Yukio Kuroiwa

Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with ETS-1 in endothelial cells.

The purpose of this study was to examine the effect of hydrogen peroxide (H2O2) on angiogenesis in cultured endothelial cells. Endothelial cells obtained from bovine thoracic aorta (BAECs) were cultured between two layers of collagen type I to measure the tube formation which is a marker for angiogenesis. Addition of H2O2 (0.1-10 microM) to endothelial cells for various periods increased the rate of tube formation. The maximum stimulation of the tube formation was obtained when cells were exposed to 1 microM H2O2 for 30 min, and the enhancement of tube formation was blocked by catalase (10 U/ml). Both proliferation and migration of BAEC which are known to affect angiogenesis, were also stimulated by the addition of H2O2 (0.1 and 1 microM). Thus relatively low concentrations of H2O2 stimulated angiogenesis, proliferation and migration. Ets-1 is a member of the ets gene family of transcription factors, which binds to the ets binding motif in the cis-acting elements and regulates the expression of certain genes such as proteases including urokinase plasminogen activator (u-PA) and matrix metalloproteinase-1 (MMP-1). Interestingly, H2O2 increased the ets-1 mRNA level in BAECs compared with the basal level. The H2O2-stimulated angiogenesis was completely blocked by an ets-1 antisense oligonucleotide, but not by a mismatched oligonucleotide. These findings indicate that low concentrations of H2O2 stimulate angiogenesis in BAECs, and the stimulation mechanisms may partially involve the enhancement of proliferation and migration. Moreover, the H2O2-induced angiogenesis is likely to be mediated by the transcription factor ets-1.

Relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans

Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. Previously, we found that the CYP2A6 gene was deleted homozygously in one subject who was deficient in cotinine formation from nicotine.

Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chansu, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans

Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. Large interindividual differences in nicotine metabolism have been reported in humans. The purpose of this study was to clarify the relationship between the poor metabolism of nicotine and the existence of the CYP2A6v1 and CYP2A6v2 alleles, and a whole deletion allele of the CYP2A6 gene. The plasma concentrations of nicotine and cotinine were measured in 10 healthy subjects after each smoked one cigarette or chewed one piece of nicotine gum. One subject showed no detectable cotinine level in plasma when smoking and the lowest cotinine level when receiving nicotine gum. The subject was regarded as a poor metabolizer of nicotine by a probit analysis and was found to carry a homozygous whole deletion allele of the CYP2A6 gene. This is the first report to show that deficient cotinine formation in humans is attributed to the whole deletion of the CYP2A6 gene.

Nicotine metabolism and CYP2A6 allele frequencies in Koreans

CYP2A6 is a major catalyst of nicotine metabolism to cotinine. Previously, we demonstrated that the interindividual difference in nicotine metabolism is related to a genetic polymorphism of the CYP2A6 gene in Japanese. To clarify the ethnic differences in nicotine metabolism and frequencies of CYP2A6 alleles, we studied nicotine metabolism and the CYP2A6 genotype in 209 Koreans. The cotinine/nicotine ratio of the plasma concentration 2 h after chewing one piece of nicotine gum was calculated as an index of nicotine metabolism. The genotypes of CYP2A6 gene (CYP2A6*1A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4 and CYP2A6*5) were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific (AS)-PCR. There were ethnic differences in the allele frequencies of CYP2A6*1A, CYP2A6*1B, CYP2A6*4 and CYP2A6*5 between Koreans (45.7%, 42.8%, 11.0% and 0.5%, respectively) and Japanese (42.4%, 37.5%, 20.1% and 0%, respectively, our previous data). Similar to the Japanese, no CYP2A6*2 and CYP2A6*3 alleles were found in Koreans. The homozygotes of the CYP2A6*4 allele (four subjects) were completely deficient in cotinine formation, being consistent with the data among Japanese. The heterozygotes of CYP2A6*4 tended to possess a lower metabolic ratio (CYP2A6*1A/CYP2A6*4, 4.79 +/- 3.17; CYP2A6*1B/CYP2A6*4, 7.43 +/- 4.97) than that in subjects without the allele (CYP2A6*1A/CYP2A6*1A, 7.42 +/- 6.56; CYP2A6*1A/CYP2A6*1B, 9.85 +/- 16.12; CYP2A6*1B/CYP2A6*1B, 11.33 +/- 9.33). The subjects who possess the CYP2A6*1B allele appeared to show higher capabilities of cotinine formation. It was confirmed that the interindividual difference in nicotine metabolism was closely related to the genetic polymorphism of CYP2A6. The probit plot of the metabolic ratios in Koreans (8.73 +/- 11.88) was shifted to a higher ratio than that in the Japanese (3.78 +/- 3.09). In each genotype group, the Korean subjects revealed significantly higher metabolic ratios than the Japanese subjects. The ethnic difference in cotinine formation might be due to environmental and/or diet factors as well as genetic factors.

Metabolism of deprenyl, a selective monoamine oxidase (MAO) B inhibitor in rat: relationship of metabolism to MAO-B inhibitory potency

Deprenyl, a selective inhibitor of monoamine oxidase type B (MAO-B), was metabolized in rats to methamphetamine (MAP), amphetamine (AP) and their corresponding p-hydroxylated metabolites, p-hydroxy-MAP and p-hydroxy-AP. Recovery of metabolites in 24 h urine was 25% of the dose, and there was no urinary excretion of unchanged deprenyl. Deprenyl was converted into MAP, AP and nordeprenyl when incubated in vitro with rat-liver microsomes in the presence of NADPH. This metabolism was inhibited in an atmosphere of N2 and by CO, and by SKF 525-A, but to a lesser extent by methimazole. Liver microsomes from phenobarbital (PB)-treated rats, but not 3-methylcholanthrene (3-MC)-treated rats, stimulated the metabolism of deprenyl in vitro to MAP and AP, but not to nordeprenyl. In contrast, microsomes from SKF 525-A-treated rats showed decreased activity in the metabolism of deprenyl to all three metabolites. The inhibitory effect of the drug on hepatic MAO-B activity was annulled by pretreatment of rats with PB, but not 3-MC, and augmented by pretreatment with SKF 525-A.

Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs

When human leukemia HL-60 cells were treated with 10(-7) M bufalin, the amounts of both topoisomerase (topo) II alpha and II beta and the activity of topo II decreased markedly and were almost undetectable 18 h after the start of treatment. The level of topo II mRNA started to decrease immediately after the start of treatment with bufalin, with a subsequent decrease in the amount of topo II alpha protein. These changes preceded the fragmentation of DNA, a typical feature of apoptosis. The results suggest that bufalin caused a marked decrease in the steady-state level of topo II alpha mRNA, which led to a decrease in the amount and activity of the enzyme and to the induction of apoptosis. A reduction in the level of topo II alpha by bufalin was also observed in other lines of human leukemia cells such as ML1 and U937. The results were exploited to potentiate the effects of cisplatin and retinoic acid (RA) on HL-60 cells: pretreatment of HL-60 cells with 10(-7) M bufalin for 6 h increased the inhibitory effects of cisplatin and RA on cell growth and enhanced the induction of cell death.

Selective Inhibitory Effect of Bufalin on Growth of Human Tumor Cells in vitro: Association with the Induction of Apoptosis in Leukemia HL‐60 Cells

We found that bufalin, an active principle of the Chinese medicine chansu, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth‐inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL‐60, ML1, and U937 leukemia cells treated with bufalin at 10−8M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL‐60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL‐60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL‐60 cells was 10−8M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all‐trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10−6M bufalin for 24 h. These results indicate the association of the growth‐inhibitory effect of bufalin with the induction of apoptosis, at least in HL‐60 cells, and suggest the usefulness of bufalin for differentiation‐apoptosis‐inducing therapy for cancer.

INTERINDIVIDUAL DIFFERENCES IN NICOTINE METABOLISM AND GENETIC POLYMORPHISMS OF HUMAN CYP2A6

Nicotine is widely consumed throughout the world, and exerts a number of physiological effects. After nicotine is absorbed through the lungs by cigarette smoking, it undergoes extensive metabolism in humans. Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. CYP2A6 can metabolize some pharmaceutical agents such as halothane, valproic acid, and fadrozole, and activate tobacco-specific nitrosamines. There are large interindividual differences in nicotine metabolism, and it has been found that the interindividual differences are attributed to the genetic polymorphisms of CYP2A6 gene. This review describes the techniques for determination of in vivo nicotine metabolism, characteristics of each human CYP2A6 alleles, and ethnic differences. The relationship between CYP2A6 genetic polymorphism and potency of nicotine metabolism, smoking behavior, and cancer risk are extensively reviewed. Finally, the usefulness of nicotine metabolism for phenotyping of CYP2A6 in individuals and implication of the significance of CYP2A6 genetic polymorphism in a clinical perspective are discussed.

Bufalin as a potent inducer of differentiation of human myeloid leukemia cells

Bufalin was found to be a potent inducer of differentiation in human erythroleukemia K562 cells by examination of various differentiation markers (as assessed by the morphology, histochemistry, and the abilities to phagocytose latex particles, to reduce nitro-blue tetrazolium and to develop Fc receptors). Bufalin, at a concentration as low as 10 nM, also produced a strong differentiation-inducing activity in three other human leukemia-derived cell lines (human promyelocytic HL60, monoblastic U937 and myeloblastic ML1). Treatment of K562 cells with other cardiotonic steroids, such as cinobufagin, ouabain and digitoxigenin, at the concentration of 10 nM for four days resulted in weak or no effect on the cells. These findings suggest that bufalin might have potentiality as a new agent in the differentiation therapy for human myelogenous leukemia.