Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells.

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.

Oral administration of paclitaxel affects the distribution and metabolism of 2-aminofluorene in various tissues of Sprague-Dawley rats.

To evaluate the question of whether or not paclitaxel affects the distribution and metabolism of chemical carcinogens such as 2-aminofluorene (AF) on Sprague-Dawley rats were examined. The AF, acetylated AF and AF metabolites were determined and examined by using high performance liquid chromatography. After having received AF only, AF with paclitaxel at the same time and paclitaxel pretreated for 24 h then treated with AF for 24 h, 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.

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells.

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.

Differential effects of allyl sulfides from garlic essential oil on cell cycle regulation in human liver tumor cells.

In this study, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), which are major organosulfur compounds (OSCs) of garlic, were used as experimental materials to investigate their modulation effects on cell viability and cell cycle in human liver tumor cells (J5). According to the results of cell viability assay, 50 or 100 microM DATS significantly decreased the cell viability as compared with the control (P < 0.05) in dose and time dependent relations. Phenomena of cell number loss, shape deformation and lysis were observed after treatment with 100 microM DATS for 24 h. Cell cycle studies showed that J5 cells were significantly arrested in G2/M phase as the cells were treated with 100 microM DADS, 10, 50 or 100 microM DATS for 24 h (P < 0.05). DATS was more effective in arresting cells in G2/M phase than DADS, and the phenomena of arresting J5 cells in G2/M phase increased obviously in dose and time dependent relations. According to the Western blot analysis, DATS decreased cyclin-dependent kinase (Cdks)-Cdk7 (i.e. Cdc2 activate kinase) protein levels in J5 cells but increased cyclin B1 protein level. The modulation potency to cyclin B1 and Cdk7 expressions was in the order of DATS > DADS > DAS. The modulation potency to cyclin B1 and Cdk7 protein levels increased with increasing in DATS concentration and culture time. In conclusion, DATS might affect cell viability and cell morphological changes in J5 cells and lead cells to be arrested in G2/M phase via controlling the expression of cyclin B1 and Cdk7 in J5 cells, and the controlling action might relate to the sulfuric atom numbers in the structures of all these allyl sulfides.

Inhibition of N-acetyltransferase activity and gene expression in human colon cancer cell lines by diallyl sulfide.

Diallyl sulfide (DAS) is one of the major components of garlic (Allium sativum) and is widely used in the world for food. In this study, DAS was selected for testing the inhibition of arylamine N-acetyltransferase (NAT) activity (N-acetylation of 2-aminofluorene) and gene expression (mRNA NAT) in human colon cancer cell lines (colo 205, colo 320 DM and colo 320 HSR). The NAT activity was examined by high performance liquid chromatography and indicated that a 24 h DAS treatment decreases N-acetylation of 2-aminofluorene in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The NAT enzymes (protein) were analyzed by western blotting and flow cytometry and it indicated that DAS decreased the levels of NAT in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The gene expression of NAT (mRNAT NAT) was determined by polymerase chain reaction (PCR), it was shown that DAS affect mRNA NAT expression in examined human colon cancer cell lines. This report is the first to demonstrate that DAS does inhibit human colon cancer cell NAT activity and gene expression.

Effects of Shao-Fu-Zhu-Yu-Tang on motility of human sperm.

Shao-Fu-Zhu-Yu-Tang (SFZYT) is reportedly beneficial to sperm. In this study, we examined sperm acrosomal activity and serum free radical changes to evaluate the possible mechanism of SFZYT. A clinical study evaluated the sperm count and motility in 36 patients with chronic prostatitis before and after treatment for 60 days. The results revealed a significant increase in sperm motility after treatment as evaluated by computer-assisted semen analysis (17.27 +/- 9.00 versus 28.29 +/- 10.00, p < 0.01). An increase in sperm quantity and quality was observed by count and morphology with a high-powered intravital microscope. To gain an understanding of the mechanisms that caused this effect, we assessed sperm acrosin activity levels before (10.6 micro lu/10(6)) and after medication (28.6 micro lu/10(6)) (p < 0.01). The levels of the free radicals was relatively higher before medication, 2144, compared to a normal value of 780 after medication (p < 0.01). In conclusion, SFZYT increased the motility and quality of human semen and this increase is related to an increase in sperm acrosin activity. SFZYT also works as a sperm antioxidant and antiaging agent.

Quercetin glucuronides inhibited 2-aminofluorene acetylation in human acute myeloid HL-60 leukemia cells.

Our earlier study has demonstrated that following the exposure of rat to the arylamine carcinogen 2-aminofluorene, DNA-2-aminofluorene adducts were found in the target tissues liver, bladder, colon, lung and also in circulating leukocytes (lymphocytes and monocytes). The result also demonstrated that orally treated antioxidants decreased N-acetylation of 2-aminofluorene in target tissues and leukocytes. Therefore, this study investigated whether quercetin glucuronides could affect N-acetylation of 2-aminofluorene in human acute myeloid leukemia HL-60 cells. Evidence is presented here that human leukemia cells are capable of acetylating 2-aminofluorene. Quercetin glucuronides did inhibit 2-aminofluorene acetylation in intact cells. The results also indicated that quercetin glucuronides induced cytotoxicity in dose-dependent manner in the examined human acute myeloid leukemia HL-60 cells.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells.

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

Effects of (-)-menthol on arylamine N-acetyltransferase activity in human liver tumor cells.

To evaluate whether or not (-)-menthol affects arylamine N-acetyltransferase (NAT) activity, we selected human liver tumor cell line (J 5) for examination. By using high performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene (AF) was determined. (-)-Menthol displayed a dose-dependent inhibition to cytosolic NAT activity. Time-course experiments showed that NAT activity measured from intact human liver tumor cells was inhibited by (-)-menthol for up to 24 hrs. But in human liver tumor intact cells, the low doses (0.0032 and 0.032 mM) of (-)-menthol promoted the NAT activity and the high doses (3.2 and 32 mM) of (-)-menthol inhibited NAT activity and the 0.32 mM (-)-menthol did not show any significant differences between control and (-)-menthol treated groups. Using standard steady-state kinetic analysis, it was demonstrated that (-)-menthol was a possible uncompetitive inhibitor (decrease Km and Vmax) to NAT activity in cytosols. This report is the first demonstration which showed (-)-menthol affect on human liver tumor cells NAT activity.

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.

Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells.

The inhibition ofarylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated. By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined. Two assay systems were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions. The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e. the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities. This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.

Effects of berberine on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adduct formation in human leukemia cells.

Berberine is an alkaloid occurring in the plant genera Berberis and Coptis. Although berberine had been demonstrated to have antineoplastic function by inhibiting DNA-synthesis in activated lymphocytes, there is no available information to address berberine affects on human leukemia cell N-acetyltransferase (NAT) activity and 2-aminofluorene (AF)-DNA adduct formation. Thus, berberine was tested for inhibition of arylamine NAT activity and AF-DNA adduct formation in human leukemia cells. The NAT activity was measured by a high performance liquid chromatography assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and the remaining AF and p-aminobenzoic acid (PABA). The NAT activity and AF-DNA adduct formation in human leukemia cells were inhibited by berberine in a dose-dependent manner, i.e. the higher the concentration of berberine, the higher the inhibition of NAT activity and AF-DNA adduct. The data also indicate that berberine decreased the apparent values of Km and Vmax from human leukemia cells in both cytosol and intact cells.

Effects of luteolin on arylamine N-acetyltransferase activity in rat blood and liver.

In this study we investigated inhibition of Arylamine N-acetyltransferase (NAT) activity in rat blood and liver tissue cytosols by luteolin. Using high-performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene and remaining unacetylated 2-aminofluorene were examined. The NAT activity in rat blood and liver tissue was inhibited by luteolin in a dose-dependent manner: higher concentrations of luteolin in the reaction resulted in greater inhibition of NAT activities in both examined tissues. The data also indicated that luteolin decreased apparent Km and Vmax of NAT enzymes from rat blood and liver tissue cytosols. This report is the first demonstration that luteolin can affect rat blood and liver tissue NAT activity.

The stem of sinomenium acutum inhibits mast cell-mediated anaphylactic reactions and tumor necrosis factor-alpha production from rat peritoneal mast cells.

The aqueous extract of Sinomenium acutum stem (SSAE) (0.1-1000 mg/kg) dose-dependently inhibited systemic anaphylactic reaction induced by compound 48/80 in mice. In particular, SSAE reduced compound 48/80-induced anaphylactic reaction with 50% at the dose of 1000 mg/kg. SSAE (100-1000 mg/kg) also significantly inhibited local anaphylactic reaction activated by anti-dinitrophenyl (DNP) IgE. When mice were pretreated with SSAE at a concentration ranging from 0.1 to 1000 mg/kg, the plasma histamine levels were reduced in a dose-dependent manner. SSAE (1-1000 microg/ml) dose-dependently inhibited histamine release from the rat peritoneal mast cells (RPMCs) activated by compound 48/80 or anti-DNP IgE. In addition, SSAE (0.1 microg/ml) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha (TNF-alpha) production. These results indicate that SSAE inhibits mast cell-mediated anaphylactic reactions and TNF-alpha production from mast cells.

Effects of berberine on arylamine N-acetyltransferase activity in human colon tumor cells.

Berberine was used to determine loss of viable cells and inhibition of arylamine Nacetyltransferase (NAT) activity in a human colon tumor (adenocarcinoma) cell line. The viable cells were determined by trypan blue exclusion under a light microscope. The NAT activity was measured by high performance liquid chromatography for the amounts of N-acetyl-2-aminofluorene (AAF), N-acetyl-p-aminobenzoic acid (N-Ac-PABA), and the remaining 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). The viability and NAT activity in a human colon tumor cell line was inhibited by berberine in a dose-dependent manner, i.e., the higher the concentration of berberine, the higher the inhibition of NAT activity and cell death. The NAT activities measured in the intact human colon tumor cells were decreased over 50% by AAF and NAc-PABA production from acetylation of AF and PABA. The apparent values of Kmoff and Vmax of NAT from colon tumor cells were also inhibited by berberine in cytosols and in intact cells. This report is the first to show that berberine did affect human colon tumor cell NAT activity.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human bladder tumor cells.

Diallyl sulfide (DAS) and diallyl disulfide (DADS) were used to determine viability and inhibition of arylamine N-acetyltransferase (NAT) activity in human bladder tumor cells. The NAT activity was measured by high performance liquid chromatography assaying for the amounts of N-acetyl-2-aminofluorene (2-AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA). The viability, NAT activity and 2-AAF-DNA adduct formation in human bladder tumor cells was inhibited by DAS and DADS in a dose-dependent manner, i.e. the higher the concentration of DAS and DADS, the higher the inhibition of NAT activity and cell death. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human bladder tumor cells in both systems examined. This report is the first demonstration to show garlic components did affect human bladder tumor cell NAT activity.

Effects of the garlic compounds diallyl sulphide and diallyl disulphide on arylamine N-acetyltransferase activity in Klebsiella pneumoniae.

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) were determined in the bacterium Klebsiella pneumoniae. Cytosols or suspensions of K. pneumoniae with or without specific concentrations of diallyl sulphide (DAS) or diallyl disulphide (DADS) as co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was decreased NAT activity associated with increased levels of DAS or DADS in K. pneumoniae. In growth studies on K. pneumoniae it was demonstrated that DAS or DADS elicited a dose-dependent bacteriocide effect on K. pneumoniae. For the cytosol examinations, the apparent values of Km and Vmax were 0.96+/-0.09 mM and 7.87+/-0.79 nmol min(-1) mg(-1) protein, respectively, for 2-AF. However, when DAS or DADS was added to the reaction mixtures, the apparent values of Km and Vmax were 0.16+/-0.04 mM and 0.99+/-0.16 nmol min(-1) mg(-1) protein with DAS, respectively, and 0.14+/-0.18 mM and 0.85+/-0.10 nmol min(-1) mg(-1) protein with DADS, respectively, for 2-AF. For the intact bacteria examination, the apparent values of Km and Vmax were 0.57+/-0.06 mM and 2.00+/-0.14 nmol min(-1) per 10x10(10) CFU, respectively, for 2-AF. However, when DAS or DADS was added to the reaction mixtures, the apparent of values of Km and Vmax were 0.41+/-0.04 mM and 1.30+/-0.10 nmol min(-1) per 10x10(10) CFU with DAS, respectively, and 0.34+/-0.04 mM and 1.08+/-0.08 nmol min(-1) per 10x10(10) CFU with DADS, respectively, for 2-AF. This report is the first demonstration to show that the garlic components DAS and DADS would affect K. pneumoniae growth and NAT activity.

Gypenoside induces apoptosis in human Hep3B and HA22T tumour cells.

The effect of gypenoside, an active component of the Chinese herb Gynostemma pentaphyllum (Thumb) Makino, on human hepatoma cell lines (Hep3B and HA22T) was investigated. Results demonstrated that gypenoside inhibited the proliferation or viability of the Hep3B and HA22T cells in a dose-dependent manner. The Hep3B and HA22T cells treated with gypenoside for 2 days were less DNA stainable and formed a sub-G1 peak. The treated cells increased cell numbers in the A0 region as well as shifting the ordinary S phase to the final S phase (D1 region), and induced a ladder pattern of fragmented DNA of about 200 base pairs. These data suggest that the cell death of the hepatoma cell lines Hep3B and HA22T induced by gypenoside was via apoptosis, and this was confirmed by morphological studies.

Effects of garlic compounds diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients.

Arylamine N-acctyltransferase (NAT) activities with p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Two assay systems were performed, one with cellular cytosols, the other with intact cell suspensions. Cytosols or suspensions of H. pylori with or without specific concentrations of diallyl sulfide (DAS) or diallyl disulfide (DADS) co-treatment showed different percentages of 2-AF and PABA acetylation. The data indicated that there was decreased NAT activity associated with increased levels of DAS or DADS in H. pylori cytosols and suspensions. Viability studies on H. pylori demonstrated that DAS or DADS elicited dose-dependent bactericide affects on H. pylori cultures. The data also indicated that DAS and DADS decreased the apparent values of K(m) and Vmax of NAT enzyme from H. pylori in both systems examined. This report is the first demonstration that garlic components can affect H. pylori growth and NAT activity.

Effects of the garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human colon tumour cells.

Diallyl sulfide (DAS) and diallyl disulfide (DADS), major components of garlic, were used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line. Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact bacterial cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by DAS and DADS in a dose-dependent manner in both system: that is, the greater the concentration of DAS and DADS in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax of NAT enzymes from human colon tumour cells in both systems examined. This is the first report to demonstrate that garlic components do affect human colon tumour cell NAT activity.