Chi Fu Hung

Berberine inhibited arylamine N-acetyltransferase activity and gene expression and DNA adduct formation in human malignant astrocytoma (G9T/VGH) and brain glioblastoma multiforms (GBM 8401) cells

Studies have demonstrated that berberine exhibits the antineoplastic action in rat model. Rat glial tumor cells also have been shown to have N-acetyltransferase activity. In this study, we reported the effects of berberine on arylamine N-acetyltransferase (NAT) activity, gene expression, and DNA adduct formation in human brain tumor cell lines (G95/VGH and GBM 8401). The activity of NAT (N-acetylation of substrate) was measured and determined by high-performance liquid chromatography (HPLC) assaying for the amounts of acetylated 2-aminofluorene (AF) and nonacetylated AF. Human brain tumor cells (G9T/VGH and GBM 8401) were used for examining NAT activity and gene expression and AF-DNA adduct formation. NAT gene expression was determined by polymerase chain reaction (PCR) for the levels of mRNA NAT in both examined cells lines. The amounts of AF-DNA adducts were also determined and quantities by HPLC. The results demonstrated that NAT activity, levels of mRNA NAT1 and AF-DNA adduct formation in both examined cell were inhibited and decreased by berberine in a dose-dependent manner. The apparent values of Km and Vmax from NAT of both examined cells were also determined with or without berberine cotreatment. The data also indicated that berberine decreased the apparent values of Km and Vmax. These effects also indicate that berberine is a uncompetitive inhibitor.

Inhibition of N-Acetyltransferase activity and DNA–2-Aminofluorene adducts by glycyrrhizic acid in human colon tumour cells

Glycyrrhizic acid (GA) was tested for inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line (colo 205). Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact colon tumour cell cultures. The NAT activity in a human colon tumour cell line was inhibited by GA in a dose-dependent manner in both types of systems examined. The data also indicated that GA decreased the apparent values of K(m) and V(max) of NAT enzymes from human colon tumour cells in both examined systems. The DNA-2-aminofluorene adduct formation in human colon tumour cells were inhibited by GA. This report is the first to demonstrate that GA does inhibit human colon tumour cell NAT activity and DNA adduct formation.

Curcumin inhibited the arylamines N-acetyltransferase activity, gene expression and DNA adduct formation in human lung cancer cells (A549)

It is well known that N-acetyltransferase (NAT) plays an important role in the arylamine metabolism. We analysed the response of A549 human lung cancer cells for N-acetylation of 2-aminofluorene (AF) to curcumin. After curcumin treatment, the NAT activity was examined by HPLC, AF-DNA adduct formation was examined by HPLC, and NAT gene expression by polymerase chain reaction were detected. The NAT activity in the human A549 cells and cytosols was suppressed by curcumin in a dose-dependent manner. The results also demonstrated that gene expression (NAT1 mRNA) in human lung A549 tumor cells was inhibited and decreased by curcumin. After the incubation of human lung A549 tumor cells with AF with or without curcumin co-treatment, the cells were recovered and DNA was prepared and hydrolyzed to nucleotides. The adducted nucleotides were extracted into butanol and analyzation of AF-DNA adducts was done by HPLC. The results also demonstrated that curcumin decreases AF-DNA adduct formation in the human lung A549 tumor cells.

Inhibitory actions of luteolin on the growth and arylamine N-acetyltransferase activity in strains of Helicobacter pylori from ulcer patients

Helicobacter pylori is now recognized as an important cause of type B gastritis, which is strongly associated with gastric and duodenal ulcer disease. H. pylori may be a causative factor in patients with gastric cancer. The growth inhibition and N-acetylation of 2-Aminofluorene (AF) or P-aminobenzoic acid (PABA) by arylamine N-acetyltransferase (NAT) in H. pylori were inhibited by luteolin, a component in herbal medicine. The growth inhibition was based on the changes of optical density (OD) by using a spectrophotometer. The N-acetylation of AF or PABA by NAT from H. pylori were assayed by the amounts of acetylated and non-acetylated AF or PABA in cytosols and intact bacteria of H. pylori by using HPLC. An inhibition of growth on H. pylori demonstrated that luteolin elicited a dose-dependent growth inhibition in the H. pylori cultures. Cytosols and suspensions of H. pylori with or without specific concentrations of luteolin co-treatment showed different percentages of AF or PABA acetylation. The data indicated that there was decreased NAT activity associated with increased levels of luteolin in H. pylori cytosols and suspensions. Using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor to NAT enzyme in H. pylori. This report is the first demonstration to show that luteolin can inhibit H. pylori growth and NAT activity.

Luteolin-inhibited arylamine N-acetyltransferase activity and DNA-2-aminofluorene adduct in human and mouse leukemia cells.

N-Acetyltransferase enzyme is an important enzyme in the first step of arylamine compounds metabolism. Luteolin has been shown to exit antibacterial and antineoplastic activity. The purpose of this present study is to evaluate the question of whether luteolin could affect arylamine N-acetyltransferase (NAT) activity and DNA-2-aminofluorene adduct formation in human (HL-60) and mouse (L1210) leukemia cells. By using HPLC, N-acetylation of 2-aminofluorene was determined. Luteolin displayed a dose-dependent inhibition to cytosolic NAT activity and intact human and mice leukemia cells. Time-course experiments showed that N-acetylation of 2-aminofluorene measured from intact human and mice leukemia cells were inhibited by luteolin for up to 24 hours. Using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA-2-aminofluorene adduct formation in human and mouse leukemia cells were inhibited by luteolin. This report is the first demonstration to show that luteolin affects human and mice leukemia cells NAT activity and DNA-2-aminofluorene on adduct formation.

The effect of paclitaxel on 2-aminofluorene-DNA adducts formation and arylamine N-acetyltransferase activity and gene expression in human lung tumor cells (A549).

In this study, paclitaxel was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity, gene expression and 2-aminofluorene-DNA adduct formation in a human lung tumor cell line (A549). The activity of NAT was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Human lung tumor cell cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity, gene expression (NAT1 mRNA) and 2-AF-DNA adduct formation in human lung tumor cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the values of the apparent Km and Vmax of NAT from human lung tumor cells were also determined in both examined systems. The result also indicated that paclitaxel decreased the apparent values of Km and Vmax from human lung tumor cells in both cytosol and intact cells. Thus, paclitaxel is an uncompetitive inhibitor to NAT enzyme.

Inhibition by Vitamin C of DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumor cells

Abstract Previous studies have already demonstrated the protective role of vitamin C (ascorbic acid) in certain types of cancer. This study reports on the effects of vitamin C on arylamineN-acetyltransferase (NAT) activity and DNA adduct formation in a human bladder tumor cell (T24) line. The activity of NAT was measured using high-performance liquid chromatography (HPLC), by assaying for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and the remaining amounts of AF and PABA. T24 cells were used for examining NAT activity and carcinogen–DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene–DNA adduct formation in T24 cells were inhibited and decreased by vitamin C in a dose-dependent manner. The apparent kinetic parameters (apparent values ofKm and Vmax) from T24 cells were also determined with and without vitamin C cotreatment. The data also indicated that vitamin C decreased the apparent values of Km and Vmax from T24 cells.

Effects of aspirin on arylamine N -acetyltransferase activity and DNA adducts in human bladder tumour cells.

Aspirin (acetylsalicylic acid) was used to determine the inhibition of arylamine N‐acetyltransferase (NAT) activity and DNA adduct formation in a human bladder tumour cell line (T24). The activity of NAT was measured by high‐performance liquid chromatography, assaying for the amounts of N‐acetyl‐2‐aminofluorene and N‐acetyl‐p‐aminobenzoic acid and remaining 2‐aminofluorene and p‐aminobenzoic acid. Two assay systems were used: one with cytosol and the other with intact cells. High‐performance liquid chromatography was also used to analyse for the 2‐aminofluorene–DNA adducts. Intact bladder tumour cells were used. The results demonstrated that NAT activity and 2‐aminofluorene–DNA adduct formation in human bladder tumour cells were inhibited by acetylsalicylic acid in a dose‐dependent manner. The effects of acetylsalicylic acid on the values of the apparent Km and Vmax were also determined in both examined systems. The data also indicated that acetylsalicylic acid decreased the apparent values of Km and Vmax from human bladder tumour cells in both cytosol and intact cells. Copyright

Ellagica acid inhibits arylamine N-acetyltransferase activity and DNA adduct formation in human bladder tumor cell lines (T24 and TSGH 8301)

Abstract The fact that vitamin C (ascorbic acid) exhibits a protective effect in certain types of cancer is well documented. Our previous studies demonstrated that human bladder tumor cell line (T24) has N-acetyltransferase (NAT) activity in cytosols and intact cells. The present studies examined the inhibition of arylamine NAT activity and carcinogen (2-aminofluorene)-DNA adduct formation by ellagic acid (EA) in human bladder tumor cell lines (T24 and TSGH 8301). Two assay systems were performed, one with cellular cytosols (9,000xa0g supernatant), the other with intact bladder tumor cell suspensions. NAT activity and 2-aminofluorene-DNA adduct formation in T24 and TSGH 8301 cells was inhibited by EA in a dose-dependent manner in both systems, i.e., the greater the concentration of EA in the reaction the greater the inhibition of NAT activity (dose-and time-course dependent effects). The data also indicated that EA decreased the apparent Km and Vmax of NAT enzymes from T24 and TSGH 8301 cells in cytosols. NAT activity and 2-aminofluorene-DNA adducts in T24 is higher than in TSGH 8301. This report is the first to demonstrate that EA affects human bladder tumor cell NAT activity.

Effects of ibuprofen on arylamine n ‐acetyltransferase activity in human colon tumor cells

The inhibition of arylamine N ‐acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose‐dependent manner in both systems, i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ibuprofen decreases the apparent Km and Vmax of NAT enzyme from human colon tumour cells in both systems examined. This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.