Masaya Watarai

Aichi Medical University, Okazaki, Aichi, Japan

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Publications (11)33.64 Total impact

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    ABSTRACT: We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid β-oxidation.
    Leukemia research 06/2013; · 2.36 Impact Factor
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    ABSTRACT: The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.
    Oncology Reports 02/2013; · 2.30 Impact Factor
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    ABSTRACT: In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.
    Genes Chromosomes and Cancer 10/2011; 51(1):42-53. · 3.55 Impact Factor
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    ABSTRACT: For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC(50): 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a "glycolytic" leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (-) medium showed more growth and survival than glucose (-) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC(50): 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (-) FCS (+) medium showed more growth and survival than glucose (+) FCS (-) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid β-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.
    Leukemia & lymphoma 11/2010; 51(11):2112-9. · 2.61 Impact Factor
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    ABSTRACT: By using neutralizing monoclonal antibodies to vascular endothelial growth factor receptor type 1 (VEGFR1) and VEGFR2, we have shown that acute myelogenous leukemia (AML) cells with specific chromosome abnormalities are dependent on VEGF/VEGFR system. AML with t(8;21) is the most dependent subtype on VEGF with both VEGFR1 and VEGFR2. t(15;17)AML cells depend on VEGF with VEGFR1. AML cells with 11q23 abnormalities showed variable dependence on VEGF. The growth of t(11;19)AML cells are most extensively inhibited by anti-VEGFR1 antibody. Then, the growth of Kasumi-1, a t(8;21) cell line was suppressed by either anti-VEGFR1 antibody (p=0.0022) or anti-VEGFR2 antibody (p=0.0029) in a dose-dependent manner. The growth of NB4, a t(15;17) cell line was more potently suppressed by anti-VEGFR1 antibody (p=0.0111) than by anti-VEGFR2 antibody (p=0.0477). These results are quite concordant with the results of clinical samples with t(8;21) or t(15;17). In addition, anti-VEGFR2 monoclonal antibody significantly potentiated the growth inhibitory effect of idarubicin for Kasumi-1. As for downstream signals, we have shown that VEGFR2 transduce growth and survival signals through phosphorylation of Akt and MEK in leukemia cells (Kasumi-1). However, VEGFR1 transduce growth and survival signals through pathways other than MEK and Akt (NB4), although Akt phosphorylation may account for some of the VEGFR1 signals (Kasumi-1). Finally, our data suggested that autocrine pathway of VEGF and VEGFRs observed in AML cells with specific chromosomal translocations have contributed to leukemogenesis as activated signaling of receptor tyrosine kinase.
    Leukemia research 04/2009; 33(12):1650-7. · 2.36 Impact Factor
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    ABSTRACT: Several anti-angiogenic drugs have recently been clinically tested for haematological malignancies. To improve the efficacy of molecular target therapy against angiogenic molecules in acute myeloid leukaemia (AML), we examined the dependency of AML cells on the vascular endothelial growth factor (VEGF)/VEGF receptor type2 (VEGFR2) system by using VEGFR2 kinase inhibitor. Nineteen patient AML samples were cultured with or without VEGFR2 kinase inhibitor. All four t(8;21) viable AML cells showed significant reductions when treated with VEGFR2 kinase inhibitor, although VEGFR2 kinase inhibitor did not affect the cell proliferation of five t(15;17) AML samples. Other AML cases showed variable responses. VEGFR2 kinase inhibitor greatly suppressed the growth of Kasumi-1, a t(8;21) cell line in a dose-dependent manner through induction of apoptosis, but did not show any significant influence on NB4, a t(15;17) cell line. In addition, VEGFR2 kinase inhibitor potentiated the growth inhibitory effect of cytarabine in Kasumi-1. Finally, it was shown that the Akt phosphorylation was augmented by VEGF(165) in Kasumi-1, which was abrogated by VEGFR2 kinase inhibitor. NB4 showed undetectable Akt phosphorylation even with VEGF(165). These data demonstrated that t(8;21) AML cells are dependent on VEGF through VEGFR2, resulting in the phosphorylation of Akt.
    British Journal of Haematology 01/2007; 135(5):673-82. · 4.94 Impact Factor
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    ABSTRACT: Various angiogenic factors, such as vascular endothelial growth factor (VEGF) and an associated molecule, placenta growth factor (PlGF), are thought to be important for normal and malignant hematopoiesis. This study examined mRNA expression of VEGF, PlGF and receptors for these molecules in AML cells and identified the disease-specific patterns of expression. AML M3 having t(15;17) abnormality showed highest expression of VEGF and VEGF receptor type 1 (VEGFR1), suggesting the autocrine pathway of VEGF-VEGFR1. Then, t(8;21) AML demonstrated augmented expression of VEGF and VEGF receptor type 2 (VEGFR2), suggesting VEGF-VEGFR2 autocrine pathway. Then, addition of VEGFR2 kinase inhibitor in Kasumi-1, a t(8;21) AML cell line, resulted in marked inhibition of cell growth, although growth inhibitory effect of R2 kinase inhibitor to HL-60 was marginal. In addition, cell cycle analysis study showed S-phase cell population reduction by R2 kinase inhibitor in Kasumi-1, but not in HL-60. This observation is thought to be the rationale for novel molecular target therapy directed to angiogenic molecules.
    Leukemia and Lymphoma 02/2006; 47(1):89-95. · 2.61 Impact Factor
  • Nihon Naika Gakkai Zasshi 01/2006; 94(12):2606-8.
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    ABSTRACT: P-glycoprotein (P-gp)/multi-drug resistance 1 (MDR1) gene is recognized to be, at least in part, responsible for the refractoriness to chemotherapy of leukemia. The transcriptional mechanism of MDR1 gene is largely unknown. However, recent reports have clarified that early growth response 1 gene (Egr1) positively regulates MDR1 transcription, while Wilms' tumor suppressor gene (WT1) does negative regulation of MDR1 gene expression in 12-O-tetradecanoylphorbol-13-acetate treated K562 cells. In addition, Egr1 and WT1 are structurally related transcription factors and bind to quite similar DNA sequences. Our study of mRNA expression profile of Egr1, WT1 and MDR1 in fresh AML samples demonstrated that there are disease-specific patterns. Egr1 mRNA was frequently and strongly expressed in monocytic leukemia cells, especially in AML M4 cells. WT1 mRNA was undetectable in t(8;21) AML cells. mRNA expression of MDR1 was frequent in AML M1 and t(8;21) AML cells, in which the expression level was highest in AML M1 and was low in monocytic leukemia (M4 and M5). Then, expression level of MDR1 was inversely correlated with Egr1. By liquid culture of leukemia cell lines and fresh AML cells with the addition of all-trans retinoic acid (ATRA), modulation of P-gp/MDR1 and Egr1 was observed and the pattern of modulation was divided into four groups: (1) blastic AML type, in which distinct expression of P-gp/MDR1 and CD34 was not influenced by ATRA; (2) t(8;21)AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 was kept high; (3) AML M3 type, in which P-gp/MDR1 expression was reduced with granulocytic differentiation by ATRA; (4) monocytic AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 expression decreased, and strong Egr1 expression was downregulated just prior to the augmentation of P-gp/MDR1 expression. WT1 expression was not influenced by the addition of ATRA in each group. Previous reports have suggested that P-gp/MDR1 plays an important role in resistance to chemotherapy, and is recognized as one of the stem cell marker. However, P-gp/MDR1 expression augmented by ATRA, which was observed in monocytic AML, was recognized as a functional molecule of mature monocyte/macrophage, because CD34 expression decreased and CD13 expression increased by ATRA. Finally, expression of P-gp/MDR1 in monocytic leukemia, which was functionally confirmed by Rh123 efflux study, was thought to be closely related to the characteristic modulation of Egr1 expression by ATRA.
    Leukemia Research 02/2002; 26(1):29-36. · 2.76 Impact Factor
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    ABSTRACT: Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).
    Leukemia 02/2002; 16(1):112-9. · 10.16 Impact Factor
  • Nihon Naika Gakkai Zasshi 07/2001; 90(6):1070-2.