Takanori Ueda

Azithromycin Reduces Tumor Necrosis Factor-Alpha Production in Lipopolysaccharide-Stimulated THP-1 Monocytic Cells by Modification of Stress Response and p38 MAPK Pathway

Abstract Macrolide antibiotics are known to have a variety of immunomodulatory effects in addition to antimicrobial activity, but the mechanisms of immunomodulation are still unclear. We investigated in vitro the effect of azithromycin on tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated THP-1 cells, a human monocytic cell line, and compared the results with those for other macrolides, minocycline and ofloxacin. In the presence of LPS, treatment with azithromycin (AZM) resulted in a significant decrease in LPS-induced TNF-α production compared to that with other antimicrobial agents. The results of phosphorylation of three MAPKs, ERK, JNK and p38, indicated that the phospho-p38 level was reduced by AZM. IκB-α, an inhibitor of NFκB, was not disrupted by the antibiotics. LPS-induced TNF-α release from THP-1 cells was inhibited in the presence of KNK437, a potent 70-kDa heat shock protein (HSp-70) inhibitor. Interestingly, the induction of HSp-70 by LPS was attenuated with the concurrent addition of AZM in the cells. AZM was found to restrain TNF-α production by monocytes at least in part by modifying the HSP-70 and p38 related signaling pathways to LPS stimulation.

Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO‐406 in newly established K562 variants with BCR‐ABL gene amplification

Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re‐establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM‐R1 and IM‐R2 cells, which were developed having 7‐ and 27‐fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR‐ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR‐ABL gene amplification was observed in IM‐R2 cells than in IM‐R1 cells, which was consistent with the higher mRNA and protein levels of Bcr‐Abl, and ultimately correlated with the greater IM resistance in IM‐R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10‐fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM‐R1 and IM‐R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO‐406 (formerly NS‐187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR‐ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients leukemia.

Clinical and prognostic significance of cytokine receptor expression in adult acute lymphoblastic leukemia : interleukin-2 receptor α-chain predicts a poor prognosis

We quantitatively assessed the expression of cytokine receptors (interleukin-2 receptor (IL-2R), IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, granulocyte-macrophage colony-stimulating factor R (GM-CSFR), G-CSFR, c-fms, c-mpl, c-kit and FLT3) in cells from 211 adults with acute lymphoblastic leukemia (ALL) by flow cytometry and determined their prevalence and clinical significance. Although all cytokine receptors were expressed to various degrees, the levels of IL-3R α-chain (IL-3Rα), IL-2Rα, IL-2Rβ, IL-7Rα, common-Rγ(γc), c-mpl, c-kit and FLT3 exhibited a wide spectrum ⩾2000u2009sites/cell. Among them, IL-3Rα, IL-2Rα and FLT3 were highly expressed in B-lineage ALL, whereas IL-7Rα, γc and c-kit predominated in T-lineage ALL. Higher levels of IL-3Rα, IL-2Rα, c-kit and FLT3 correlated with the expression of CD13/33. Increased IL-2Rα levels related to the presence of Philadelphia chromosome (Ph), leukocytosis and shorter event-free survival (EFS). C-kit preferred in male. Elevated FLT3 levels correlated with age ⩾60 years. Multivariate analysis in B-lineage ALL revealed only IL-2Rα (P=0.028) and Ph (P=0.020) as independent factors for EFS. These findings suggest that several cytokine receptors associated with certain cellular and clinical features, but IL-2Rα solely had a prognostic value and should be considered as a major prognostic factor for adult ALL that is comparable with Ph.

Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses

Cytarabine (ara-C) is the key drug for treatment of acute myeloid leukemia. Since intracellular cytarabine triphosphate (ara-CTP) is an active metabolite of ara-C, factors that reduce the amount of ara-CTP are known to induce drug resistance. However, these factors do not fully explain the development of resistance to ara-C. The present study was conducted to search for new candidate ara-C resistance factors, including those that are unrelated to ara-CTP production. For this purpose, we newly established five ara-C-resistant leukemic clones from different blood cell lineage leukemic cell lines (HL-60, K562, CEM, THP1 and U937). The resistant subclones were 5-58-fold more ara-C-resistant than their parental counterparts. All of the ara-C-resistant subclones, except for ara-C-resistant CEM cells, displayed alteration of ara-CTP-related factors such as ara-C membrane transport capacity, deoxycytidine kinase activity or cytosolic nucleotidase II activity. To identify new candidate factors, we used two comprehensive approaches: DNA microarray and proteome analyses. The DNA microarray analysis revealed eight genes (C19orf2, HSPA8, LGALS1, POU4F3, PSAP, AKT1, MBC2 and CACNA2D3) that were altered in all five ara-C-resistant lines compared to parental cells. Both proteome and DNA microarray analyses further detected a reduced protein level of stathmin1 in the ara-C-resistant CEM subclone compared to its parental line. Thus, the present findings suggested the involvement of novel multiple mechanisms in mediating the ara-C resistance of leukemic cells. The role of some of these molecules in resistance is still unclear.

Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

(Cancer Sci 2010; 101: 767–773)

Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells

The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5’-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5’-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

Establishment of an Arsenic Trioxide—Resistant Human Leukemia Cell Line That Shows Multidrug Resistance

We have established an arsenic trioxide (As2O3)-resistant cell line (K562/AS-3) derived from the human leukemia cell line K562. K562/AS-3 was sequentially cultured with increasing concentrations of As2O3 up to 3.5 µM and then cloned by the limiting dilution method. K562/AS-3 was found to be about 7-fold more resistant to As2O3 than the parent cells (IC50 = 12.9 µM for K562/AS-3 and 1.8 µM for K562), and also showed cross resistance to VP-16 and vincristine. The multidrug resistance—associated protein (MRP1) gene was found to be overexpressed, but the MDR gene was not detected. MRP1 function was evaluated by measuring calcein acetoxymethyl ester (calcein-AM) efflux, and by verifying its inhibition by MK571, a potent MRP inhibitor. In addition, an increase of the total intracellular glutathione content was found in K562/AS-3. The resistance of K562/AS-3 to As2O3 was reversed by the addition of MK571, but not by verapamil. K562/AS-3 may be useful for studying the mechanism of the anticancer effect of As2O3 and how to overcome As2O3-resistance.

Multiple inflammatory cytokine-productive ThyL-6 cell line established from a patient with thymic carcinoma

Thymic epithelial cells can produce many kinds of cytokines, and interleukin (IL)‐6‐producing thymic carcinoma cases have been reported. However, a cytokine‐producing human thymic tumor cell line has not previously been established. In this paper, we report a novel, multiple inflammatory cytokine‐productive cell line that was established from a patient with thymic carcinoma. This cell line, designated ThyL‐6, positively expressed epithelial membrane antigen, cytokeratins, vimentin intermediate filament and CD5, although hematological markers were not present in the cells. Cytokine antibody array analysis showed that the cells secreted several cytokines including IL‐1α, IL‐6, IL‐8, RANTES, soluble TNFα‐receptor 1, VEGF and CTLA into the culture medium. The addition of ThyL‐6‐cultured supernatant supported the growth of human myeloma ILKM‐3 cells, which require the presence of IL‐6 in the culture medium for the maintenance of cell growth, suggesting that the secreted IL‐6 from ThyL‐6 cells was biologically active. Chromosome analysis demonstrated that ThyL‐6 cells had complex karyotype anomalies, including der(16)t(1;16); the latter has been recognized in thymic squamous cell carcinoma and thymic sarcomatoid carcinoma cases, as well as in several other kinds of malignancies. Heterotransplantation of the cells into nude mice showed tumorigenesis with neutrophil infiltration and liquefactive necrosis. These findings suggest that ThyL‐6 cells will provide us with a new experimental tool for investigating not only the pathogenesis, biological behavior, chromo‐somal analysis and therapeutic reagents of human thymic carcinoma, but also for studying cytokine–chemokine network systems. (Cancer Sci 2008; 99: 1778–1784)

Acute hyperglycemia causes microvascular damage, leading to poor functional recovery and remodeling in patients with reperfused ST-segment elevation myocardial infarction.

BackgroundAlthough acute hyperglycemia (AHG) is associated with poor outcomes in ST-segment elevation myocardial infarction (STEMI) patients, underlying mechanisms have not been fully elucidated. We investigated the influence of AHG on myocardial microcirculation in reperfused STEMI patients.Methods and ResultsThirty-four STEMI patients were divided into 2 groups according to the presence (Group H, nxa0=xa011) or the absence (Group L, nxa0=xa023) of AHG. Myocardial blood flow (MBF) and myocardial flow reserve (MFR) in the infarct-related area were compared between 2 groups, using 13N-ammonia positron emission tomography. Wall motion abnormality scores (WMASs) and end-diastolic volume indices (EDVI) were also assessed at 1 and 6xa0months after the onset. Although resting MBF was similar, MFR was lower in Group H than in Group L (1.69xa0±xa00.37 vs 2.39xa0±xa00.56, Pxa0=xa0.001). WMAS was greater in Group H than in Group L at both 1xa0month (7.4xa0±xa03.7 vs 3.7xa0±xa03.0, Pxa0=xa0.011) and 6xa0months (7.3xa0±xa03.9 vs 3.1xa0±xa03.4, Pxa0=xa0.015). EDVI tended to be greater in Group H than in Group L at 6xa0months (103.8xa0±xa042.9 vs 73.9xa0±xa016.0xa0mL/m2, Pxa0=xa0.071). Multivariate analysis showed AHG to be independently associated with low MFR.ConclusionsIn STEMI patients, AHG impaired myocardial microcirculation, leading to poor functional recovery and remodeling despite successful reperfusion.

Beneficial effects of statin treatment on coronary microvascular dysfunction and left ventricular remodeling in patients with acute myocardial infarction

BACKGROUNDnStatin treatment has been shown to improve coronary endothelial function, irrespective of lipid-lowering effects. This studys aim was to elucidate the effects of statin treatment on coronary microvascular dysfunction and left ventricular remodeling in acute myocardial infarction (AMI) patients.nnnMETHODSnThirty-five patients undergoing successful reperfusion following AMI were assigned to a statin-treated (Group S, 16) or a non-statin-treated (Group NS, 19) group, according to fasting serum low-density lipoprotein-cholesterol. (13)N-ammonia positron emission tomography was performed to assess myocardial flow reserve (MFR) in the infarct area.nnnRESULTSnInfarct sizes and lipid profiles during the chronic period were similar between the two groups. At 2 weeks after AMI onset, mean MFR in the infarct area was significantly higher in Group S than in Group NS (2.34 ± 0.63 vs. 1.91 ± 0.43, p=0.0214). At 6 months post-AMI, Group S had a smaller left-ventricular end-diastolic volume index (69.4 ± 11.7 mL/m(2) vs. 88.5 ± 32.5 mL/m(2), p=0.0328) and higher left-ventricular ejection fraction (67.7 ± 9.2% vs. 59.2 ± 13.3%, p=0.0394) than Group NS. Serum asymmetric dimethylarginine was significantly increased in Group NS at 1 month post-AMI (0.43 ± 0.12 μmol/L (baseline) vs. 0.52 ± 0.14 μmol/L, p=0.0186), but unchanged in Group S.nnnCONCLUSIONSnStatin treatment appears to beneficially attenuate left ventricular remodeling after AMI, which may be associated with restoring coronary endothelial function via endogenous nitric oxide.