km for AF in the examined tissues was not affected by the paclitaxel. However, the Vmax estimate of liver NAT activity was significantly decreased after paclitaxel pretreatment. Following exposure of rats to the AF with and without pretreatment with paclitaxel, DNA-AF adducts were examined in the target tissue liver, colon and bladder, and also in non-target tissue lung and circulating leukocytes. The DNA-AF adducts in liver, bladder, lung, colon, and leukocytes were decreased by pretreatment with paclitaxel. This is the first finding to show paclitaxel affecting AF distribution and N-acetylation and DNA adduct in SD rats in vivo.
To evaluate the question of whether or not paclitaxel affects the distribution and metabolism of chemical carcinogens such as AF on SD rats were examined. The AF, acetylated AF and AF metabolites were determined and examined by using HPLC. After received AF only, AF with paclitaxel at the same time and paclitaxel pretreated for 24 hours then treated with AF for 24 hours, urine, stool and tissues such as liver, kidneys, stomach, colon, bladder and blood were collected and assayed for AF and its metabolites. Compared to the control group, paclitaxel caused an increase of the metabolites excreted in urine and stool. The major metabolite excreted in urine and stool was 9-OH-AAF. The liver is the major metabolism center and the major residual metabolite of AF in the liver was also 9-OH-AAF. |
