Science.gov

Sample records for hl-60 cells exposed

  1. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900MHz radiofrequency fields.

    PubMed

    Sun, Yulong; Zong, Lin; Gao, Zhen; Zhu, Shunxing; Tong, Jian; Cao, Yi

    2017-03-01

    HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120μW/cm(2) power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Zhang, X. F.; Han, L. H.; Harrison, G. H.; Davis, C. C.; Zhou, X. J.; Ioffe, V.; McCready, W. A.; Abraham, J. M.; Meltzer, S. J.

    1998-01-01

    We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

  3. BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Zhang, X. F.; Han, L. H.; Harrison, G. H.; Davis, C. C.; Zhou, X. J.; Ioffe, V.; McCready, W. A.; Abraham, J. M.; Meltzer, S. J.

    1998-01-01

    We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

  4. BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields.

    PubMed

    Balcer-Kubiczek, E K; Zhang, X F; Han, L H; Harrison, G H; Davis, C C; Zhou, X J; Ioffe, V; McCready, W A; Abraham, J M; Meltzer, S J

    1998-12-01

    We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

  5. Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

    SciTech Connect

    Holian, O.; Reyes, H.M.; Attar, B.M.; Walter, R.J.; Astumian, R.D.; Lee, R.C.

    1996-12-31

    The authors examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5--1,000 mV/cm p-p) were applied for 1 h to cells (10{sup 7}/ml) in Teflon chambers at 37 C in the presence or absence of 2 {micro}M phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100--1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA, EFs (33--330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents.

  6. Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions.

    PubMed

    Gee, David J; Wright, L Kate; Zimmermann, Jonathan; Cole, Kayla; Soule, Karen; Ubowski, Michelle

    2012-08-01

    Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin-Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96 h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72 h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96 h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.

  7. Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions

    PubMed Central

    Gee, David J.; Wright, L. Kate; Zimmermann, Jonathan; Cole, Kayla; Soule, Karen; Ubowski, Michelle

    2012-01-01

    Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin–Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96 h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72 h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96 h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo. PMID:22494057

  8. [Preparation of cytoplasts from HL-60 cells].

    PubMed

    Wang, Lili; Yu, Huangfei; Fang, Ning; Chen, Daixiong

    2013-06-01

    This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for

  9. [The examination of ultraweak luminescence of HL-60 cells and adriamycin-resistant HL-60/ADM cells].

    PubMed

    Ma, Shi-rong; Hui, Yan-ping; Xu, Yu-qiao; Li, Yan

    2007-07-01

    To explore the relationship between the ultraweak luminescence and adriamycin resistantance in human leukemic cells (HL-60) and human adriamycin resistant leukemic cells (HL-60/ADM). MTT colorimetry, flow cytometry (FCM), growth curve and immunohistochemical staining were used to detect the sensitivity of HL-60 and HL-60/ADM cells to ADM, the change of cell cycle and the expression of P-glycoprotein(P-gp170). The intensity of the ultraweak luminescence of HL-60 cells and HL-60/ADM cells was examined by the IFFM-D chemiluminescence instrument. The sensitivity of HL-60/ADM cells to ADM was distinctly lower than that of HL-60 cells. The percentage of HL-60/ADM cells in G(0)/G(1) was 44.80%+/-1.97% and that in G(2)/M phase was 9.90%+/-0.27%, which was both higher than that of HL-60 cells. The percentage of HL-60/ADM cells in S phase was 45.30%+/-1.93%, which was lower than that of HL-60 cells. The doubling time of HL-60/ADM cells was 1.8 times as long as that of HL-60 cells. The expression of P-gp170 was positive in HL-60/ADM cells but negative in HL-60 cells.Treated with 10(-4) mol/L luminol and 3 mL/L H(2)O(2) and under four different cell concentration (8x10(7)/L,1x10(8)/L,1.26x10(8)/L, 2.73x10(8)/L), the intensity of ultraweak luminescence of HL-60/ADM cells was distinctly lower than that of HL-60 cells (P<0.05). The lower sensitivity to ADM, longer doubling time and the expression of P-gp170 showed the drug resistance of HL-60/ADM cells. With the obtaining of drug resistance, ultraweak luminescence intensity was lower in HL-60/ADM cells than that of HL-60 cells. It showed that the decreasing of intensity of ultraweak luminescence could be an index of examining drug-resistantce of tumor cells.

  10. Importance of inducible multidrug resistance 1 expression in HL-60 cells resistant to gemtuzumab ozogamicin.

    PubMed

    Matsumoto, Taichi; Jimi, Shiro; Hara, Shuuji; Takamatsu, Yasushi; Suzumiya, Junji; Tamura, Kazuo

    2012-07-01

    Resistance to gemtuzumab ozogamicin (GO) hampers the effective treatment of refractory acute myeloid leukemia (AML). To clarify the mechanism of resistance to GO, HL-60 cells were persistently exposed to GO in order to establish GO-resistant HL-60 (HL-60/GOR) cells. Multidrug resistance 1 (MDR-1) was strongly expressed in HL-60/GOR cells, but not in HL-60 cells. Although withdrawal of GO after the chronic exposure of HL-60/GOR cells to this compound gradually decreased MDR-1 expression to trace levels, reintroducing GO restored high MDR-1 expression in HL-60/GOR cells, but not in HL-60 cells. These results indicate that HL-60/GOR cells acquired the ability to induce MDR-1 expression in response to GO. U0126, a MEK1/2 inhibitor, prevented GO-inducible MDR-1 expression and abrogated GO resistance in HL-60/GOR cells. These results suggest that in the clinical use of GO, inducible MDR-1 expression in tumor cells should be investigated before treatment with GO. If the cells are positive then MEK1/2 inhibitors may be effective in overcoming resistance to GO.

  11. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  12. Effect of ozone on platelet activating factor metabolism in phorbol-differentiated HL60 cells

    SciTech Connect

    Samet, J.M.; Friedman, M. )

    1992-11-01

    The mechanisms of ozone (O3) toxicity in the lung may involve the formation of lipid inflammatory mediators. We have previously demonstrated that exposure to O3 in vitro results in increased accumulation and release of platelet activating factor (PAF) in the macrophage-like cell line HL60 differentiated with phorbol ester (dHL60). In the present study we have examined possible biochemical mechanisms responsible for the O3-induced increase in PAF levels in dHL60 cells. Specifically, we studied the effect of O3 on phospholipase A2 (PLA2), acetyltransferase, acetylhydrolase, and reacylation activities. dHL60 cells were exposed to 1.0 ppm O3 or air alone. O3 exposure was found to significantly decrease dHL60 cell acetylhydrolase activity by 36%. Additional experiments demonstrated that extracellular acetylhydrolase activity, but not intracellular acetylhydrolase activity, was inhibited by O3 exposure of dHL60 cells. O3 exposure resulted in a small (13%) but statistically significant reduction in reacylation activity in dHL60 cells. In addition, a significant (22%) contribution of PLA2 activation to the O3-induced increase in PAF levels was also found. Basal and calcium ionophore-induced acetyltransferase activity was found to be unaffected by exposure of dHL60 cells to O3. These data suggest that in vitro exposure to O3 affects both synthetic and degradative pathways of PAF metabolism in dHL60 cells.

  13. [Inhibitory effect of 14-3-3ζ on the proliferation of HL-60 cells and HL-60/VCR cells].

    PubMed

    Liang, Rong; Chen, Xie-Qun; Wang, Zhe; Xiong, Hua; Bai, Qing-Xian; Gao, Guang-Xun; Dong, Bao-Xia; Zhu, Hua-Feng

    2013-08-01

    This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

  14. DNA repair in human promyelocytic cell line, HL-60.

    PubMed Central

    Farzaneh, F; Feon, S; Lebby, R A; Brill, D; David, J C; Shall, S

    1987-01-01

    The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity. Images PMID:3106934

  15. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  16. Modulation of p53, c-fos, RARE, cyclin A, and cyclin D1 expression in human leukemia (HL-60) cells exposed to arsenic trioxide

    PubMed Central

    Yedjou, Clement G.; Tchounwou, Paul B.

    2010-01-01

    Arsenic trioxide (As2O3) has recently been successfully used to treat all-trans retinoic acid (ATRA) resistant relapsing acute promyelocytic leukemia. However, its molecular mechanisms of action are poorly understood. In the present study, we used the human leukemia (HL-60) cell line as a test model to study the cellular and molecular mechanisms of anti-cancer properties of As2O3. We hypothesized that As2O3-induced expression of stress genes and related proteins may play a role in the cellular and molecular events leading to cell cycle modulation in leukemic cells. To test this hypothesis, we performed Western blot analysis to assess the expression of specific cellular response proteins including p53, c-fos, RARE, Cyclin A, and Cyclin D1. Densitometric analysis was performed to determine the relative abundance of these proteins. Western Blot and densitometric analyses demonstrated a strong dose-response relationship with regard to p53 and RARE expression within the dose range of 0-8μg/mL. Expression of c-fos was slightly up-regulated at 2μg/mL, and down-regulated within the dose-range of 4-8 μg/mL. A statistically significant down-regulation of this protein was detected at the 6 and 8 μg/mL dose levels. No statistically significant differences (p>0.05) in Cyclin D1 expression was found between As2O3-treated cells and the control. Cyclin A expression in As2O3-treated HL-60 cells was up-regulated at 6μg/mL, suggesting that it is required for S phase and passage through G2 phase in cell cycle progression. Taken together, these results indicate that As2O3 has the potential to induce cell cycle arrest through activation of the 53-kDa tumor suppressor protein and repression of the c-fos transcription factor. Up-regulation of RARE by As2O3 indicates that its cytotoxicity may be mediated through interaction/binding with the retinoic acid receptor, and subsequent inhibition of growth and differentiation. PMID:19444595

  17. C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation.

    PubMed

    Hashimoto, Ken-ichiro; Sonoda, Yoshiko; Yamakado, Masakazu; Funakoshi-Tago, Megumi; Yoshida, Naomi; Rokudai, Akiko; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2006-07-01

    We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.

  18. Ascorbic acid modulates cell migration in differentiated HL-60 cells and peripheral blood leukocytes.

    PubMed

    Schwager, Joseph; Bompard, Albine; Weber, Peter; Raederstorff, Daniel

    2015-08-01

    The impact of L-ascorbic acid (L-AA) on the chemokinesis (CK) and chemotaxis (CT) of HL-60 cells and polymorphonuclear cells (PMN) was investigated. HL-60 cells were differentiated with DMSO, retinoic acid (RA), vitamin D, or L-AA. Chemokinesis and chemotaxis of differentiated HL-cells were assayed. Vitamin D3-treated HL-60 cells (dHL-60vitD3 cells) and RA-treated cells (dHL-60RA cells) acquired monocyte/macrophage-like and neutrophil-like phenotypes, respectively. DMSO induced the differentiation of an intermediate phenotype (dHL-60DMSO cells), whereas L-AA downregulated neutrophil markers (dHL-60L-AA cells). dHL-60DMSO cells had increased CK and potent CT in gradients of IL-8 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). dHL-60RA cells and dHL-60L-AA cells migrated less toward IL-8 and fMLP; dHL-60vitD3 cells preferably responded to fMLP. L-AA enhanced CK of dHL-60DMSO cells and was a weak chemo-attractant. In human leukocytes, IL-8 and fMLP triggered receptor-mediated chemotaxis. CXCR2 and fMLPR were downregulated by IL-8 and fMLP, respectively. L-AA stimulated chemotaxis although significantly less than IL-8 and fMLP. IL-8 targeted chemotaxis was enhanced both in HL-60 cells and leukocytes when cells were incubated with L-AA. L-AA modulated chemokinesis and had significant chemo-attractant properties, which were independent on fMLP or IL-8 receptors. The results suggest that L-AA improves leukocyte function in innate immune responses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Influence of galangin on HL-60 cell proliferation and survival.

    PubMed

    Bestwick, Charles S; Milne, Lesley

    2006-11-08

    The effect of galangin, a flavonol component of India root spice and the 'herbal' medicine propolis, on HL-60 human leukaemia cell survival is characterised. Galangin (1-100 microM) exerted an antiproliferative effect that, with dose and exposure longevity, was progressively associated with an elevated hypodiploid DNA content and expression of the active form of caspase-3, principally prior to membrane damage. At >or=50 microM, plasmamembrane phosphatidylserine exposure was observed. There was no evidence for intracellular oxidative stress as an orchestrator of cytotoxicity and significant phagocyte-like differentiation was not detected. We discuss whether such cytotoxicity will be therapeutically exploitable or contribute to cancer prevention within a pharmacological or dietary context.

  20. Analogs of farnesylcysteine induce apoptosis in HL-60 cells.

    PubMed

    Pérez-Sala, D; Gilbert, B A; Rando, R R; Cañada, F J

    1998-04-24

    S-Farnesyl-thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL-60 cells and induced apoptosis, as evidenced by the development of increased annexin-V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs.

  1. Diallyl disulfide induces apoptosis in human leukemia HL-60 cells through activation of JNK mediated by reactive oxygen.

    PubMed

    Yi, Lan; Ji, Xiao-Xia; Lin, Min; Tan, Hui; Tang, Yi; Wen, Ling; Ma, Yan-Hua; Su, Qi

    2010-09-01

    Diallyl disulfide (DADS) is a chemopreventive agent that can induce apoptosis in many tumor cells. Reactive oxygen species (ROS) are important mediators in apoptosis induced by various stimuli, including chemopreventive agents. The phosphotransferase c-JUN N-terminal kinase (JNK) has been shown to regulate apoptosis. In this study, we found that DADS-induced apoptosis in human leukemia HL-60 cells is mediated by ROS-activated JNK. The DADS-treated HL-60 cells showed a dose- and time-dependent decrease in cell viability and proliferation. Agarose gel electrophoresis of cells treated with 10.0 or 20.0 mg/L DADS for 24 h showed a characteristic ladder pattern in their DNA. Levels of DADS-induced ROS, as measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence, also showed dose- and time-dependent increases in HL-60 cells. Activity of JNK was induced by DADS in a dose-dependent manner; HL-60 cells exposed to 10.0 mg/L DADS for 8 h showed maximum levels of phosphorylated JNK, which decreased when exposed for additional 4h. In contrast, Sp600125, a specific inhibitor of JNK, blocked apoptosis of HL-60 cells exposed to DADS. N-Acetylcysteine (NAC), a known antioxidant, also decreased ROS generation, effectively blocked apoptosis, and decreased DADS-induced phosphorylated JNK levels. These results suggest that JNK is involved in DADS-induced ROS-mediated apoptosis in HL-60 cells.

  2. [Human umbilical cord mesenchymal stem cells reduce the sensitivity of HL-60 cells to cytarabine].

    PubMed

    Cui, Jun-Jie; Chi, Ying; Du, Wen-Jing; Yang, Shao-Guang; Li, Xue; Chen, Fang; Ma, Feng-Xia; Lu, Shi-Hong; Han, Zhong-Chao

    2013-06-01

    This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

  3. [Mechanism of HL-60 cells apoptosis induced by proteasome inhibitor MG132].

    PubMed

    Zhou, Yong-Ming; Yu, Mei-Xia; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2013-08-01

    The purpose of this study was to elucidate the apoptosis, apoptotic pathway of HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Apoptosis of HL-60 cells was detected by flow cytometry, the expression of P21, P27 and P53 proteins in HL-60 cells treated with MG132 was assayed by Western blot. The HL-60 cells were treated with 1 µmol/L MG132 for 48 h, and irradiated by 75 Gy of (60)Co γ-ray, but their antigenicity was preserved. The effect of irradiated HL-60 cells treated with MG132 on proliferation of peripheral blood mononuclear cells (PBMNC) was measured by CCK-8 method. The results showed that the apoptotic rate of MG132-treated HL-60 cells increased in dose-and time-dependent manner. No significant changes in MG132-induced apoptosis were observed after inhibiting caspase-8 and caspase-9 pathway. The expression of P21 and P27 protein increased after treatment of HL-60 cells with MG132. CCK-8 test showed that HL-60 cells induced with low-dose of MG132 displayed the enhancing effect on proliferation of PBMNC. It is concluded that high dose of MG132 can induce the apoptosis of HL-60 cells, and has direct killing effect on HL-60 cells, but this inducing apoptotic effect on HL-60 cells can not be realized through caspase-8 and caspase-9 pathway. The P21 and P27 protein may be involved in MG132 induced HL-60 cell apoptosis. Low dose of MG132 promotes the proliferation of PBMNC in healthy individuals and enhance the immunity of organism.

  4. AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells.

    PubMed

    Zou, Wenying; Chu, Xinwei; Cai, Chunqing; Zou, Mengchen; Meng, Xiaojing; Chen, Haiyang; Zou, Fei

    2012-08-01

    Neutrophil polarization is critical for the inflammatory response. AKT is a serine/threonine protein kinase and has been implicated in cell migration. However, it is not completely clear whether AKT affects neutrophil polarization. In this study, we tested the hypothesis that AKT regulates the polarization of neutrophil-like differentiated HL-60 cells (dHL-60) in response to fMLP. HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1.3 % DMSO for up to 6 days. Polarization of dHL-60 cells and primary human neutrophils were measured by Zigmond chamber. Phospho-Akt was analyzed by immunofluorescence and Western blot analysis. F-actin polymerization was detected by Rhodamine-Phalloidine staining. Rac2 activation was evaluated using GST Pull-down assay. We found that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pretreated with the PI3 kinase inhibitors or the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases was also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells. This study demonstrates that AKT activation plays a crucial role in dHL-60 cell polarization.

  5. [Effect of 5-aza-2'-deoxycytidine on DAPK gene expression in human HL-60 cells].

    PubMed

    Wang, Chun-Yan; Liu, Wen-Jun

    2014-06-01

    This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.

  6. [Drug resistance reversal of HL-60/ADR cells by simultaneous suppression of XIAP and MRP].

    PubMed

    Wang, Xiao-Fang; Wang, Chun; Qin, You-Wen; Yan, Shi-Ke; Gao, Yan-Rong

    2006-12-01

    This study was purposed to explore the mechanisms of drug resistance of HL-60/ADR cells and to compare the reversal drug-resistance effects of antisense oligonucleotides (AS ODN) of XIAP (X-linked inhibitor of apoptosis protein) and AS ODNs of MRP (multidrug resistance-associated protein) by use alone or in combination. Reverse transcription-PCR and Western blot were applied to detect the expression of XIAP, BCL-2, MRP and MDR1 in mRNA and protein levels of HL-60 cells and HL-60/ADR cells, respectively. Fully phosphorothioated AS ODN of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine 2000 in the form of liposome-ODN complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODN of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to daunorubicin (DNR). Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP, MRP in mRNA and protein levels respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than those in HL-60 cells. AS ODN of XIAP and MRP down-regulated the expression of XIAP and MRP in HL-60/ADR cells and increased the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODN of XIAP + MRP did not enhance the inhibition expression of XIAP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly as compared with AS ODN of XIAP (P < 0.05). AS ODN of XIAP + MRP did not increase the concentration of DNR nor enhanced the inhibition expression of MRP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P < 0.05), as compared with AS ODN of MRP. It is concluded that both XIAP and MRP may be involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells can be reversed significantly when antisense oligonucleotides of XIAP and MRP were used in combination.

  7. [Influence of TIEG1 on apoptosis of HL-60 cells and expression of Bcl-2/Bax].

    PubMed

    Yao, Kun; Yang, Ying; Hu, Rong; Miao, Miao; Liao, Ai-Jun; Yang, Wei; Liu, Zhuo-Gang

    2013-06-01

    This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax. Different concentration of TIEG1 were used to treat HL-60 cells, the cell growth inhibition rate was detected by MTT method. After treating HL-60 cells with 12.03 ng/ml TIEG1, cell apoptosis was detected with flow cytometry. Bcl-2 and Bax was detected with RT-PCR. The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation, and in time-and dose-dependent manners. The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml. During the course of cell apoptosis, Bax expression increased, but Bcl-2 expression decreased (P < 0.05). It is concluded that TIEG1 inhibits HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners. During the course of HL-60 cells apoptosis induced by TIEG1, Bcl-2/Bax are associated with HL-60 cell apoptosis induced by TIEG1.

  8. PML(NLS(-)) inhibits cell apoptosis and promotes proliferation in HL-60 cells.

    PubMed

    Gao, Yuan-Mei; Zhong, Liang; Zhang, Xi; Hu, Xiu-Xiu; Liu, Bei-Zhong

    2013-01-01

    Promyelocytic leukemia (PML) is a cell-growth suppressor, and PML-retinoic acid receptor α (PML-RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Studies have reported that neutrophil elastase(NE) cleaved bcr-1-derived PML-RARα in early myeloid cells leading to the removal of nuclear localization signal (NLS) from PML. The resultant PML without NLS named PML(NLS(-)). PML(NLS(-)) mainly locates and functions in the cytoplasm. PML(NLS(-)) may act in different ways from PML, but its biological characteristics have not been reported. In this study, the PML (NLS(-)) was silenced with shRNA [HL-60/pPML(NLS(-))-shRNA] and over-expressed by preparation of recombinant adenovirus [HL-60/pAd-PML(NLS(-))]. The mRNA and protein expression of PML(NLS(-)) were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect apoptotic cells. The transcription of BCL-2, BAX and C-MYC was detected in HL-60/pAd-PML(NLS(-)) cells. Our results showed that compared to the control group, the expression of PML(NLS(-)) was significantly reduced in the HL-60/pPML(NLS(-))-shRNA cells, and increased significantly in the HL-60/pAd-PML(NLS(-)) cells. The proliferation was significantly inhibited in the HL-60/pPML(NLS(-))-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-PML(NLS(-)) cells treated with 60 μmol/L emodin. FCM revealed the apoptosis increased in HL-60/pPML(NLS(-))-shRNA cells, and decreased in the HL-60/pAd-PML(NLS(-)) cells. The expression of BAX decreased significantly, while that of BCL-2 and C-MYC increased significantly in HL-60/ pAd-PML(NLS(-)) cells. Down-regulation of PML(NLS(-)) expression inhibits the proliferation and induces the apoptosis of HL-60 cells. On the contrary, over-expression of PML(NLS(-)) promotes the proliferation and reduce the emodin-induced apoptosis of HL-60 cells.

  9. [(Sp)-8-chloroadenosine 3',5'-cyclophosphate induced differentiation on human leukemia HL-60 cells].

    PubMed

    Su, J; Yang, X B; Wang, L X; Xu, B; Zhang, L H

    1996-01-01

    (Sp)-octyl 8-chloroadenosine 3',5'-cyclophosphate(OCC), a newly synthesized cAMP analog, strongly induced growth inhibition and differentiation in human leukemia HL-60 cells. The effects were dose- and time-dependent and irreversible. In flow cytometry, OCC brought about a block at the G1 phase of HL-60 cell cycle. Determined by incorporation assay, OCC was shown to strongly inhibit DNA synthesis without affecting the synthesis of RNA and protein in HL-60 cells. OCC activated the protein kinase A(PKA) in the cytosol of HL-60 cells and inhibited its binding to cAMP. The activities of PKA in the cytosol of HL-60 cells treated with OCC were more significantly increased than those in control cells. It can be concluded that OCC binds itself to PKA in competition with cAMP and, as a result, activates PKA.

  10. Possible involvement of calcineurin in retinoic acid-induced inhibition of leukemic HL-60 cell proliferation.

    PubMed

    Kihira, H; Hiasa, A; Yamamoto, M; Katayama, N; Kuno, T; Ohtsuka, K; Shiku, H; Nishikawa, M

    1998-03-01

    Differentiation of leukemic HL-60 cells by all transretinoic acid (ATRA) resulted in a reduced rate of growth. Cyclosporin A and FK506, at concentrations that inhibited calcineurin activity, abrogated the ATRA-induced inhibition of HL-60 cell growth but these immunosuppressants had no effect on the ATRA-induced granulocytic differentiation. Treatment with 1 microM ATRA led to a progressive increase in calcineurin phosphatase activity of HL-60 cells; the increase in this activity appeared to parallel the functional change of HL-60 cells during granulocytic differentiation. Increase in calcineurin activity was concordant with the increased expressions of calcineurin A and calcineurin B subunit proteins. The FKBP12 expression increased during ATRA-induced differentiation and expression of cyclophilin A remained unchanged. We propose that the increased expression of calcineurin is involved in the ATRA-induced inhibition of HL-60 cell proliferation, as in the case with 1,25alpha-dihydroxy-vitamin D3.

  11. Effects of static magnetic field on human leukemic cell line HL-60.

    PubMed

    Sabo, J; Mirossay, L; Horovcak, L; Sarissky, M; Mirossay, A; Mojzis, J

    2002-05-15

    A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.

  12. Study on human promyelocytic leukemia HL-60 cells apoptosis induced by fucosterol.

    PubMed

    Ji, Yu-Bin; Ji, Chen-Feng; Yue, Lei

    2014-01-01

    In this study, we investigated the effect of fucosterol on HL-60 and the molecular mechanism. HL-60 Cells were treated with fucosterol, and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method was used to study fucosterol anti-tumor activity. Morphology of HL-60 cells was observed. Flow cytometry (FCM) was employed to detect the cell cycle. Laser scanning confocal microscope (LSCM) was used to analyze mitochondrial membrane potential (MMP) and the expressions of Fas, FasL, Fadd and Caspase-8. Western blot was performed to analyze the expressions of Cyt-C, Pro-Caspase-9 and Pro-Caspase-3. Caspase activity kits were used to determine the activity of Caspase-9, Caspase-8 and Caspase-3. The results showed fucosterol could inhibit the growth of HL-60 cells, and the cell cycle was arrested at G2/M phase. HL-60 cells showed obvious apoptosis morphology. After being treated with fucosterol for 24 h, HL-60 cells decreased MMP, induced Cyt-C release and Caspase-9, Caspase-3 activation. Fucosterol also increased the protein expression of Fas, FasL, Fadd and Caspase-8. Moreover, the activity of Caspase-9, Caspase-8 and Caspase-3 was increased significantly. In conclusion, Fucosterol can induce HL-60 cells apoptosis, suggesting that it may be a potent agent for cancer prevention and treatment.

  13. Apoptosis of HL-60 cells induced by extracts from Narcissus tazetta var. chinensis.

    PubMed

    Liu, Jing; Li, Yan; Ren, Wei; Hu, Wei-Xin

    2006-10-08

    Narcissus tazetta var. chinensis is one member of the Amaryllidaceae family. We found that extracts from N. tazetta var. chinensis (ENT) strongly decreased the survival rate of the following tumor cell lines: HL-60, K562, KT1/A3, and A3R. The cytotoxic effects of ENT on non-cancer cells lines (NHBE and NIH3T3) were smaller than on leukemia cell lines. AO/EB staining and flow cytometry assays showed that ENT induced HL-60 cell apoptosis. Furthermore, the release of cytochrome c and the increase of caspase-8, -9, and -3 activities were tested after HL-60 cells were treated with ENT, which indicated that the mitochondrial pathway and cell death receptor pathway were both involved in the apoptosis signal pathways induced by ENT. Upregulation of Bax showed that the Bcl-2 family was involved in the control of apoptosis. Our results suggest that apoptosis activity can be mediated by ENT in HL-60 cells.

  14. PBK/TOPK expression during TPA-induced HL-60 leukemic cell differentiation.

    PubMed

    Liu, Yu-Hong; Gao, Xue-Mei; Ge, Fan-Mei; Wang, Zhe; Wang, Wen-Qing; Li, Xiao-Yong

    2012-01-01

    This study concerns expression of PBK/TOPK during differentiation of HL-60 leukemic cells induced by tetradecanoyl phorbol acetate (TPA). Wright-Giemsa staining was performed to observe morphological changes in the HL-60 cells, and flow cytometry was used to assess the cell cycle and CD11b, CD14, CD13, and CD33 expression. PBK/TOPK levels were determined by Western blot analysis. After treating HL60 cells with 5.1×10⁻⁹ mmol/L of TPA for three days, the number of nitroblue-tetrazolium-positive cells and CD11b, CD13, and CD14 expression increased, whereas the PBK/TOPK levels decreased. TPA can inhibit proliferation and induce differentiation of HL60 cells of the granulocytic or monocytic lineage. PBK/TOPK expression was downregulated during this process, whereas the Pho-PBK/TOPK expression was increased.

  15. Differential impact of bortezomib on HL-60 and K562 cells.

    PubMed

    Kliková, Katarína; Štefaniková, Andrea; Pilchová, Ivana; Hatok, Jozef; Chudý, Peter; Chudej, Juraj; Dobrota, Dušan; Račay, Peter

    2015-01-01

    Bortezomib (PS-341, or Velcade), reversible inhibitor of 20S proteasome approved for the treatment of multiple myeloma and mantle cell lymphoma, exhibited a cytotoxic effect toward other malignancies including leukaemia. In this study, we have documented that incubation of both HL-60 and K562 leukaemia cells with nanomolar concentrations of bortezomib is associated with the death of HL-60 cells observed within 24 hours of incubation with bortezomib and the death of K562 cells that were observed after 72 hours of incubation with bortezomib. The relative resistance of K562 cells to bortezomib correlated well with significantly higher expression of HSP27, HSP70, HSP90α, HSP90β and GRP75 in these cells. Incubation of both HL-60 and K562 cells with bortezomib induced a cleavage of HSP90β as well as expression of HSP70 and HSP90β but bortezomib did not affect levels of HSP27, HSP90α, GRP75 and GRP78. The death of both types of cells was accompanied with proteolytic activation of caspase 3 that was observed in HL-60 cells and proteolytic degradation of procaspase 3 in K562 cells. Our study has also pointed to essential role of caspase 8 in bortezomib-induced cleavage of HSP90β in both HL-60 and K562 cells. Finally, we have shown that bortezomib induced activation of caspase 9/caspase 3 axis in HL-60 cells, while the mechanism of death of K562 cells remains unknown.

  16. Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

    NASA Astrophysics Data System (ADS)

    Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

    2017-04-01

    Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

  17. The Effect of Combined Exposure of 900 MHz Radiofrequency Fields and Doxorubicin in HL-60 Cells

    PubMed Central

    Jiang, Bingcheng; Zhou, Zhen; Tong, Jian; Cao, Yi

    2012-01-01

    Human promyelocytic leukemia HL-60 cells were pre-exposed to non-ionizing 900 MHz radiofrequency fields (RF) at 12 µW/cm2 power density for 1 hour/day for 3 days and then treated with a chemotherapeutic drug, doxorubicin (DOX, 0.125 mg/L). Several end-points related to toxicity, viz., viability, apoptosis, mitochondrial membrane potential (MMP), intracellular free calcium (Ca2+) and Ca2+-Mg2+ -ATPase activity were measured. The results obtained in un-exposed and sham-exposed control cells were compared with those exposed to RF alone, DOX alone and RF+DOX. The results indicated no significant differences between un-exposed, sham-exposed control cells and those exposed to RF alone while treatment with DOX alone showed a significant decrease in viability, increased apoptosis, decreased MMP, increased Ca2+ and decreased Ca2+-Mg2+-ATPase activity. When the latter results were compared with cells exposed RF+DOX, the data showed increased cell proliferation, decreased apoptosis, increased MMP, decreased Ca2+ and increased Ca2+-Mg2+-ATPase activity. Thus, RF pre-exposure appear to protect the HL-60 cells from the toxic effects of subsequent treatment with DOX. These observations were similar to our earlier data which suggested that pre-exposure of mice to 900 MHz RF at 120 µW/cm2 power density for 1 hours/day for 14 days had a protective effect in hematopoietic tissue damage induced by subsequent gamma-irradiation. PMID:23029402

  18. [Effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of HL-60 cells and its mechanism].

    PubMed

    Xie, Xia; Li, Jie; Wang, Rui-Cang; Geng, Rui-Li; Wang, Su-Yun; Wang, Chao; Zhao, Xiao-Yun; Hao, Hong-Ling

    2014-06-01

    This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.

  19. Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

    PubMed

    Bhakkiyalakshmi, Elango; Suganya, Natarajan; Sireesh, Dornadula; Krishnamurthi, Kannan; Saravana Devi, Sivanesan; Rajaguru, Palanisamy; Ramkumar, Kunka Mohanram

    2016-02-05

    The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

  20. [Mechanisms of Chansu Injection in reversing multidrug resistance of HL60/ADM cells].

    PubMed

    Xiong, Xin; Zhou, Yuan-da

    2012-03-01

    To investigate the mechanisms underlying the effect of Chansu Injection (CHS) in reversing multi-drug resistance (MDR) of HL60/ADM cells. MTT assay was used to investigate the effect of CHS on adramycin (ADM) sensitivity of HL-60/ADM cells. Flow cytometry was used to observe the effect of CHS on the cell cycle of HL60/ADM cell. The expressions of NF-κB, MRP, GST-π, and iNOS were detected by immunocytochemistry. Treatment with CHS lowered the IC(50) of ADM in HL60/ADM cells from 34.1971 µmol/L to 17.4393 µmol/L, and caused an increase in G(0)/G1 and G(2)/M phase cells with decreased S phase cells. CHS decreased the expressions of MRP mRNA and GST-π and MRP proteins but increased the expressions of iNOS and NF-κB proteins in the cells. CHS can partly reverse MDR in HL60/ADM cells possibly by down-regulating MRP and GST-π, up-regulating NF-κB and iNOS, and promoting cell apoptosis, thereby increase ADM sensitivity of HL-60/ADM cells.

  1. Terpinen-4-ol induces autophagic and apoptotic cell death in human leukemic HL-60 cells.

    PubMed

    Banjerdpongchai, Ratana; Khaw-On, Patompong

    2013-01-01

    Terpinen-4-ol, a monoterpene, is found as the main component of essential oil extracts from many plants. In this study apoptotic and autophagic types of cell death induced by terpinen-4-ol and associated mechanisms were investigated in human leukemic HL-60 cells. The cytotoxicity of human leukemic U937 and HL-60 cells was determined by MTT assay. Cytochrome c release, expression of Bax, Bcl-2, Bcl-xl and cleaved Bid were determined by Western blotting. Cell morphology was examined under a transmission electron microscope. LC3-I/II, ATG5 and Beclin-1 levels were detected by immunoblotting. Terpinen-4-ol exhibited cytotoxicity to human leukemic HL-60 but not U937 cells. The apoptotic response to terpinen-4-ol in HL-60 cells was due to induction of cytochrome c release from mitochondria and cleavage of Bid protein after the stimulation of caspase-8. There was a slightly decrease of Bcl-xl protein level. The characteristic cell morphology of autophagic cell death was demonstrated with multiple autophagosomes in the cytoplasm. At the molecular level, the results from Western blot analysis showed that terpinen-4-ol significantly induced accumulation of LC3-I/II, ATG5 and Beclin-1, regulatory proteins required for autophagy in mammalian cells. Terpinen-4-ol induced-human leukemic HL-60 cell death was via both autophagy and apoptosis.

  2. Induction of differentiation of human promyelocytic leukemia cells (HL60) by metabolites of hexamethylene bisacetamide.

    PubMed

    Snyder, S W; Egorin, M J; Geelhaar, L A; Hamburger, A W; Callery, P S

    1988-07-01

    We studied the ability of five metabolites of hexamethylene bisacetamide (HMBA), which we had previously identified in patient urine, to induce differentiation or to influence differentiation induced by HMBA of a human promyelocytic cell line. Differentiation of HL60 cells was quantified by morphological changes and by the ability to reduce nitroblue tetrazolium. N-Acetyl-1,6-diaminohexane (NADAH), the deacetylated, first metabolite of HMBA, was a more potent inducer of HL60 differentiation than was HMBA. NADAH produced 20-30% differentiation at 0.25 mM and 30-40% differentiation at 0.5 mM. NADAH (1 mM) induced 2-3-fold more differentiation than did 1 mM HMBA. HL60 differentiation, induced by various combinations of HMBA and NADAH, reflected a combined effect of the two compounds. In contrast, 1,6-diaminohexane, at 0.5-5 mM, failed to induce HL60 differentiation. Similarly, 0.5-5 mM 6-acetamidohexanoic acid, the major metabolite of HMBA, and 6-aminohexanoic acid failed to induce differentiation of HL60 cells. However, 6-acetamidohexanoic acid, when combined with HMBA or NADAH at various concentrations and ratios, enhanced the differentiation of HL60 cells induced by these two compounds. This enhancement was most apparent with addition of 0.50-3.0 mM 6-acetamidohexanoic acid to HL60 cells incubated with 1.0-3.0 mM HMBA or 0.25-1.0 mM NADAH. 6-Aminohexanoic acid similarly enhanced HMBA-induced differentiation of HL60 cells. These in vitro results have implications in terms of the clinical application of HMBA and interpretation of the results of clinical trials performed to date and may provide some insight into the mechanism of HMBA-induced cellular differentiation.

  3. α-tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells

    PubMed Central

    HUANG, HUARONG; CHEN, SHAOHUA; VAN DOREN, JEREMIAH; LI, DONGLI; FARICHON, CHELSEA; HE, YAN; ZHANG, QIUYAN; ZHANG, KUN; CONNEY, ALLAN H; GOODIN, SUSAN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    α-tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α-tomatine on human myeloid leukemia HL-60 cells were investigated. Treatment of HL-60 cells with α-tomatine resulted in growth inhibition and apoptosis in a concentration-dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL-60 cells. Growth inhibition and apoptosis induced by α-tomatine in HL-60 cells was partially abrogated by addition of cholesterol indicating that interactions between α-tomatine and cell membrane-associated cholesterol may be important in mediating the effect of α-tomatine. Activation of nuclear factor-κB by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate failed to prevent apoptosis in HL-60 cells treated with α-tomatine. In animal experiments, it was found that treatment of mice with α-tomatine inhibited the growth of HL-60 xenografts in vivo. Results from the present study indicated that α-tomatine may have useful anti-leukemia activities. PMID:25625536

  4. Effects of curine in HL-60 leukemic cells: cell cycle arrest and apoptosis induction.

    PubMed

    Dantas, Bruna Braga; Faheina-Martins, Gláucia Veríssimo; Coulidiati, Tangbadioa Hervé; Bomfim, Caio César Barbosa; da Silva Dias, Celidarque; Barbosa-Filho, José Maria; Araújo, Demetrius Antônio Machado

    2015-04-01

    Curine is a natural alkaloid isolated from Chondrodendron platyphyllum and it has been reported that this alkaloid has vasodilatory and anti-inflammatory effects. The aim of this study is to analyze the cytotoxic effects of curine in cancer cell lines HL-60, K562, and HT-29, and in primary cultures of peripheral blood mononuclear cells (PBMC). Cells were treated with curine (from 3 to 15 µM) for 24 and 48 h. Cell viability was analyzed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry with propidium iodide (PI) assay. To assess the type of cell death induced in HL-60, the cell cycle, morphological, and biochemical alterations were analyzed, which were determined by differential staining with acridine orange/ethidium bromide, and annexin V/PI double-labeling and change in mitochondrial membrane potential assays. Curine demonstrated a potent cytotoxic effect on leukemic cell lines (HL-60 and K562). Its cytotoxic effects in HL-60 cells was related to plasma membrane damage and cell cycle arrest at the G1 phase from 43.4 ± 1.0 to 56.7 ± 1.4 % (p < 0.05). Curine (15 μM) also increased the apoptotic cells number by around 60 % in HL-60 cells and caused phosphatidylserine externalization, inducing about 57 % of apoptosis. Moreover, this alkaloid provoked 20 % of mitochondrial membrane depolarization. We conclude that curine presented a cytotoxic effect and induced apoptosis in HL-60 cells. Thus, it can be considered a promising pharmacological drug.

  5. Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines.

    PubMed

    Wagner, Brett A; Britigan, Bradley E; Reszka, Krzysztof J; McCormick, Michael L; Burns, C Patrick

    2002-05-15

    We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product. (c) 2002 Elsevier Science (USA).

  6. [Differentiation of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin and its mechanism].

    PubMed

    Xie, Zhao-Yang; Wu, Bin-Hua; Yang, Zhi-Gang; Chen, Xiao-Fang; Chen, Qiu-Shen

    2013-08-01

    This study was purposed to investigate the proliferation, differentiation and apoptosis of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin (PAC). HL-60 cells were incubated with 20 mg/L PAC for 24 h, the cell growth was evaluated by CCK-8 assay. the effect of PAC on HL-60 cells was evaluated and the cells morphology was observed by optical microscopy. Expression of CD14 and CD11b, and cell cycle were analyzed by flow cytometry. The results showed that the growth of HL-60 cells was inhibited after treatment with PAC of different concentration in a dose-dependent manner (P < 0.05). 20 mg/L PAC displayed significant effect on HL-60 cells with inhibition ratio (72.3 ± 1.8)% for 24 h. Microscopy displayed that some cells differentiated to relative mature cells after treating for 48 h. Expression of CD14 increased and the expression of CD11b increased a little after treating with 20 mg/L PAC for 24 h, the ratio of cells in G0/G1 phase increased, but the ratio of cells in S phase decreased. The mRNA and protein expression of P21 gene increased, but the protein expression of CDK4 and Cyclin D1 decreased. It is concluded that PAC may inhibit the proliferation of HL-60 cells in vitro, induces the differentiation of HL-60 cells, and arrests the cells in G0/G1 phase. The possible mechanism may be related to up-regulation of P21 gene expression and down-regulation of the protein expression of CDK4 and Cyclin D1.

  7. Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells.

    PubMed

    Lin, Minhui; Hu, Jianda; Liu, Tingbo; Li, Jing; Chen, Buyuan; Chen, Xinji

    2013-09-01

    Nucleophosmin, a multifunctional nucleolar phosphoprotein, is involved in many cellular activities. However, the role of NPM in drug-resistance of leukemia has not yet been explored. We designed and selected one shRNA targeting on NPM gene transduction into HL-60 and HL-60/ADR cell lines (an adriamycin resistant cell line) by lentivirus. Cell proliferation, apoptosis and differentiation were assessed. The expressions of the related genes and proteins were detected by real-time quantitative RT-PCR and Western blotting. The results showed obvious down-regulation of NPM mRNA and protein levels after NPM RNAi. NPM-targeted RNAi also resulted in many cellular changes, such as, suppressing cell proliferation and inducing cell differentiation. Down-regulation of NPM gene could arrest the cell cycle progression, an increase in the proportion of G0/G1 phase in knockdown groups. NPM gene silencing could also induce pro-apoptotic genes and proteins expression, and inhibit anti-apoptotic genes/proteins expression. Furthermore, IC50 of two chemotherapeutic agents (adriamycin and ADR; daunorubicin and DNR) to HL-60 and HL-60/ADR cells decreased, especially more remarkable on HL-60/ADR cells. IC50 of ADR on HL-60/ADR cells was reduced from 12.544 ± 0.851 μmol/L (before NPM RNAi) to 6.331 ± 0.522 μmol/L (after NPM RNAi), IC50 of DNR was reduced from 2.152 ± 0.143 μmol/L (before NPM RNAi) to 1.116 ± 0.093 μmol/L (after NPM RNAi). The relative reversal rate of HL-60/ADR cells on ADR was 50.2%, and on DNR was 48.9%. In conclusion, our results demonstrated that shRNA expression vectors could effectively reduce NPM expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents.

  8. Analysis of mechanisms of TPA induced differentiation in HL-60 cells by expression profiling with DNA microarray.

    SciTech Connect

    Zheng, X.; Ravatn, R.; Lin, Y.; Shih, W.-C.; Rabson, A.; Strair, R.; Huberman, E.; Conney, A.; Chin, K.-V.; Biochip Technology Center

    2002-10-15

    The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of differentiation in human promyelocytic leukemia cells. Recently, TPA has been successfully administered to patients with myelocytic leukemia and has produced therapeutic effects that led to temporary remission. These studies demonstrated the potential efficacy of TPA in cancer chemotherapy. We now seek to understand the biological effects and molecular mechanisms of differentiation in response to TPA treatment in leukemia cells by expression profiling using DNA microarray. Our results show distinct temporal and coordinated gene changes that are consistent with differentiation and activation of multiple biochemical pathways in HL-60 cells exposed to TPA. Alterations of gene expression in HL-60 cells include various transcription factors, cytokines and protein markers that are consistent with the induction of differentiation elicited by TPA. These temporal patterns of gene expression were abolished or greatly diminished in an HL-60 derived TPA- resistant variant cell line (HL-525), thus revealing transcriptional and consequential biochemical changes that may be required for TPA-induced differentiation. In addition, certain genes were upregulated by TPA in TPA-resistant HL-525 cells but not in TPA-sensitive HL-60 cells suggesting that these genes may play a role in the resistant pheno type. These patterns of gene expression may be important for predicting response to TPA.

  9. [Reversing effects of emodin on multidrug resistance in resistant HL-60/ADR cells].

    PubMed

    Chen, Ying-Yu; Li, Jing; Hu, Jian-Da; Zheng, Jing; Zheng, Zhi-Hong; Zhu, Liang-Fang; Chen, Xin-Ji; Lin, Zhen-Xing

    2013-12-01

    This study was aimed to investigate the reversing effects of emodin on multidrug resistance (MDR) in resistant HL-60/ADR cells, and to explore the underlying mechanisms. The MTT assay was used to assess the chemoresistance of HL-60/ADR cells to emodin and 8 chemotherapeutic agents commonly used in clinic. The reversal effects of emodin on MDR of HL-60/ADR cells were also evaluated by MTT method. DNA ploidy analysis and DNA Ladder assay were used to detect apoptosis-induced effects on HL-60/ADR cells via the adriamycin (ADR) and emodin combination. The expression changes of the drug resistance-associated genes and proteins were detected by RT-PCR and Western Blot respectively. The intracellular accumulation and subcellular distribution of ADR and DNR were measured by flow cytometry and confocal laser scanning microscopy. The results showed that emodin inhibited HL-60/ADR cell proliferation with an average IC50 value of 24.09 ± 1.72 µmol/L, which was similar to that of the parental HL-60 cells (average IC50 = 23.18 ± 0.87 µmol/L). HL-60/ADR cells were resistant to a variety of chemotherapeutic agents, such as ADR, DNR, VP16, VCR,Ara-C, HHT, MTZ and THP. The reversal multiple were between 1.58 and 4.12 after the treatment with low concentration of emodin combined with the above mentioned different agents. The combination of ADR with emodin showed the best reversal effects, and the typical hypodiploid peak (apoptotic peak) and DNA ladder could be detected after the co-treatment.In addition, emodin down-regulated the mRNA and protein expression levels of MRP1, TOPOIIβ, GST π and BCL-2. Furthermore, the addition of emodin enhanced ADR and DNR intracellular accumulation and subcellular distribution in HL-60/ADR cells in dose-dependent manner. It is concluded that the emodin shows reversing effects on the multidrug resistant HL-60/ADR cells, possibly via decreasing the expression levels of drug resistance-associated genes, increasing the intracellular accumulation of

  10. Microfluidic switching system for analyzing chemotaxis responses of wortmannin-inhibited HL-60 cells

    PubMed Central

    Liu, Yuxin; Sai, Jiqing; Richmond, Ann

    2009-01-01

    The chemotaxis of phosphoinositide kinase-3 (PI3K)-inhibited differentiated HL-60 cells stably expressing CXCR2 was studied in a microfluidic switching gradient device that can generate stable and well-defined forward and reverse gradients. Wortmannin, a widely used PI3K inhibitor, was added during cell preparation and the experiment process. The studies quantify the chemotaxis gradient and the effects of a change in the direction of a CXCL-8 gradient on cell migration. PI3K-inhibited HL-60 cells migrated more efficiently toward the gradient before gradient switching than after, as measured by the effective chemotactic index. The inhibited HL-60 cells also showed that inadequate polarization, slower response time, and reduced cell populations can follow the gradient change. We observed that the role of PI3K in directing cellular response to gradient reversal was important in cell polarization and directional sensing associated with gradient switching. PMID:18205049

  11. Synergistic Effect of Baicalin and Adriamycin in Resistant HL-60/ADM Leukaemia Cells.

    PubMed

    Zheng, Jing; Asakawa, Tetsuya; Chen, Yingyu; Zheng, Zhihong; Chen, Buyuan; Lin, Minhui; Liu, Tingbo; Hu, Jianda

    2017-08-31

    The present study was designed to investigate the expression of multidrug resistance (MDR)-related genes, verify the synergistic effects of baicalin and Adriamycin (ADM) and investigate the related mechanisms in ADM-resistant leukaemic HL-60/ADM cells. We used a HL-60/ADM cell line. Cytotoxicity and flow cytometry assays were employed to verify the cytotoxic effects of baicalin. Real-time polymerase chain reaction and Western blotting assays were used to assess the expression of MDR-related genes and the changes in gene expression (both MDR-related and PI3K/Akt pathway-related) induced by administration of baicalin. We found that only multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and Bcl-2 genes were expressed in both HL-60 and HL-60/ADM cells. HL-60/ADM cells exhibited significantly higher expression (p < 0.05). We also observed that low-dose baicalin (5 and 10 µmol/L) can induce growth inhibition and apoptotic effects on HL-60/ADM cells by increasing the intracellular accumulation of ADM. The synergistic effect of baicalin and ADM was verified. Concerning the potential mechanisms involved in this process, we showed that baicalin down-regulated the expression of several MDR-related and PI3K/Akt pathway-related genes. We confirmed the increased expression of MRP1, LRP and Bcl-2 genes in HL-60/ADM cells compared to regular HL-60 cells, which are recommended for future investigation on MDR. The present study provided evidence of the synergistic effect of baicalin and ADM in HL-60/ADM cells. Therefore, baicalin may be considered as a potential therapeutic agent against resistant leukaemia. Suppression of the PI3K/Akt signalling pathway, followed by inhibition of the expression of MDR-related genes may be a common mechanism in combination treatments with ADM for the reduction of resistance to ADM. © 2017 The Author(s). Published by S. Karger AG, Basel.

  12. [Effect of honokiol on proliferation and apoptosis in HL-60 cells and its potential mechanism].

    PubMed

    Fan, Jia-Xin; Zeng, Ying-Jian; Weng, Guang-Yang; Wu, Jian-Wei; Li, Zhang-Qiu; Li, Yuan-Ming; Zheng, Rong; Guo, Kun-Yuan

    2014-12-01

    This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P < 0.05). The expression of cyclin D1, cyclin A, cyclin E and CDK2/4/6 were significantly down-regulated (P < 0.05), the expression of P53 and P21 was significantly upregulated after treating for 24 h with HNK (P < 0.05). After 24 h treatment with HNK, HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P < 0.05). It is concluded that HNK arrestes the cells at G0/G1 phase and induces HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.

  13. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

    PubMed Central

    Nagy, L; Thomázy, V A; Shipley, G L; Fésüs, L; Lamph, W; Heyman, R A; Chandraratna, R A; Davies, P J

    1995-01-01

    Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines. PMID:7791761

  14. NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

    PubMed

    Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

    2014-01-01

    A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

  15. [Effect of astragalus polysaccharide on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism].

    PubMed

    Zeng, Peng-Yun; Deng, Li-Li; Yue, Ling-Ling; Zhang, Lian-Sheng

    2012-08-01

    The objective of this study was to explore the effect of astragalus polysaccharide (APS) on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism. The cytotoxicities of NK cells against HL-60 cells were analyzed by LDH releasing assay at different effect-to-target cell ratios (E:T) before and after treated with APS. The gene expression of MHC class I chain-related (MICA) in HL-60 cells before and after APS treatment was assayed with RT-PCR. Protein expression of MICA in HL-60 cells was assayed by flow cytometry before and after treated by APS. The results showed that after treated with APS 15 mg/ml for 48 h, the cytotoxicities of NK cells against HL-60 cells enhanced at different effect-to-target (P < 0.05), and the gene and protein expressions in MICA of HL-60 cells were up-regulated (P < 0.05). It is concluded that the APS can obviously up-regulate the expression of MICA in HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.

  16. Aloe-emodin induced in vitro G2/M arrest of cell cycle in human promyelocytic leukemia HL-60 cells.

    PubMed

    Chen, H C; Hsieh, W T; Chang, W C; Chung, J G

    2004-08-01

    In this study, we have evaluated the chemopreventive role of aloe-emodin in human promyelocytic leukemia HL-60 cells in vitro by studying the regulation of proliferation, cell cycle and apoptosis. Aloe-emodin inhibited cell proliferation and induced G2/M arrest and apoptosis in HL-60 cells. Investigation of the levels of cyclins B1, E and A by immunoblot analysis showed that cyclin E level was unaffected, whereas cyclin B1 and A levels increased with aloe-emodin in HL-60 cells. Investigation of the levels of cyclin-dependent kinases, Cdk1 and 2, showed increased levels of Cdk1 but the levels of Cdk2 were not effected with aloe-emodin in HL-60 cells. The levels of p27 were increased after HL-60 cells were cotreated with various concentrations of aloe-emodin. The increase of the levels of p27 may be the major factor for aloe-emodin to cause G2/M arrest in these examined cells. Flow cytometric assays and DNA fragmentation gel electrophoresis also confirmed aloe-emodin induced apoptosis in HL-60 cells. The levels of caspase-3 were increased after HL-60 cells were cotreated with 10 microM aloe-emodin for 12, 24, 48, and 72 hours. Taken together, aloe-emodin therefore appears to exert its anticarcinogenesis properties by inhibiting proliferation and inducing cell cycle arrest and apoptosis underwent activation of caspase-3 in human leukemia HL-60 cells.

  17. Manipulation of neutrophil-like HL-60 cells for the study of directed cell migration.

    PubMed

    Millius, Arthur; Weiner, Orion D

    2010-01-01

    Many cells undergo directed cell migration in response to external cues in a process known as chemotaxis. This ability is essential for many single-celled organisms to hunt and mate, the development of multicellular organisms, and the functioning of the immune system. Because of their relative ease of manipulation and their robust chemotactic abilities, the neutrophil-like cell line (HL-60) has been a powerful system to analyze directed cell migration. In this chapter, we describe the maintenance and transient transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays (micropipette and EZ-TAXIS). Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging.

  18. Induction of apoptosis in human promyelocytic leukemia HL60 cells by panaxynol and panaxydol.

    PubMed

    Yan, Zhonghong; Yang, Ruolin; Jiang, Yi; Yang, Zhihui; Yang, Junrui; Zhao, Qian; Lu, Yang

    2011-06-29

    Panaxynol and panaxydol are naturally occurring polyacetylenes, isolated from the lipophilic fractions of Panax notoginseng, that exert anti-proliferative effects against malignant cells. However, to the best of our knowledge, no study concerning the inhibitory effects of the two polyacetylenes on cell growth of human promyelocytic leukemia cells has been reported. In this paper, we examined the antiproliferation and proapoptotic effects of panaxynol and panaxydol on HL60 cells and investigated their mechanism of action. Cell growth inhibition of panaxynol and panaxydol were determined by trypan blue dye exclusion assays. Apoptosis of cells was revealed by morphological observation, analysis for nuclear DNA distribution and by annexin V-FITC/ PI staining using flow cytometry. It was found that panaxynol and panaxydol markedly inhibited proliferation of HL60 cells in a time- and dose-dependent manner via an apoptotic pathway. In concern with these findings, Western blot analysis showed proteolytic activation of PKCδ, caspase-3 activation and cleavage of poly (ADP [adenosine diphosphate]-ribose) polymerase in HL60 cells treated by panaxynol and panaxydol. In conclusion, panaxynol and panaxydol have profound effects on growth and apoptosis of HL60 cells, suggesting those substances are worthy of further exploration as potential anti-cancer agents.

  19. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-05-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.

  20. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

    PubMed Central

    Rodríguez, P; Viñuela, J E; Alvarez-Fernández, L; Buceta, M; Vidal, A; Domínguez, F; Gómez-Márquez, J

    1998-01-01

    Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate. PMID:9560301

  1. Viability study of HL60 cells in contact with commonly used microchip materials.

    PubMed

    Wolbers, Floor; ter Braak, Paul; Le Gac, Severine; Luttge, Regina; Andersson, Helene; Vermes, Istvan; van den Berg, Albert

    2006-12-01

    This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing.

  2. Pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 human leukemia cells.

    PubMed

    Siedlecka-Kroplewska, K; Jozwik, A; Boguslawski, W; Wozniak, M; Zauszkiewicz-Pawlak, A; Spodnik, J H; Rychlowski, M; Kmiec, Z

    2013-10-01

    Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

  3. Pycnogenol induces differentiation and apoptosis in human promyeloid leukemia HL-60 cells.

    PubMed

    Huang, W W; Yang, J S; Lin, C F; Ho, W J; Lee, M R

    2005-06-01

    Pycnogenol, rich of many phytochemicals of medical value, is a commercialized nutrient supplement extracted from the bark of European coastal pine. In this study, we investigated the anti-tumor effects of Pycnogenol on HL-60, U937 and K562 human leukemia cell lines. We found that Pycnogenol inhibited cell proliferation dose- and time-dependently, and the IC(50)s of Pycnogenol on HL-60, U937 and K562 cells were 150, 40 and 100 microg/ml, respectively. When HL-60 cells were incubated with low concentrations of Pycnogenol (50, 100 and 125 microg/ml) for 24 h, a prominent G0/G1 arrest was observed, followed by gradual accumulation of sub-G0/G1 nuclei. At 48 h of treatment, 50-70% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, induction of NSE activity, and increases of cell surface expression of CD11b. However, results from Annexin V/PI staining, DAPI staining and DNA fragmentation assay indicated that Pycnogenol induced HL-60, U937 and K562 cell apoptosis at their respective IC(50)s after 24 h of treatments. Pretreatment of z-DEVD-fmk, a caspase-3 specific inhibitor, not only decreased caspase-3 activity but also reduced the percentage of apoptotic cells induced by Pycnogenol. This indicated that caspase-3 activation was involved in Pycnogenol induced-apoptosis. In conclusion, Pycnogenol induced differentiation and apoptosis in leukemia cells. Our data suggest that Pycnogenol could serve as a potent cancer chemopreventive or chemotherapeutic agent for human leukemia.

  4. [Effects of baicalin on HL-60 cell xenografts in nude mice and its mechanism].

    PubMed

    Zheng, Jing; Hu, Jian-Da; Huang, Yi; Chen, Ying-Yu; Li, Jing; Chen, Bu-Yuan

    2012-10-01

    This study was aimed to investigate the effects of baicalin on HL-60 cell xenografts in nude mice in vivo and explore its mechanism. Xenograft tumor model of HL-60 cells in nude mice was established, which was divided randomly into 6 groups: negative control group (injection of 5% NaHCO(3)), 25, 50 and 100 mg/kg baicalin groups, combination group (50 mg/kg baicalin + 2 mg/kg VP16) and positive control group (VP16 4 mg/kg). The nude mice with HL-60 cell xenografts were treated with drugs via intraperitoneal injection daily. After treatment for 14 days average weigh and inhibitory rate of transplanted tumor stripped from 5 nude mice in each group were calculated, and the ultrastructure change of xenografts cells were tested by transmission electron microscopy. Histopathologic examination was used to observed the change of main organs in nude mice. The expression of signaling molecular PI3K/Akt proteins extracted from xenografts was detected by Western blot. The effects of baicalin on overall survival time in nude mice with HL-60 cell xenografts were evaluated. The results showed that baicalin could inhibit the growth of transplanted tumors in dose-dependent manner. There were more necrotic and apoptotic cells in mice of baicalin-treated groups and combination group than that in mice of negative control group. Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/mTOR signal pathway, where the expressions of p-Akt, mTOR and p-mTOR proteins decreased compared with negative control group, and no significant difference of Akt expression was found between different groups. Compared with negative control group, the median survival time of mice in combination group was more prolongated (P < 0.05). It is concluded that baicalin can inhibit growth and induce apoptosis of HL-60 cell xenografts in nude mice, and prolong median survival time of nude mice. The possible mechanisms may be related to inhibition of Akt activity and down

  5. Sucutinirane-diterpene derivatives induce apoptosis via oxidative stress in HL-60 cells.

    PubMed

    Deguchi, Jun; Horiguchi, Kaori; Wong, Chin Piow; Hosoya, Takahiro; Iihoshi, Akane; Kaneda, Toshio; Morita, Hiroshi

    2014-10-01

    Previously, we reported the isolation of cassane-type diterpenes, sucutiniranes A-F, from the seeds of Bowdichia nitida. In this study, a series of sucutinirane derivatives was prepared, and their in vitro toxicity in the HL-60 cell line was evaluated. Then the action mechanism of a representative compound that induces cell death was investigated. Whereas C-6 or C-7 diol esters and ether decreased the activity against the HL-60 cell line, furan-oxidized derivatives 12 and 13 showed improvement or retention of the activity compared with those of the natural products sucutinirane A (11), E (1), and F (2). Treatment with sucutinirane derivative 13 elevated caspase 3/7 activity and also decreased expression of Bcl-2 family proteins, Mcl-1, and Bid. Derivative 13 generated reactive oxygen species in HL-60 cells, whose apoptotic effects were attenuated by the addition of an antioxidant, N-acetyl-L-cysteine. These results suggest that cassane butenolide 13 induces apoptosis in HL-60 via its oxidative effects.

  6. IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils.

    PubMed Central

    Bober, L A; Waters, T A; Pugliese-Sivo, C C; Sullivan, L M; Narula, S K; Grace, M J

    1995-01-01

    IL-4 is a T-helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL-4 on neutrophilic maturation using the cell line HL-60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL-60 cells with recombinant hu IL-4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL-60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL-4 increased surface expression of the neutrophil-lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL-4 treated HL-60 cells demonstrated enhanced Fc- and complement-mediated phagocytic capacity and increased hexose-monophosphate shunt activity. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pre-treated for 24 h with DMSO. To investigate the effect of IL-4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL-4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti-human IL-4 abolished phagocytic stimulation. Finally, IL-4 treatment also stimulated resting neutrophils to migrate toward zymosan-activated serum (ZAS) and human IL-5. The results demonstrate that IL-4 is a potent maturation factor for myelocytes to become neutrophils and that IL-4 can stimulate resting mature neutrophils. PMID:7529148

  7. [Restorative effect of quercetin on subcellular distribution of daunorubicin in multidrug resistant leukemia cell lines K562/ADM and HL-60/ADM].

    PubMed

    Cai, Xun; Chen, Fang-Yuan; Han, Jie-Ying; Gu, Chun-Hong; Zhong, Hua; Ouyang, Ren-Rong

    2004-12-01

    Quercetin, a widely distributed natural flavonoid with a variety of biological functions, can reverse multidrug resistance (MDR) in leukemia according to recent researches. This study was to investigate the mechanism of quercetin restoring subcellular distribution of daunorubicin (DNR) in multidrug resistant leukemia cell lines, K562/ADM and HL-60/ADM, and reversing their MDR. MTT cell viability assay was used to verify the sensitization of DNR by quercetin in K562/ADM and HL-60/ADM cells,and determine the reverse concentration extent,confocal laser scanning microscope was used to observe the subcellular distribution of DNR in K562/ADM and HL-60/ADM cells,and relevant sensitive cell lines, K562/S and HL-60/S,before and after quercetin exposion. Compared with K562/S and HL-60/S cells,20-40 micromol/L of quercetin in vitro remarkably enhanced the sensitivity of K562/ADM and HL-60/ADM cells to DNR, restore the subcellular distribution of DNR, so as to reverse MDR. quercetin could be a candidate of effective multidrug resistance-reversing agent in leukemia chemotherapy.

  8. Effects and action mechanism of Diospyros kaki on the differentiation of human leukemia HL-60 cells.

    PubMed

    Kim, Seung Hyun; Cho, Seung Sik; Simkhada, Jaya Ram; Park, Sung Ju; Lee, Hyo Jeong; Kim, Tae Sung; Yoo, Jin Cheol

    2010-01-01

    Diospyros kaki Thunb. (Ebenaceae) is widely distributed in North-East Asian countries. Almost all parts of this plant have been traditionally used as medicine. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid (ATRA). Combination of low doses of ATRA or 1,25-dihydroxyvitamin D3 that do not induce toxicity with another drug is a useful strategy for acute promyelocytic leukemia therapy. Our main aim was to investigate the effect of an acetone extract of D. kaki leaves (KV-1) on HL-60 cell differentiation in combination of ATRA or 1,25-dihydroxyvitamin D3. Treatment of HL-60 cells with zero to 100 microg/ml of KV-1 for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when the HL-60 cells were treated with ATRA or 1,25-(OH)2D3 and the extract. The inhibitors of protein kinase C (PKC) (alpha and betaI) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K) and c-Jun N-terminal kinase (JNK) inhibited the HL-60 differentiation induced by the extract in combination of ATRA or 1,25-(OH)2D3, suggesting that PKC and ERK were involved in the cell differentiation enhancement by the extract. The results indicate that the acetone extract of D. kaki leaves has the ability to enhance HL-60 cell differentiation and suggest that it may be useful in acute promyelocytic leukemia therapy.

  9. Involvement of Mcl1 in diallyl disulfide-induced G2/M cell cycle arrest in HL-60 cells.

    PubMed

    Yi, Lan; Ji, Xiao-Xia; Tan, Hui; Feng, Mei-Yan; Tang, Yi; Wen, Ling; Su, Qi

    2012-06-01

    Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, we found that myeloid cell leukemia sequence 1 (Mcl1) was downregulated in DADS-induced cell cycle arrest in HL-60 human leukemia cells. Here, we investigated the role of this protein in DADS-induced G2/M cell cycle arrest in HL-60 cells. We demonstrated that DADS treatment significantly increased the proportion of G2/M phase HL-60 cells (P<0.05) and caused a time-dependent significant downregulation of Mcl1 and the cell cycle-related proteins PCNA and CDK1 (P<0.05). Small interfering RNA-mediated knockdown of Mcl1 expression in HL-60 cells arrested the cell cycle in G2/M phase. By co-immunoprecipitation, we demonstrated that Mcl1 associated with PCNA and CDK1 in G2/M cell cycle arrest in DADS-treated HL-60 cells. DADS decreased the interaction of Mcl1 with PCNA and CDK1, leading to G2/M cell cycle arrest in HL-60 cells. Mcl1 plays an important role in DADS-induced G2/M cell cycle arrest in HL-60 human leukemia cells.

  10. [Effect of overexpression of CAV1 mediated by lentivirus on proliferation and apoptosis of HL-60 cells].

    PubMed

    Ma, Wei; Wang, Di-Di; Wang, Zhao; Zhu, Gui-Ming; Zhang, Peng-Xia

    2013-08-01

    This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells. Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed, and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids. Then collecting virus suspension infects the HL-60 cells, which make CAV1 gene stable transfection and high expression in the cells. The CAV1 protein expression status in HL-60 cells transfected was evaluated through Western blot method. Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry, respectively. The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly, recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90%. The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection. CCK-8 result indicated that cell proliferation activity increased at 48 h after transfection (P < 0.05), flow cytometry testing results displayed that apoptosis rate of HL-60 cells obviously decreased after transfection (P < 0.01). It is concluded that the overexpression of CAV1 in HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

  11. The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells.

    PubMed

    Xiao, Jie; Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2014-05-01

    Our previous studies have shown that quercetin inhibits Cox-2 and Bcl-2 expressions, and induces human leukemia HL-60 cell apoptosis. In order to investigate the role of AMP-activated protein kinase (AMPK) on quercetin-induced apoptosis of HL-60 cells, we used flow cytometry to detect cell apoptosis. The expressions of LKB1, phosphorylated AMPK (p-AMPK), and Cox-2 protein were detected in HL-60 cells and normal peripheral blood mononuclear cells (PBMCs) by western blot. The expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin. The expressions of p-AMPK were detected in HL-60 cells after culture with AMPK inhibitor Compound C. Then, the expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin alone or quercetin + Compound C. It was found that there was no significant difference in LKB1 between PBMCs and HL-60. p-AMPK in PBMCs was higher than that in HL-60, while Cox-2 was lower. After culture of HL-60 with quercetin, p-AMPK was increased, Cox-2 was decreased, but LKB1 remained unchanged. After culture of HL-60 with Compound C, p-AMPK was decreased. There was no significant difference in LKB1 between the quercetin-alone and the quercetin + Compound C groups. p-AMPK decreased more significantly, while Cox-2 increased more significantly in the quercetin + Compound C groups than those in the quercetin-alone groups. Taken together, these findings suggested that quercetin activates AMPK expression in HL-60 cells independent of LKB1 activation, inhibits Cox-2 expression by activating AMPK, and further regulates the Bcl-2-dependent pathways of apoptosis to exert its anti-leukemia effect.

  12. Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells.

    PubMed

    Yi, Lan; Shan, Jian; Chen, Xin; Li, Guoqing; Li, Linwei; Tan, Hui; Su, Qi

    2016-09-01

    Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

  13. Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells

    PubMed Central

    Yi, Lan; Shan, Jian; Chen, Xin; Li, Guoqing; Li, Linwei; Tan, Hui; Su, Qi

    2016-01-01

    Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells. PMID:27588133

  14. Ethanolic rhizome extract from Kaempferia parviflora Wall. ex. Baker induces apoptosis in HL-60 cells.

    PubMed

    Banjerdpongchai, Ratana; Suwannachot, Kittiphan; Rattanapanone, Viboon; Sripanidkulchai, Bungorn

    2008-01-01

    Kaempferia parviflora Wall. ex. Baker is a Thai herb containing many flavonoids that have anti-inflammatory, anti-allergic and antioxidant activities. The objective of this study was to demonstrate apoptotic effects of Kaempferia parviflora Wall. ex. Baker rhizome ethanolic extract on HL-60 cells in vitro. The extract suppressed HL-60 cell growth and decreased cell viability in a dose- and time-dependent manner. Apoptotic cell death was demonstrated by changes in cell morphology, externalization of phosphatidylserine on the cell surface, loss in mitochondrial transmembrane potential and activation of caspase 3. Apoptosis induced by K. parviflora Wall. ex. Baker rhizome ethanolic extract was enhanced by treatment with paclitaxel or doxorubicin, and inhibitors of Akt, PI3-K and MEK.

  15. Caveolin-1 plays a key role in the oleanolic acid-induced apoptosis of HL-60 cells.

    PubMed

    Ma, Wei; Wang, Di-Di; Li, Li; Feng, Yu-Kuan; Gu, Hong-Mei; Zhu, Gui-Ming; Piao, Jin-Hua; Yang, Yu; Gao, Xu; Zhang, Peng-Xia

    2014-07-01

    Our previous study found that caveolin-1 (CAV-1) protein expression is upregulated during oleanolic acid (OA)-induced inhibition of proliferation and promotion of apoptosis in HL-60 cells. CAV-1 is the main structural protein component of caveolae, playing important roles in tumorigenesis and tumor development. It has been shown that cav-1 expression is lower in leukemia cancer cell lines SUP-B15, HL-60, THP-1 and K562 and in chronic lymphocytic leukemia primary (CLP) cells when compared with normal white blood cells, with the lowest cav-1 expression level found in HL-60 cells. To study the effects of cav-1 in HL-60 cells and the effects of cav-1 overexpression on OA drug efficacy, cav-1 was overexpressed in HL-60 cells using lentiviral-mediated transfection combined with OA treatment. The results showed that cav-1 overexpression inhibited HL-60 cell proliferation, promoted apoptosis, arrested the cell cycle in the G1 phase and inhibited activation of the PI3K/AKT/mTOR signaling pathway. Overexpression of CAV-1 also increased HL-60 cell sensitivity to OA. To further verify whether OA affects HL-60 cells via the activation of downstream signaling pathways by CAV-1, cav-1 gene expression was silenced using RNAi, and the cells were treated with OA to examine its efficacy. The results showed that after cav-1 silencing, OA had little effect on cell activity, apoptosis, the cell cycle and phosphorylation of HL-60 cells. This study is the first to show that CAV-1 plays a crucial role in the effects of OA on HL-60 cells.

  16. Gemtuzumab ozogamicin and olaparib exert synergistic cytotoxicity in CD33-positive HL-60 myeloid leukemia cells.

    PubMed

    Yamauchi, Takahiro; Uzui, Kanako; Nishi, Rie; Shigemi, Hiroko; Ueda, Takanori

    2014-10-01

    Gemtuzumab ozogamicin (GO) consists of the cluster of differentiation 33 (CD33) antibody linked to calicheamicin. The binding of GO to the CD33 antigen on leukemic cells results in internalization and subsequent release of calicheamicin, thereby inducing DNA strand breaks. We hypothesized that the poly (ADP-ribose) polymerase inhibitor olaparib might inhibit DNA repair initiated by GO-induced DNA strand breaks, thereby increasing cytotoxicity. The human myeloid leukemia cell line HL-60 and a GO-resistant variant (HL/GO20) were used. The 50% growth-inhibitory concentrations (IC50) were 24 ng/ml for HL-60 cells and 550 ng/ml for GO-resistant variant HL/GO20 cells. HL/GO20 cells were also refractory to GO-induced apoptosis. CD33 positivity was reduced in HL/GO20 cells. Olaparib-alone did not inhibit the cell growth and did not induce apoptosis in either HL-60 cells or HL/GO20 cells at concentrations of up to 10 μM. When cells were treated with different concentrations of GO in the presence of 10 μM olaparib, the IC50 of GO for HL-60 cells was 13 ng/ml. The combination index was 0.86, indicating synergistic cytotoxicity of GO and olaparib in combination. Such a combination was ineffective for HL/GO20 cells. GO and olaparib exerted synergistic cytotoxicity in CD33-positive myeloid leukemia cells in vitro. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  17. Pyranocoumarins isolated from Peucedanum praeruptorum as differentiation inducers in human leukemic HL-60 cells.

    PubMed

    Zhang, Jin-Xia; Fong, Wang-Fun; Wu, Jimmy Yiu-Cheong; Yang, Mengsu; Cheung, Hon-Yeung

    2003-03-01

    Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (+/-)-4'- O-acetyl-3'- O-angeloyl- cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 microg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1, but not p21 WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.

  18. Characterization of Regulatory Volume Decrease in the THP-1 and HL-60 Human Myelocytic Cell Lines

    DTIC Science & Technology

    1994-01-01

    swelling-induced changes transporters (for review, Sarkadi and Parker, 1991). in surface receptor numbers associated with inhibition Volume regulation...neutral transporter(s) ( Sarkadi and Parker, 1991), of the monocytic-macrophage lineage or in macroph- while in other cells, such as lymphocytes, K and C1...exchanger of the ing RVD in these cells is different from the pathway HL-60 cell. J. Gen. Physiol., 100:825-846. gRVD in lymphocytes. Sarkadi , B., and

  19. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment.

    PubMed

    Wang, Cong; Zhang, Qun; Gou, Bao-Di; Zhang, Tian-Lan; Wang, Kui

    2014-06-01

    Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-trans retinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of "differentiation induced by differentiated cells", we explored the feasibility of ex vivo therapy.

  20. Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells.

    PubMed

    Lin, Xin; Chen, Qiang; Xue, Li-Ying; Ma, Xiao-Jun; Wang, Rui

    2004-11-01

    Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.

  1. Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells.

    PubMed

    Hata, Tomona; Sakaguchi, Ikuyo; Mori, Masahiro; Ikeda, Norikazu; Kato, Yoshiko; Minamino, Miki; Watabe, Kazuhito

    2003-01-01

    Limonene is a primary component of citrus essential oils (EOs) and has been reported to induce apoptosis on tumor cells. Little is known about induction of apoptosis by citrus EOs. In this study, we examined induction of apoptosis by Citrus aurantium var. dulcis (sweet orange) EO, Citrus paradisi (grapefruit) EO and Citrus limon (lemon) EO. These EOs induced apoptosis in HL-60 cells and the apoptosis activities were related to the limonene content of the EOs. Moreover, sweet orange EO and grapefruit EO may contain components besides limonene that have apoptotic activity. To identify the components with apoptotic activity, grapefruit EO was fractionated using silica gel columns, and the components were analyzed by GC-MS. The n-hexane fraction contained limonene, and the dichloromethane fraction (DF) contained aldehyde compounds and nootkatone. Decanal, octanal and citral in the DF showed strong apoptotic activity, suggesting that the aldehyde compounds induced apoptosis strongly in HL-60 cells.

  2. Dehydrocostus lactone enhances tumor necrosis factor-alpha-induced apoptosis of human leukemia HL-60 cells.

    PubMed

    Oh, G S; Pae, H O; Chung, H T; Kwon, J W; Lee, J H; Kwon, T O; Kwon, S Y; Chon, B H; Yun, Young Gab

    2004-05-01

    Sesquiterpene lactones have raised considerable interest because of their ability to block the activation of nuclear transcription factor-kappaB (NF-kappaB). NF-kappaB plays an important role in the resistance of cancer cells to the induction of apoptosis by anticancer drugs and tumor necrosis factor-alpha (TNF-alpha). Pharmacological inhibition of NF-kappaB offers the promise of enhancing the efficacy of anticancer therapies. Here, we demonstrate that dehydrocostus lactone (DL), the major sesquiterpene lactone isolated from the roots of Saussurea lappa, inhibits NF-kappaB activation by preventing TNF-alpha-induced degradation and phosphorylation of its inhibitory protein I-kappaB alpha in human leukemia HL-60 cells and that DL renders HL-60 cells susceptible to TNF-alpha-induced apoptosis by enhancing caspase-8 and caspase-3 activities.

  3. Targeting thioredoxin reductase by plumbagin contributes to inducing apoptosis of HL-60 cells.

    PubMed

    Zhang, Junmin; Peng, Shoujiao; Li, Xinming; Liu, Ruijuan; Han, Xiao; Fang, Jianguo

    2017-04-01

    Plumbagin (PLB), a natural naphthoquinone from the traditional folk medicines Plumbago zeylanica, Dionaea muscipula, or Nepenthes gracilis, has been documented possessing a wide variety of pharmacological activities. Although PLB demonstrates anticancer activity in multiple types of malignant cells, the cellular targets of PLB have not been well defined and remained only partially understood. We reported here that PLB selectively inhibits TrxR and elicits reactive oxygen species in human promyelocytic leukemia HL-60 cells, which leads to elevation of GSSG/GSH ratio and decrease of cellular thiol pool. As a consequence, PLB disturbs the cellular redox homeostasis, induces oxidative stress-mediated apoptosis and eventually selectively kills HL-60 cells. Inhibition of TrxR by PLB thus discloses an unprecedented mechanism underlying the anticancer efficacy of PLB, and sheds light in considering the usage of PLB as a promising cancer therapeutic agent.

  4. Disodium cromoglycate suppresses the induction of cysteinyl leukotriene synthesis during granulocytic differentiation in HL-60 cells.

    PubMed

    Zaitsu, Masafumi; Honjo, Kinji; Ishii, Eiichi; Hamasaki, Yuhei

    2004-03-01

    Disodium cromoglycate (DSCG) and nedocromil sodium are anti-asthma drugs that have a variety of physiological and biological effects. We examined whether DSCG affects the induction of cysteinyl Leukotriene (cysLT) synthesis during dimethyl sulfoxide (DMSO)-induced granulocytic differentiation in HL-60 cells. HL-60 cells were differentiated to mature granulocyte-like cells by DMSO in the presence or absence of DSCG for 5 days. Then, we measured A23187-stimulated production of LTC4, an initial product of cysLTs. We also examined the mRNA expression and enzyme activity of LTC4 synthase and other LT-synthetic enzymes. The amount of LTC4 production was 732.0+/-19.0 pg/10(6) cells in DMSO-differentiated HL-60 cells. The value was significantly decreased to 420.7+/-22.7 pg/10(6) cells in the presence of DSCG at 100 microg/ml. The DMSO-induced mRNA expression and enzyme activity of LTC4 synthase was also suppressed by DSCG. Our results indicate that DSCG suppresses the DMSO-induced LTC4 synthase-activity by inhibiting mRNA expression of LTC4 synthase, which might be a novel anti-allergic action of DSCG.

  5. Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells

    SciTech Connect

    Boulay, F.; Mery, L.; Tardif, M.; Brouchon, L.; Vignais, P. )

    1991-03-26

    A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formlypeptide receptor is 34%.

  6. Increase of caspase-3 activity by lignans from Machilus thunbergii in HL-60 cells.

    PubMed

    Park, Bo-Young; Min, Byung-Sun; Kwon, Ok-Kyoung; Oh, Sei-Ryang; Ahn, Kyung-Seop; Kim, Tae-Jin; Kim, Doo-Young; Bae, KiWhan; Lee, Hyeong-Kyu

    2004-08-01

    Nine lignans and two butanolides were isolated from the stem bark of Machilus thunbergii and their structures were identified as machilin A (1), licarin B (2), zuonin B (3), macelignan (4), secoisolancifolide (5), isolancifolide (6), oleiferin C (7), meso-dihydroguaiaretic acid (8), licarin A (9), machilin F (10), and nectandrin B (11) by spectroscopic means. These compounds were assessed for their abilities to activate a caspase-3 activity in human promyeloid leukemic HL-60 cells. The intracellular caspase-3 activity of macelignan (4), oleiferin C (7), meso-dihydroguaiaretic acid (8), and licarin A (9) increased approximately 3.04, 6.16, 2.10, and 3.10-fold at 100 microM over that of untreated control. In addition, compounds 4, 7, 8, and 9 induced internucleosomal DNA fragmentation in HL-60 cells.

  7. Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

    PubMed Central

    Wang, Ming-Fu; Liao, Ya-Fan; Hung, Ying-Cheng; Lin, Chih-Li; Hour, Tzyh-Chyuan; Lue, Ko-Huang; Hung, Hui-Chih

    2011-01-01

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψm), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway. PMID:21372632

  8. The impact of Tegillarca granosa extract haishengsu on HL-60 cell

    PubMed Central

    Wang, Jinshen; Wei, Lixia; Han, Yueqin; Qin, Daogang; Yang, Qiaozhi

    2015-01-01

    Haishengsu (Hss) is a purified protein from Tegillarca granosa that has been used as a traditional Chinese medicine to treat cancer for more than a century. In this study, we observed the impact of Haishengsu (Hss) on the proliferation and differentiation of HL-60 cells in the leukemic cell line by taking tretinoin and AS2O3 as a positive control and making a comparative analysis between the effect of Hss and tretinoin and AS2O3. We found that Hss could significantly inhibit the proliferation of HL-60 cells and caused most of the cells to stay in the G0/G1 phase. Its effect was much stronger than that of tretinoin and AS2O3, and the ability of Hss to induce differentiation was close to tretinoin. Hss functions probably by inhibiting the expression of the Bcl-2 and MPO genes and further promoting the expression of the Bax gene. Hss has a significant effect on both inhibiting the proliferation and inducing the differentiation of HL-60 cells. It is possible that Hss may be a new kind of clinical differentiation inducer.

  9. The association between oxidative stress-induced galectins and differentiation of human promyelocytic HL-60 cells.

    PubMed

    Vinnai, James R; Cumming, Robert C; Thompson, Graham J; Timoshenko, Alexander V

    2017-06-15

    Galectins are multifunctional β-galactoside-binding proteins that are involved in the regulation of cellular stress responses and differentiation. The relationship between these processes is unclear and we report here that galectins display oxidative-stress specific expression patterns in neutrophil-like differentiated HL-60 cells. Three galectins (-1, -3, and -10) are upregulated in response to either menadione or DMSO exposure whereas galectins -9 and -12 exhibited a stimulus-dependent downregulation. Changes in galectin expression are oxidant dependent based on the observations that 1) oxidative stress biomarkers HMOX1 (heme oxygenase-1) and NCF1 (neutrophil cytosolic factor 1, which is also a biomarker of neutrophil differentiation) are elevated in both cases, and 2) the antioxidant N-acetyl-L-cysteine restores basal expression of galectin-3 following oxidant exposure. In addition, our results suggest that the regulation of oxidative stress-sensitive galectins involves DNA hypomethylation mechanisms. Expression of galectin-3 and galectin-12 exhibits an opposite relationship to the expression of HMOX1/NCF1, suggesting a stimulatory and inhibitory role of these galectins in neutrophil-like differentiation of HL-60 cells. We also show that the inhibition of galectins reduces the growth rate of HL-60 cells, and facilitates their neutrophil-like differentiation. Collectively, our findings indicate that the process of cellular differentiation implicates, in part, oxidative stress-sensitive galectins, which further highlights a biological significance of galectin network remodeling in cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Apoptosis inducing activity of 4-substituted coumarins from Calophyllum brasiliense in human leukaemia HL-60 cells.

    PubMed

    Ito, Chihiro; Murata, Tomiyasu; Itoigawa, Masataka; Nakao, Keisuke; Kaneda, Norio; Furukawa, Hiroshi

    2006-07-01

    With the objective of identifying anti-tumour-promoting agents, we carried out a primary screening of ten 4-substituted coumarins isolated from Calophyllum brasiliense Camb. (Guttiferae), to determine the ability of these compounds to inhibit proliferation of the human leukaemia cell line HL-60. Among the 4-substituted coumarins isolated, calophyllolide (2) and mammea B/BB (3) showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time-dependent manner after treatment with calophyllolide (2) or mammea B/BB (3). In addition, the activity of caspase-9 and caspase-3 was also enhanced in a time-dependent manner upon treatment with the 4-substituted coumarins 2 and 3. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by 4-substituted coumarins 2 and 3. These results suggest that calophyllolide (2) and mammea B/ BB (3) induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, which is triggered by mitochondrial dysfunction.

  11. Activation of caspase-3 in HL-60 cells treated with pyrithione and zinc.

    PubMed

    Kondoh, Masuo; Tasaki, Emi; Takiguchi, Masufumi; Higashimoto, Minoru; Watanabe, Yoshiteru; Sato, Masao

    2005-04-01

    The transition metal zinc (Zn) is an endogenous regulator of apoptosis. The ability of Zn to modulate apoptosis is believed to be mediated by the regulation of caspase activity. Previously, we reported that an acute influx of labile Zn induced apoptosis via activation of caspase in human leukemia HL-60 cells treated with a Zn ionophore (Py, pyrithione) and Zn at 1 and 25 microM, respectively. In the present study, we investigated the involvement of caspase-3 in Py (1 microM)/Zn (25 microM)-induced apoptosis in HL-60 cells. Pro-caspase-3 is an inactive form of caspase-3. The processing of pro-caspase-3, a sign of caspase-3 activation, occurred 6 h after treatment with Py/Zn. Proteolysis of poly (ADP-ribose) polymerase (PARP), a substrate of caspase-3, was also observed 6 h after treatment with Py/Zn. We also confirmed the elevation of caspase-3 activity as an index of the cleavage of amino acid sequences recognized by activated caspase-3. An inhibitor of caspase-3 attenuated the appearance of the DNA ladder. Taken together, these results indicate that the activation of caspase-3 is partly responsible for the induction of apoptosis in Py/Zn-treated HL-60 cells.

  12. Studies of leukotoxin from Actinobacillus actinomycetemcomitans using the promyelocytic HL-60 cell line.

    PubMed Central

    Zambon, J J; DeLuca, C; Slots, J; Genco, R J

    1983-01-01

    The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease. PMID:6572616

  13. Induction of apoptotic cell death in HL-60 cells by jacaranda seed oil derived fatty acids.

    PubMed

    Yamasaki, Masao; Motonaga, Chihiro; Yokoyama, Marino; Ikezaki, Aya; Kakihara, Tomoka; Hayasegawa, Rintaro; Yamasaki, Kaede; Sakono, Masanobu; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

    2013-01-01

    Various fatty acids are attracting considerable interest for their anticancer effects. Among them, fatty acids containing conjugated double bonds show one of the most potent cytotoxic effects on cancer cells. Here, we focused on the cancer cell killing activity of jacaranda seed oil. The seed oil of jacaranda harvested from Miyazaki in Japan contained 30.9% cis-8, trans-10, cis-12 octadecatrienoic acid, called jacaric acid (JA). Fatty acid prepared from this oil (JFA) and JA strongly induced cell death in human leukemia HL-60 cells. On the other hand, linoleic acid and trans-10, cis-12 conjugated linoleic acid (<10 μM) did not affect cell proliferation and viability. An increase in the sub-G₁ population and internucleosomal fragmentation of DNA was observed in JA- and JFA-treated cells, indicating induction of apoptotic cell death. Finally, the cytotoxic effects of JA and JFA were completely abolished by α-tocopherol. Taken together, these data suggest that jacaranda seed oil has potent apoptotic activity in HL-60 cells through induction of oxidative stress.

  14. Effects of PCB126 and PCB153 on telomerase activity and telomere length in undifferentiated and differentiated HL-60 cells

    PubMed Central

    Xin, Xing; Senthilkumar, PK; Schnoor, Jerald L.; Ludewig, Gabriele

    2015-01-01

    PCBs are persistent organic pollutants with carcinogenic, immune- and developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiate to granulocytes and monocytes. We exposed HL-60 cells to the PCB126 (dioxin-like) and PCB153 (non-dioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were down-regulated. The telomere shelterins TRF1, TRF2, and POT were upregulated in granulocytes, TRF2 was up- and POT1 downregulated in monocytes. Both PCBs reduced telomerase activity in undifferentiated cells and further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Long term treatment of undifferentiated HL-60 cells with PCB126 produced a downregulation of telomerase activity and decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153 the effects were less pronounced and some shelterin genes were increased after 30 days exposure. With each PCB no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell type and PCB congener specific effects on telomeres/telomerase-related genes and, although PCBs do not seem to strongly affect differentiation, they could influence the longevity of stem or progenitor cells through telomere attrition with potential long-term consequences for health. PMID:26330309

  15. Effects of PCB126 and PCB153 on telomerase activity and telomere length in undifferentiated and differentiated HL-60 cells.

    PubMed

    Xin, Xing; Senthilkumar, P K; Schnoor, Jerald L; Ludewig, Gabriele

    2016-02-01

    PCBs are persistent organic pollutants that are carcinogenic and immunotoxic and have developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex, thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiated into granulocytes and monocytes. We exposed HL-60 cells to PCB126 (dioxin-like) and PCB153 (nondioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were downregulated. The telomere shelterins TRF1, TRF2, and POT1 were upregulated in granulocytes, and TRF2 was upregulated and POT1 downregulated in monocytes. Both PCBs further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Treatment of undifferentiated HL-60 cells for 30 days with PCB126 produced a downregulation of telomerase activity and a decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153, the effects were less pronounced and some shelterin genes were increased after 30 days of exposure. With each PCB, no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell-type and PCB congener-specific effects on telomere/telomerase-related genes. Although PCBs do not seem to strongly affect differentiation, they may influence stem or progenitor cells through telomere attrition with potential long-term consequences for health.

  16. [Effect of mPGES-1 inhibitor MK886 on apoptosis and drug resistance of HL-60/A cells].

    PubMed

    Li, Yi-Qing; Yin, Song-Mei; Nie, Da-Nian; Xie, Shuang-Feng; Ma, Li-Ping; Wang, Xiu-Ju; Wu, Yu-Dan

    2012-08-01

    This study was aimed to investigate the effect of MK886, a mPGES-1 inhibitor, on apoptosis and drug resistance of leukemia HL-60/A cell line. Expression of mPGES-1 was assayed by QT-PCR and Western blot. The effect of MK886 on HL-60/A cell proliferation was assayed by CCK-8 method, and flow cytometry was used to detect cell apoptosis. The expression of Akt and P-Akt was detected by Western blot. PGE2 was measured by ELISA. Effect of MK886 (10 µmol/L) on the chemotherapeutic sensitivity of HL-60/A cells and expression of mdr-1 mRNA and P170 protein were investigated too. The results indicated the expression of mPGES-1 was higher in HL-60/A cells. MK886 inhibited HL-60/A cell proliferation and induced apoptosis in a time- and concentration-dependent manner. Expression of mPGES-1 and P-Akt and synthesis of PGE2 decreased significantly. MK886 reduced expression of mdr-1 and P170 protein and enhanced the sensitivity of HL-60/A cells to chemotherapeutic drugs. It is concluded that MK886 can inhibit HL-60/A cell proliferation, induce apoptosis and enhance sensitivity to chemotherapeutic drugs, the mechanism of which possibly associates to down-regulation of mPGES-1/PGE2 synthesis, reduction P-Akt expression and decreasing mdr-1 and P170 protein expression.

  17. Store-operated Ca²⁺ entry mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells under hypoxia.

    PubMed

    Ma, Shuang; Cai, Chunqing; Ma, Yan; Bai, Zhengzhong; Meng, Xiaojing; Yang, Xinyi; Zou, Fei; Ge, Rili

    2014-03-01

    The regulation of neutrophil polarization by calcium entry is critical for maintaining an effective host response. Hypoxia has a major effect on the apoptosis of neutrophils, however the role of store-operated Ca2+ entry (SOCE) in neutrophil polarization under hypoxia remains to be elucidated. In the present study, we examined the polarization of differentiated human neutrophil-like HL-60 (dHL-60) cells exposed to hypoxia (3% O2) and the results demonstrated that the percentage of polarized cells following exposure to an N-formyl-Met-Leu-Phe (fMLP) gradient in the Zigmond chamber was increased. We examined stromal interaction molecule 1 (STIM1) and Orai1 expression in dHL-60 cells during hypoxia, and it was observed that the expression of STIM1 and Orai1 was significantly reduced at day 2. However, no apparent change was observed on the first day, indicating that this effect is dependent on stimulation time. Fluo-4/acetoxymethyl (AM) ester imaging also demonstrated that SOCE was decreased in dHL-60 cells. The plasmid overexpression assay demonstrated that the response of polarization was returned to the control level. We demonstrated the inhibitory role of SOCE on the polarization of dHL-60 cells under hypoxic conditions, which may be the mechanism for the adaptation of neutrophils to hypoxia. SOCE is also suggested to be a key modulator of immune deficiency under hypoxic conditions and is potentially a therapeutic target.

  18. Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells.

    PubMed

    Honda, A; Yamamoto, Y; Mori, Y; Yamada, Y; Kikuchi, H

    1985-07-31

    To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.

  19. 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces accelerated production of chemokines in HL-60 cells.

    PubMed

    Kishimoto, Seishi; Kobayashi, Yuriko; Oka, Saori; Gokoh, Maiko; Waku, Keizo; Sugiura, Takayuki

    2004-04-01

    2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Previously, we provided evidence that 2-arachidonoylglycerol, but not anandamide (N-arachidonoylethanolamine), is the true natural ligand for the cannabinoid receptors. In the present study, we examined in detail the effects of 2-arachidonoylglycerol on the production of chemokines in human promyelocytic leukemia HL-60 cells. We found that 2-arachidonoylglycerol induced a marked acceleration in the production of interleukin 8. The effect of 2-arachidonoylglycerol was blocked by treatment of the cells with SR144528, a cannabinoid CB2 receptor antagonist, indicating that the effect of 2-arachidonoylglycerol is mediated through the CB2 receptor. Augmented production of interleukin 8 was also observed with CP55940, a synthetic cannabinoid, and an ether-linked analog of 2-arachidonoylglycerol. On the other hand, neither anandamide nor the free arachidonic acid induced the enhanced production of interleukin 8. A similar effect of 2-arachidonoylglycerol was observed in the case of the production of macrophage-chemotactic protein-1. The accelerated production of interleukin 8 by 2-arachidonoylglycerol was observed not only in undifferentiated HL-60 cells, but also in HL-60 cells differentiated into macrophage-like cells. Noticeably, 2-arachidonoylglycerol and lipopolysaccharide acted synergistically to induce the dramatically augmented production of interleukin 8. These results strongly suggest that the CB2 receptor and its physiological ligand, i.e., 2-arachidonoylglycerol, play important regulatory roles such as stimulation of the production of chemokines in inflammatory cells and immune-competent cells. Detailed studies on the cannabinoid receptor system are thus essential to gain a better understanding of the precise regulatory mechanisms of inflammatory reactions and immune responses.

  20. [Effect of quercetin on doxorubicin-induced expression of MDR1 gene in HL-60 cells].

    PubMed

    He, Hai-Lan; Ji, Li-Juan; Li, Qi-Zhi; Zhang, Rong; Huang, Jian-Ming; Li, Ge

    2015-02-01

    Leukemia cells can acquire a multidrug resistant (MDR) phenotype in response to a wide variety of chemotherapeutic agents including doxorubicin (Dox). In addition to the constitutive expression in the leukemia prior to chemotherapy, a complex phenotype of pleiotropic resistance is presented in the residual or recurrent leukemia. Recent studies showed Dox-induced coexpression of COX2 and MDR1 genes in human leukaemia cells, and whether Dox-induced MDR1 up-regulation in acute leukaemia cells is dependent on COX2-transcriptional activity and thus might be overcome or prevented with COX2-promotor inhibitor quercetin interfering with COX2 expression and activity. This study was purposed to investigate the impacts of quercetin on Dox-induced mRNA expression of MDR1 and COX2 genes in HL-60 leukemia cells. The MDR1 and COX2 mRNA expression in HL-60 cells was detected by RT-PCR; the prostaglandin E2 (PGE2) release was measured by ELISA; the cytotoxicity of Dox was determined by MTT test. The incubation of HL-60 cells with Dox not only up-regulated MDR1 mRNA, but also COX2 mRNA expression, and after co-incubation with quercetin or celecoxib, Dox-induced overexpression of MDR1 and COX2 mRNA were reduced by quercetin, not by celecoxib, whereas PGE2 release was significantly decreased with subsequent enhancement of Dox cytotoxic efficacy by both of them. Dox-induced MDR1 up-regulation may be dependent on COX2-transcriptional activity, not PGE2, suggesting that the existence of causal link between COX2 and MDR1 expression induced by Dox, and modulation of COX2 transcriptional expression by quercetin would not only sensitize leukemia cells to Dox, but also prevent the acquisition of MDR during chemotherapy.

  1. Synergistic growth inhibition in HL-60 cells by the combination of acyclic retinoid and vitamin K2.

    PubMed

    Kitagawa, Junichi; Hara, Takeshi; Tsurumi, Hisashi; Ninomiya, Soranobu; Ogawa, Kengo; Adachi, Seiji; Kanemura, Nobuhiro; Kasahara, Senji; Shimizu, Masahito; Moriwaki, Hisataka

    2011-05-01

    The aim of this study was to assess the effects of acyclic retinoid (ACR) and vitamin K(2) (VK(2)) in HL-60 cells. We used HL-60 cells, and the Trypan Blue dye exclusion method was used for cell proliferation assays. For detection of apoptosis, the Annexin V-binding capacity of treated cells was examined by flow cytometry. To evaluate the cell cycle, we used a FITC BrdU Flow KIT and flow cytometry. Total extracted and equivalent amounts of protein were examined by Western blotting using specific antibodies. ACR and VK(2) dose dependently inhibited the proliferation of HL-60 cells. These two agents in combination synergistically inhibited cell growth and induced apoptosis. VK(2) inhibited activation of the Ras/MAPK signaling pathway, and ACR plus VK(2) cooperatively inhibited phosphorylation of RXRα and the growth of HL-60 cells. Moreover, ACR and VK(2) induced increases in G0/G1 phase HL-60 cells, alone and synergistically in combination. The synergistic effects of ACR and VK(2) on HL-60 cells may provide a novel strategy for treating leukemia.

  2. Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells.

    PubMed

    González-Aragón, David; Ariza, Julia; Villalba, José M

    2007-02-01

    Dicoumarol, a competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), increases intracellular superoxide and affects cell growth of tumor cells. This work was set to establish a mechanistic link between dicoumarol, superoxide and cell cycle alterations in HL-60 cells. Using ES936, a mechanism-based irreversible inhibitor of NQO1, we demonstrate that NQO1 inhibition is not a major factor involved in superoxide boost. Mitochondrial Complexes II, III and IV were directly inhibited by dicoumarol. Succinate, which inhibits superoxide generation by reversed electron flow in Complex II, significantly decreased superoxide boost in dicoumarol-treated cells and in isolated mitochondria incubated with dicoumarol and decylubiquinol. Superoxide generation in cells was strongly potentiated by blocking the quinone site of Complex II with thenoyltrifluoroacetone, supporting the involvement of cytochrome b560 to drive electrons for increasing superoxide. Simultaneous inhibition of the mitochondrial chain upstream ubiquinone and displacement of succinate from the Complex II active site is proposed as a major mechanism to explain how dicoumarol increases superoxide in HL-60 cells. Dicoumarol-treated cells accumulated in S phase due to the impairment of pyrimidine biosynthesis at dihydroorotate dehydrogenase step because blockade was overcome by addition of exogenous uridine or orotate, but not by dihydroorotate. We demonstrate for the first time that dicoumarol inhibits mitochondrial electron transport, induces superoxide release by reversed electron flow in Complex II, and inhibits pyrimidines biosynthesis. These actions must be taken into account when considering dicoumarol effects on cells.

  3. Effects of Simulated Microgravity on Functions of Neutrophil-like HL-60 Cells

    NASA Astrophysics Data System (ADS)

    Wang, Chengzhi; Li, Ning; Zhang, Chen; Sun, Shujin; Gao, Yuxin; Long, Mian

    2015-11-01

    Altered gravity, especially microgravity affects cellular functions of immune cells and can result in immune dysfunction for long-term, manned spaceflight and space exploration. The underlying mechanism, however, of sensing and responding to the gravity alteration is poorly understood. Here, a rotary cell culture system (RCCS) bioreactor was used to elucidate the effects of simulated microgravity on polymorphonuclear neutrophils (PMN)-like HL-60 cells. Alteration of cell morphology, up-regulation of (nitric oxide) NO production, enhancement of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein 1 (MCP-1) secretion, and diversity of cellular adhesion molecule expression were observed for the cells cultured in RCCS, leading to the up-regulated inflammatory immune responses and host defense. It was also indicated that such alterations in biological responses of PMNs mediated the reduced rolling velocity and decreased adhesion of PMN-like HL-60 cells on endothelial cells under shear flow. This work furthers the understandings in the effects and mechanism of microgravity on PMN functions, which are potentially helpful for optimizing the countermeasures to immune suppression in the future long-term, manned spaceflight.

  4. Antiproliferative activity of various Uncaria tomentosa preparations on HL-60 promyelocytic leukemia cells.

    PubMed

    Pilarski, Radosław; Poczekaj-Kostrzewska, Magdalena; Ciesiołka, Danuta; Szyfter, Krzysztof; Gulewicz, Krzysztof

    2007-01-01

    The woody Amazonian vine Uncaria tomentosa (cat's claw) has been recently more and more popular all over the world as an immunomodulatory, antiinflammatory and anti-cancer remedy. This study investigates anti-proliferative potency of several cat's claw preparations with different quantitative and qualitative alkaloid contents on HL-60 acute promyelocytic human cells by applying trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT). By standardization and statistical comparison of the obtained results pteropodine and isomitraphylline are indicated to be most suitable for standardization of medical cat's claw preparations.

  5. Lanostanoid triterpenes from Laetiporus sulphureus and apoptosis induction on HL-60 human myeloid leukemia cells.

    PubMed

    León, Francisco; Quintana, José; Rivera, Augusto; Estévez, Francisco; Bermejo, Jaime

    2004-12-01

    A new lanostanoid triterpene, 3-oxosulfurenic acid (1), together with three known triterpenes (3, 4, and 7) were isolated from the fruit bodies of Laetiporus sulphureus. Cytotoxicity of these compounds and their derivatives (2, 5, and 6) was evaluated on HL-60 cells. Further studies revealed that acetyl eburicoic acid (5) was the most potent apoptosis inducer. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase-1 and was also associated with an early release of cytochrome c from the mitochondria.

  6. Xanthones from Garcinia paucinervis with in vitro anti-proliferative activity against HL-60 cells.

    PubMed

    Li, Da-Hong; Li, Chen-Xi; Jia, Cui-Cui; Sun, Ya-Ting; Xue, Chun-Mei; Bai, Jiao; Hua, Hui-Ming; Liu, Xiao-Qiu; Li, Zhan-Lin

    2016-02-01

    Three new xanthones, paucinervins H-J (1-3), as well as eleven known compounds (4-14), were isolated from the leaves of Garcinia paucinervis. The structures of the new compounds (1-3) were elucidated by 1D, 2D NMR spectra and HR ESIMS. In vitro antiproliferative activity against human promyelocytic leukemia HL-60 cells was tested, among which, compounds 2, 5, 6 and 7 exhibited strong growth inhibitory effects with GI50 values ranging from 1.30 to 9.08 μM, respectively. Preliminary SARs were also discussed.

  7. Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes.

    PubMed Central

    Mollinedo, F; Gajate, C; Tugores, A; Flores, I; Naranjo, J R

    1993-01-01

    Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate (PMA) for 30-60 min or with dimethyl sulphoxide (DMSO) for 24 h respectively. The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels, as well as with an increase in the binding activity of transcription factor AP-1. Nevertheless, gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes. When HL-60 cells were treated with sodium butyrate, which induced monocytic differentiation, a remarkable increase in AP-1 binding activity was detected. Treatment of HL-60 cells with 1 alpha,25-dihydroxyvitamin D3, another monocytic differentiation agent, induced a weak, but appreciable, increase in AP-1 activity. Furthermore, addition of sodium butyrate or 1 alpha,25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos, c-jun, jun B and jun D proto-oncogenes. In contrast, when HL-60 cells were treated with retinoic acid, a granulocytic differentiation inducer, no enhanced AP-1 binding activity was observed, and only a weak increase in jun D mRNA level was detected. These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes, whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity. The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8363564

  8. A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress.

    PubMed

    Plantin-Carrenard, E; Bringuier, A; Derappe, C; Pichon, J; Guillot, R; Bernard, M; Foglietti, M J; Feldmann, G; Aubery, M; Braut-Boucher, F

    2003-04-01

    A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.

  9. Induction of apoptosis by bleomycin in p53-null HL-60 leukemia cells.

    PubMed

    Gimonet, Delphine; Landais, Emilie; Bobichon, Helene; Coninx, Paul; Liautaud-Roger, Françoise

    2004-02-01

    The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

  10. NF-kappaB activation by triphenyltin triggers apoptosis in HL-60 cells.

    PubMed

    Marinovich, M; Viviani, B; Corsini, E; Ghilardi, F; Galli, C L

    1996-07-10

    Trisubstituted organotin pesticides are lethal for different cell types. In this study we investigated whether triphenyltin chloride (TPT) causes apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We report that 5 microM TPT increased intracellular Ca2+ in HL-60 cells within seconds; concomitantly actin depolymerization was detected 30 s and 1 min after the treatment. This was followed 15 min later by NF-kappaB activation, and apoptotic bodies and DNA fragmentation were evident after 3 and 6 h, respectively. At these times TPT also induced the release of tumor necrosis factor-alpha (TNF-alpha). Prior treatment of the cells with a polyclonal antibody to human TNF-alpha abolished TPT-induced DNA fragmentation, which suggests that the ultimate effect of TPT may be mediated by TNF-alpha. Prior treatment of the cells with 100 microM pyrrolidine dithiocarbamate, an antioxidant and potent inhibitor of NF-kappaB activation, prevented actin depolymerization, NF-kappaB activation, and DNA fragmentation, although it did not affect TPT-induced Ca2+ mobilization. These findings suggest that TPT increases intracellular Ca2+, alters actin polymerization and the cytoskeleton, and induces NF-kappaB activation, TNF-alpha synthesis, DNA degradation, and apoptosis. Reactive oxygen species seem to be essential to NF-kappaB activation, TNF-alpha synthesis, and the subsequent steps.

  11. Extracellular stress stimuli alter galectin expression profiles and adhesion characteristics of HL-60 cells.

    PubMed

    Timoshenko, A V; Lanteigne, J; Kozak, K

    2016-02-01

    Galectins, a family of soluble β-galactoside-binding proteins, are involved in the regulation of various cellular functions, which are essential for adaptive cellular stress responses (CSRs). Although expression patterns of galectins and galectin-binding glycans change during tissue development and cancer, the requirement and role of galectin networks in the CSRs are not completely understood. In this study, we report that the treatment of human promyelocytic HL-60 cells with stimuli mimicking hypoxia (CoCl2), inducing the endoplasmic reticulum stress (tunicamycin), and stimulating cell differentiation, result in stress-specific differential expression of galectin transcripts. In addition, we show that CoCl2 increases the expression of cell surface glycans recognized by both β-galactoside- and GlcNAc-binding lectins. Thus, microenvironmental stress changes the glycobiological status of cells representing expression profiles of endogenous lectins and corresponding glycans. These findings introduce a novel classification of galectins in HL-60 cells, which suggests diverse functions of galectin members in CSRs.

  12. Scavenging of superoxide anions by lecithinized superoxide dismutase in HL-60 cells.

    PubMed

    Ishihara, Tsutomu; Shibui, Misaki; Hoshi, Takaya; Mizushima, Tohru

    2016-01-01

    Superoxide dismutase covalently bound to four lecithin molecules (PC-SOD) has been found to have beneficial therapeutic effects in animal models of various diseases. However, the mechanism underlying these improved therapeutic effects has not yet been elucidated. It has previously been shown that PC-SOD localizes on the plasma membrane and in the lysosomes of cells. In this study, we evaluated the superoxide anion-scavenging activity of PC-SOD in HL-60 human promyelocytic leukemia cells. Compared to SOD, PC-SOD had only 17% scavenging activity in cell-free systems. Nevertheless, by analyzing enzyme activities in cell suspensions containing PC-SOD or SOD, PC-SOD and SOD showed almost equal activity for scavenging extracellular superoxide anions produced by HL-60 cells. Furthermore, the activity for scavenging extracellular superoxide anions increased with increased amount of PC-SOD on the plasma membrane. Moreover, PC-SOD exhibited no obvious inhibitory effect on the scavenging of intracellular superoxide anions. These results suggested that the association of PC-SOD with the plasma membrane plays a key role in its beneficial therapeutic effects. Thus, this finding may provide a rationale for selecting target diseases for PC-SOD treatment.

  13. Multidrug resistance protein 1 (ABCC1) confers resistance to arsenic compounds in human myeloid leukemic HL-60 cells.

    PubMed

    Xu, Shi; Zhang, Yan Fang; Carew, Micheal W; Hao, Wen Hui; Loo, Jacky Fong Chuen; Naranmandura, Hua; Le, X Chris

    2013-06-01

    Arsenic trioxide (As(2)O(3)) is established as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia, as well as other types of malignant tumors. However, HL-60 cells are resistant to As(2)O(3), and little is known about the underlying resistance mechanism for As(2)O(3) and its biomethylation products, namely, monomethylarsonous acid (MMA(III)) on the treatment of tumors. In the present study, we investigated the molecular mechanisms underlying iAs(III) and its intermediate metabolite MMA(III)-induced anticancer effects in the HL-60 cells. Here, we show that the HL-60 cells exhibit resistance to inorganic iAs(III) (IC(50) = 10 μM), but are relatively sensitive to its intermediate MMA(III) (IC(50) = 3.5 μM). Moreover, we found that the multidrug resistance protein 1 (MRP1), but not MRP2, is expressed in HL-60 cells, which reduced the intracellular arsenic accumulation, and conferred resistance to inorganic iAs(III) and MMA(III). Pretreatment of HL-60 with MK571, an inhibitor of MRP1, significantly increased iAs(III) and MMA(III)-induced cytotoxicity and arsenic accumulations, suggesting that the expression of MRP1/4 may lead to HL-60 cells resistance to trivalent arsenic compounds.

  14. Inhibition of Ser/Thr phosphatase PPM1D induces neutrophil differentiation in HL-60 cells.

    PubMed

    Kamada, Rui; Kudoh, Fuki; Yoshimura, Fumihiko; Tanino, Keiji; Sakaguchi, Kazuyasu

    2017-05-09

    Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy. It has been reported that PPM1D knockout mice exhibit neutrophilia in blood and show a defective immune response. Here, we found that inhibition of PPM1D induced neutrophil differentiation of human promyelocytic leukemia cell line HL-60. The combination of a PPM1D inhibitor and all-trans retinoic acid significantly increased their differentiation efficiency. The PPM1D inhibitor also induced G1 arrest in HL-60 cells. Our results suggest that PPM1D may be a potential therapeutic target for blood cell diseases including leukemia. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. A nuclear protein associated with lethal heat shock of HL-60 cells.

    PubMed

    Pipkin, J L; Hinson, W G; Lyn-Cook, L E; Burns, E R; Sheehan, D; Casciano, D A

    1992-09-01

    The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.

  16. Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells.

    PubMed

    Vávrová, Jirina; Mareková-Rezácová, Martina; Vokurková, Doris; Szkanderová, Sylva; Psutka, Jan

    2003-10-01

    Most cell lines that lack functional p53 protein are arrested in the G(2) phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G(2) phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G(2) phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D(0) value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G(2) phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D(0)=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5-7 post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.

  17. Subcellular localization of DJ-1 in human HL-60 leukemia cells in response to diallyl disulfide treatment

    PubMed Central

    Li, Qingye; Tang, Yuxian; Qin, Jing; Yi, Lan; Yang, Yening; Wang, Juan; He, Jie; Su, Qi; Tan, Hui

    2016-01-01

    Diallyl disulfide (DADS) has been demonstrated to exert potent anticancer effects in vitro and in vivo. Previous studies indicate that DADS may induce the differentiation and/or apoptosis of human leukemia cells in vitro. However, the mechanisms underlying these anticancer effects remain elusive. The aim of the present study was to investigate alterations in the subcellular localization of protein deglycase DJ-1 (also known as Parkinsonism associated deglycase-7, PARK-7) in the cytoplasm, nucleus and mitochondria of human leukemia HL-60 cells induced by DADS, in order to provide novel experimental evidence for the molecular mechanisms underlying the anticancer mechanisms of DADS in leukemia cells. HL-60 cells induced by DADS were collected at different time points, and proteins from the cytoplasm, nucleus and mitochondria of the cells were isolated using specific cellular component isolation kits. The protein expression levels of DJ-1 in these subcellular fractions of HL60 cells following exposure to DADS for varying lengths of time, were determined using western blotting, immunocytochemistry and immunofluorescence techniques. Following exposure of HL-60 cells to 1.25 mg/l DADS for 8 h, the protein expression levels of DJ-1 were significantly decreased in the cytoplasm, while nuclear fractions exhibited a significant increase in DJ-1 expression when compared with untreated controls. The protein expression levels of DJ-1 in mitochondria of HL-60 cells were significantly decreased following treatment with 5 and 10 mg/l DADS. These results demonstrate that exposure of HL-60 cells to low concentrations of DADS may promote DJ-1 protein translocation from the cytoplasm to the nucleus, which suggests that DJ-1 may function as a transcription factor or cofactor binding protein in the process of cell differentiation. The expression of DJ-1 in mitochondria may be associated with induction of apoptosis in HL-60 cells treated with moderate doses of DADS. PMID:27748821

  18. Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

    A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low. Images PMID:2476665

  19. Targeting miR-155 suppresses proliferation and induces apoptosis of HL-60 cells by targeting Slug/PUMA signal.

    PubMed

    Liang, Hui; Dong, Ziyan; Liu, Jiang-Feng; Chuang, Wei; Gao, Li-Zhen; Ren, Yu-Guo

    2016-10-27

    Recent studies have shown that high miR-155 expression was associated with poor prognosis in patients with acute myelogeneous leukemia (AML). Furthermore, targeting miR-155 results in monocytic differentiation and apoptosis. However, the exact role and mechanisms of miR-155 in human AML remains speculative. HL-60 cells were treated with anti-miR-155 for 72 h. Cell growth and apoptosis in vitro were detected by MTT, BrdU proliferation, colony formation and flow cytometry assay. The effect of anti-miR-155 on growth of HL-60 cells was also evaluated in a leukemia mouse model. Slug cDNA and PUMA siRNA trannsfection was used to assess the signal pathway. Different protein expression was detected by western blot assay and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. The results shown that targeting miR-155 resulted in a 24-fold decrease of miR-155 expression compared to negative control in the HL-60 cells. Targeting miR-155 significantly downregulated Slug and upregulated PUMA expression, and decreased HL-60 cell growth by 70% , impaired colony formation by approximately 60%, and increased HL-60 cell apoptosis by 45%. Targeting PUMA reversed miR-155 sliencing-induced proliferation and apoptosis of HL-60 cells. Restoration of Slug decreased PUMA expression. In murine engraftment models of HL-60 cells, we showed that targeting miR-155 was able to reduce tumor growth. This was accompanied with decreased Slug expression and increased PUMA expression in these tumors. Collectively, our findings strongly suggest targeting miR-155 exhibited in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of Slug expression and increase of PUMA expression.

  20. Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.

    PubMed Central

    Nervi, C; Grippo, J F; Sherman, M I; George, M D; Jetten, A M

    1989-01-01

    Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells. Images PMID:2548190

  1. Involvement of proapoptotic Bcl-2 family members in terbinafine-induced mitochondrial dysfunction and apoptosis in HL60 cells.

    PubMed

    Yang, Kuo-Ching; Wu, Chi-Chen; Wu, Chih-Hsiung; Chen, Jur-Hao; Chu, Chien-Hwa; Chen, Chien-Ho; Chou, Yean-Hwei; Wang, Ying-Jan; Lee, Wen-Sen; Tseng, How; Lin, Shyr-Yi; Lee, Chia-Hwa; Ho, Yuan-Soon

    2006-02-01

    Terbinafine (TB, lamisil), a promising world widely used oral-anti-fungal agent, has been used in the treatment of superficial mycosis. In this study, we found that apoptosis but not cell growth arrest was induced by TB (1 microM, for 24 h) in human promyelocytic leukemia (HL60) cells. The apoptotic effect induced by TB in the HL60 cell was not through the general differentiation mechanisms evidenced by evaluation of three recognized markers, including CD11b, CD33, and morphological features. In addition, our results also revealed that TB-induced apoptosis was not through the cellular surface CD 95 receptor-mediated signaling pathway. We found that the mitochondria membrane in the TB-treated HL60 cells was dissipated by decreasing of the electrochemical gradient (DeltaPsi(m)) led to leakage of cytochrome c from mitochondria into cytosol. Such effects were completely blocked by in vitro transfection of the HL60 cells with Bcl-2 overexpression plasmid (HL60/Bcl-2). However, our data found that TB-mediated apoptosis could not be completely prevented in the Bcl-2 over expressed (HL60/Bcl-2) cells. Such results implied that additional mediators (such as caspase-9) other than mitochondria membrane permeability might contribute to the TB-induced cellular apoptosis signaling. This hypothesis was supported by the evidence that administration of caspases-9 specific inhibitor (z-LEHD-fmk) blocked the TB-induced apoptosis. Our studies highlight the molecular mechanisms of TB-induced apoptosis in human promyelocytic leukemia (HL60) cells.

  2. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

    PubMed Central

    Šalovská, Barbora; Fabrik, Ivo; Ďurišová, Kamila; Link, Marek; Vávrová, Jiřina; Řezáčová, Martina; Tichý, Aleš

    2014-01-01

    DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells. PMID:25003641

  3. Epigarcinol and isogarcinol isolated from the root of Garcinia ovalifolia induce apoptosis of human promyelocytic leukemia (HL-60 cells).

    PubMed

    Pieme, Constant Anatole; Ambassa, Pathaleon; Yankep, Emmanuel; Saxena, Ajit Kumar

    2015-11-23

    Plants from garcinia genus have been used for centuries against several diseases. This study aimed to investigate the mechanism of apoptosis induced by epigarcinol and isogarcinol isolated from the root of Garcinia ovalifolia (Clusiaceae) on human promyelocytic leukemia (HL-60 cells). Epigarcinol and isogarcinol were isolated from the root of G. ovalifolia by using column chromatography method. The antiproliferative property of these molecules and fractions were assessed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The light fluorescence microscope was utilized to observe the morphological changes of HL-60 cells after 24 h treatment. Early apoptosis and cell cycle distribution were analyzed by using flow cytometry (FCM). The results showed that epigarcinol and isogarcinol inhibited the proliferation of HL-60 and PC-3 cells in a concentration-dependent manner with IC50 varying between 4 and 76 µg/mL depending on the cell line and the molecule. The apoptosis rate and the number of apoptotic cells significantly increased with the augmentation of the concentration of the molecules. The results of flow cytometry (FCM) indicated that epigarcinol and isogarcinol induced significant G2/S arrest of HL-60 cells, the disruption of mitochondrial membrane potential and reactive oxygen species (ROS) generation. These results indicated that epigarcinol and isogarcinol demonstrated in vitro antiproliferative properties and induce apoptosis of HL-60 cells which is related to the G2/S arrest, and it exerts its apoptotic effect through the loosing of mitochondrial membrane potential.

  4. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

    PubMed

    Kachalaki, Saeed; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi; Shanehbandi, Dariush; Mohammadinejad, Sina; Mansoori, Behzad

    2015-10-01

    Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells. Copyright © 2015. Published by Elsevier Masson SAS.

  5. NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

    PubMed Central

    Kuwabara, Wilson Mitsuo Tatagiba; Zhang, Liling; Schuiki, Irmgard; Curi, Rui; Volchuk, Allen; Alba-Loureiro, Tatiana Carolina

    2015-01-01

    Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells. PMID:25668518

  6. Dual mechanism of daunorubicin-induced cell death in both sensitive and MDR-resistant HL-60 cells

    PubMed Central

    Côme, M-G; Skladanowski, A; Larsen, A K; Laurent, G

    1999-01-01

    Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance. This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation. However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis. To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity. Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells. However, at equitoxic doses, similar cell death pathways were observed. In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5–1 μM and delayed mitotic cell death at 0.1 μM. These concentrations are within the clinical dose range. Similarly, HL-60/Vinc cells underwent apoptosis at 50–100 μM daunorubicin and mitotic cell death at 10 μM. These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells. Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent. Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias. © 1999 Cancer Research Campaign PMID:10098741

  7. Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation.

    PubMed

    Chen, Hongmei; Zhang, Bo; Yuan, Xuan; Yao, Ying; Zhao, Hong; Sun, Xiling; Zheng, Quisheng

    2013-11-01

    To evaluate the role of redox homeostasis in differentiation in human promyelocytic leukemia cells (HL-60) induced by isoliquiritigenin (ISL) through modulation of the nuclear erythroid-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway. Morphological changes, cell surface markers CD11b/CD14, and nitroblue tetrazolium (NBT)-reducing ability were used to determine the differentiation of HL-60, and 2,7-dichlorofluorescein was used to detect the level of intracellular reactive oxygen species (ROS). Thiobarbituric acid test was utilised to determine the levels of malondialdehyde production in ISL-treated HL-60. The study determines and presents the redox state of the ratio of reduced/oxidised glutathione as a consequence of progression from differentiation in HL-60. Expression levels of the Nrf2/ARE downstream target genes were determined by quantitative polymerase chain reaction. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitors, apocynin (APO), and diphenyleneiodonium (DPI) were used for the preliminary study to determine the potential downstream targets regulated by NADPH oxidase in ISL-induced HL-60 differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of HL-60 differentiation, namely, morphology changes, NBT reductive activities, and expression levels of surface antigens CD11b/CD14. Intercellular redox homeostasis changes toward oxidation during drug exposure are necessary to support ISL-induced differentiation. The unique expression levels of the Nrf2/ARE downstream target genes in the differentiation of HL-60 recorded a statistically significant and dose-dependent increase (P < 0.05), which were suppressed by NADPH oxidase inhibitor, APO, and DPI. ISL as a differentiation-inducing agent with mechanisms involved in the Nrf2/ARE pathway to modulate intercellular redox homeostasis, and thus, facilitate differentiation.

  8. [Effect of the NHE-1-specific inhibitor DMA on pHi, proliferation and apoptosis of HL-60/ADM cells in vitro].

    PubMed

    Chang, Cheng; Kong, Pei-Yan; Chen, Xing-Hua; Peng, Xian-Gui; Liu, Lin; Liu, Hong; Zeng, Dong-Feng; Liang, Xue; Wang, Qing-Yu

    2006-06-01

    The aim of this study was to evaluate the effect of dimethyl amiloride (DMA), a specific inhibitor of Na(+)/H(+) exchanger-1 (NHE-1), on intracellular pH value (pHi), proliferation and apoptosis of HL-60/ADM cells in vitro. After treatment with DMA at different doses, pHi of HL-60 and HL-60/ADM cell lines were determined by using pH-sensitive fluorescence dye BECEF-AM; the rate of growth inhibition of cells was detected with MTT assay; cell cycle was detected by flow cytometric DNA analysis; cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results showed that pHi in HL-60/ADM cells was higher than that in HL-60 cells. After treatment with DMA at different doses, pHi decreased, the rate of growth inhibition and the rate of apoptotic cells in HL-60/ADM cells were all higher than those in HL-60 cells. Meanwhile, after treatment with DMA during 100 micromol/L to 150 micromol/L, the increase amplitude of G(0)/G(1) phase cells and the decrease amplitude of S + G(2)/M cells in HL-60/ADM cells were higher than those in HL-60 cells. It is concluded that by causing intracellular acidification, the NHE-1-specific inhibitor DMA inhibits proliferation of HL-60/ADM cells and induces apoptosis of HL-60/ADM cells, and the degree of this growth inhibition of HL-60/ADM cells is higher than that of HL-60 cells.

  9. Effect of bixin on DNA damage and cell death induced by doxorubicin in HL60 cell line.

    PubMed

    Santos, G C; Almeida, M R; Antunes, Lmg; Bianchi, Mlp

    2016-12-01

    Bixin is a natural red pigment extracted from annatto. Although it is widely used as a coloring agent in food, there are few studies about the effect of this carotenoid on DNA. This study aimed to investigate the effects of bixin on cytotoxicity and genotoxicity induced by doxorubicin in HL60 cells. At concentrations above 0.3 μg/mL, bixin demonstrated cytotoxic effects in HL60 cells. Furthermore, this carotenoid was neither mutagenic nor genotoxic to HL60 cells and reduced the DNA damage induced by doxorubicin. Bixin and doxorubicin showed no apoptotic effect in HL60 cells, but the simultaneous combined treatments showed an increase in the percentage of apoptotic cells. In conclusion, our results showed that bixin modulates the cytotoxicity of doxorubicin via induction of apoptosis. The results of this study provide more knowledge about the toxic effects of anticancer treatments and how the natural compounds can be useful on these therapeutic approaches. © The Author(s) 2016.

  10. [Change of oxygen free radical in reversing multidrug-resistance of human leukemic cell line HL-60/ADM by cyclosporin A].

    PubMed

    Xu, Yu-Qiao; Hui, Yan-Ping; Ma, Shi-Rong; Wang, Ying-Mei

    2008-10-01

    This study was aimed to evaluate the reversed effects of cyclosporin A (CsA) on multidrug resistance (MDR) of human leukemic cell line HL-60/ADM, and to investigate the relationship of the oxygen free radical content between HL-60/ADM cells and the reversed HL-60/ADM cells (HL-60/ADM + CsA). The cytotoxicity and the reversed effects of CsA on multidrug resistance of human leukemic cell line HL-60/ADM were studied by MTT, flow cytometry (FCM) and immunohistochemical assay; the oxygen free radical for HL-60/ADM and HL-60/ADM + CsA cell lines were detected by colorimetric method. The results showed that the CsA less than 4 microg/ml had no significant cytotoxicity on HL-60/ADM, while the cytotoxicity was rised with CsA concentration increasing; And CsA (4 microg/ml) combined with ADM (1 microg/ml) could obviously restrain the growth of HL-60/ADM cells (p < 0.001). The P-gp expression of HL-60/ADM decreased obviously after exposure to CsA (4 microg/ml) for 72 hours, at the same cell conditions, MDA concentration of the reversed groups (HL-60/ADM + CsA cells) was higher than that of the control groups (HL-60/ADM cells) (p < 0.05), while the levels of SOD and GSH in the reversed groups were significantly lower than that in control groups (p < 0.001). It is concluded that MDR of HL-60/ADM can be reversed effectively by low dose of CsA, the level of oxygen free radical increases and the activity of antioxidants decreases in the reversed cells. Oxygen free radicals may be involved in this reverse process, which thereby lead to the cell death.

  11. Ganoderma lucidum polysaccharide exerts anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

    PubMed

    Yang, Guohua; Yang, Lei; Zhuang, Yun; Qian, Xifeng; Shen, Yunfeng

    2016-01-01

    In this study, we investigated the anti-tumor activity both in vitro and in vivo of a polysaccharide obtained from Ganoderma lucidum on HL-60 acute myeloid leukemia cells, and focused on its targeting effect on mitogen-activated protein kinase (MAPK) pathways. It was found by the methods such as western blot and flow cytometry (FCM), that G. lucidum polysaccharide (GLP) blocked the extracellular signal-regulated kinase/MAPK signaling pathway, simultaneously activated p38 and JNK MAPK pathways, and therefore regulated their downstream genes and proteins, including p53, c-myc, c-fos, c-jun, Bcl-2, Bax, cleaved caspase-3 and cyclin D1. As a result, cycle arrest and apoptosis of HL-60 cells were induced. Therefore, GLP exerted anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

  12. Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

    PubMed Central

    Baud, L; Goetzl, E J; Koo, C H

    1987-01-01

    The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane. PMID:3477571

  13. Examining the Lateral Displacement of HL60 Cells Rolling on Asymmetric P-Selectin Patterns

    PubMed Central

    Lee, Chia-Hua; Bose, Suman; Van Vliet, Krystyn J.; Karp, Jeffrey M.; Karnik, Rohit

    2011-01-01

    The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand–receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm2), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P

  14. Flow-Through Electroporation of HL-60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes.

    PubMed

    Chen, Zhiqiang; Akenhead, Michael A; Sun, Xinghua; Sapper, Harrison; Shin, Hainsworth Y; Hinds, Bruce J

    2016-08-01

    A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10μm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system.

    PubMed

    Fernández-Ayala, Daniel J M; Brea-Calvo, Gloria; López-Lluch, Guillermo; Navas, Plácido

    2005-07-30

    Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q(10) and exogenous Q(9) among cell membranes in human HL-60 cells. Endogenous Q(10) synthesized using the radiolabelled precursor [(14)C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [(14)C]-Q(10). Brefeldin A prevented Q(10) incorporation in plasma membrane. Exogenous Q(9) was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.

  16. HL-60 cells can be made copper deficient by incubating with tetraethylenepentamine.

    PubMed

    Percival, S S; Layden-Patrice, M

    1992-12-01

    A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.

  17. The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro.

    PubMed

    Li, Xiao-Ling; Zhao, Yan-Xia; Sun, Li-Rong; Yang, Jing; Xu, Hui-Juan

    2012-10-01

    Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

  18. Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells.

    PubMed

    Parker, Amber; Cuddihy, Sarah L; Son, Tae G; Vissers, Margreet C M; Winterbourn, Christine C

    2011-10-01

    Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H(2)O(2) to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H(2)O(2) was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.

  19. Ultraviolet light-emitting diode irradiation-induced cell death in HL-60 human leukemia cells in vitro

    PubMed Central

    XIE, DONG; SUN, YAN; WANG, LINGZHEN; LI, XIAOLING; ZANG, CHUANNONG; ZHI, YUNLAI; SUN, LIRONG

    2016-01-01

    Ultraviolet (UV) radiation is considered to be a potent cell-damaging agent in various cell lineages; however, the effect of UV light-emitting diode (LED) irradiation on human cells remains unclear. The aim of the present study was to examine the effect of UV LED irradiation emitting at 280 nm on cultured HL-60 human leukemia cells, and to explore the underlying mechanisms. HL-60 cells were irradiated with UV LED (8, 15, 30 and 60 J/m2) and incubated for 2 h after irradiation. The rates of cell proliferation and apoptosis, the cell cycle profiles and the mRNA expression of B-cell lymphoma 2 (Bcl-2) were detected using cell counting kit-8, multicaspase assays, propidium iodide staining and reverse transcription-quantitative polymerase chain reaction, respectively. The results showed that UV LED irradiation (8–60 J/m2) inhibited the proliferation of HL-60 cells in a dose-dependent manner. UV LED at 8–30 J/m2 induced dose-dependent apoptosis and G0/G1 cell cycle arrest, and inhibited the expression of Bcl-2 mRNA, while UV LED at 60 J/m2 induced necrosis. In conclusion, 280 nm UV LED irradiation inhibits proliferation and induces apoptosis and necrosis in cultured HL-60 cells. In addition, the cell cycle arrest at the G0/G1 phase and the downregulation of Bcl-2 mRNA expression were shown to be involved in UV LED-induced apoptosis. PMID:26820261

  20. Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation.

    PubMed

    Diez-Bello, R; Jardin, I; Salido, G M; Rosado, J A

    2016-11-16

    Store-operated Ca(2+) entry (SOCE) is a major mechanism for the regulation of intracellular Ca(2+) homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca(2+) imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

  1. 18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

    PubMed

    Huang, Yi-Chang; Kuo, Chao-Lin; Lu, Kung-Wen; Lin, Jen-Jyh; Yang, Jiun-Long; Wu, Rick Sai-Chuen; Wu, Ping-Ping; Chung, Jing-Gung

    2016-07-01

    In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC50 value of 100 μM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (ΔΨm) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.

  2. In vitro antileukaemic activity of extracts from chokeberry (Aronia melanocarpa [Michx] Elliott) and mulberry (Morus alba L.) leaves against sensitive and multidrug resistant HL60 cells.

    PubMed

    Skupień, Katarzyna; Kostrzewa-Nowak, Dorota; Oszmiański, Jan; Tarasiuk, Jolanta

    2008-05-01

    The aim of the present study was to determine in vitro antileukaemic activities of extracts obtained from chokeberry (Aronia melanocarpa [Michx] Elliot) and mulberry (Morus alba L.) leaves against promyelocytic HL60 cell line and its multidrug resistant sublines exhibiting two different MDR phenotypes: HL60/VINC (overexpressing P-glycoprotein) and HL60/DOX (overexpressing MRP1 protein). It was found that the extracts from chokeberry and mulberry leaves were active against the sensitive leukaemic cell line HL60 and retained the in vitro activity against multidrug resistant sublines (HL60/VINC and HL60/DOX). The values of resistance factor (RF) found for these extracts were very low lying in the range 1.2-1.6.

  3. Enhancement of the incorporation of 5-fluorodeoxyuridylate into DNA of HL-60 cells by metabolic modulations

    SciTech Connect

    Tanaka, M.; Kimura, K.; Yoshida, S.

    1983-11-01

    The exposure of HL-60 human promyelocytic leukemia cells to 0.5 microM 5-fluoro-2'-(/sup 3/H)deoxyuridine (FdUrd) for 16 hr resulted in the incorporation of 5.14 +/- 0.31 (S.D.) X 10(-7) mol FdUrd into DNA per mol of DNA nucleotide, which corresponds to 0.146 +/- 0.082 pmol FdUrd per 10(7) cells. Pretreatment with 50 microM deoxythymidine for 24 hr led to a 2.7-fold increase in the incorporation of this analogue into newly synthesized DNA during the ensuing 16-hr exposure to 0.5 microM (/sup 3/H)FdUrd. Pretreatment with 0.5 microM methotrexate for 3 hr also increased the (/sup 3/H)FdUrd incorporation into newly synthesized DNA approximately 5-fold. The coexistence of deoxythymidine or methotrexate with (/sup 3/H)FdUrd, however, led to decreased incorporation of FdUrd into DNA. More than 50% of the radioactivity in DNA separated by Cs2SO4 equilibrium density gradient centrifugation was proven to be fluorodeoxyuridylate by means of its binding to Lactobacillus casei deoxythymidine monophosphate synthetase.

  4. Quantitative Proteomic Analysis Revealed Lovastatin-induced Perturbation of Cellular Pathways in HL-60 Cells

    PubMed Central

    Dong, Xiaoli; Xiao, Yongsheng; Jiang, Xinning; Wang, Yinsheng

    2011-01-01

    Lovastatin, a member of the statin family of drugs, is widely prescribed for treating hypercholesterolemia. Statin family of drugs, however, also show promise for cancer treatment and prevention. Although lovastatin is known to be an inhibitor for HMG-CoA reductase, the precise mechanisms underlying the drug’s antiproliferative activity remain unclearly defined. Here we utilized mass spectrometry, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to analyze the perturbation of protein expression in HL-60 cells treated with lovastatin. We were able to quantify ~3200 proteins with both forward and reverse SILAC labeling experiments, among which ~120 exhibited significant alterations in expression levels upon lovastatin treatment. Apart from confirming the expected inhibition of cholesterol biosynthesis pathway, our quantitative proteomic results revealed that lovastatin perturbed estrogen receptor signaling pathway, which was manifested by the diminished expression of estrogen receptor α, steroid receptor RNA activator 1 and other related proteins. Lovastatin also altered glutamate metabolism through down-regulation of glutamine synthetase and γ-glutamylcysteine synthetase. Moreover, lovastatin treatment led to a marked down-regulation of carbonate dehydratase II (a.k.a. carbonic anhydrase II) and perturbed the protein ubiquitination pathway. Together, the results from the present study underscored several new cellular pathways perturbed by lovastatin. PMID:21967149

  5. Regulation of shear stress on rolling behaviors of HL-60 cells on P-selectin

    NASA Astrophysics Data System (ADS)

    Ling, YingChen; Fang, Ying; Yang, XiaoFang; Li, QuHuan; Lin, QinYong; Wu, JianHua

    2014-10-01

    Circulating leukocytes in trafficking to the inflammatory sites, will be first tether to, and then roll on the vascular surface. This event is mediated through specific interaction of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), and regulated by hemodynamics. Poor data were reported in understanding P-selectin-mediated rolling. With the flow chamber technique, we herein observed HL-60 cell rolling on P-selectin with or without 3% Ficoll at various wall shear stresses from 0.05 to 0.4 dyn/cm2. The results demonstrated that force rather than transport regulated the rolling, similar to rolling on L- and E-selectin. The rolling was accelerated quickly by an increasing force below the optimal shear threshold of 0.15 dyn/cm2 first and then followed by a slowly decelerating phase starting at the optimum, showing a catch-slip transition and serving as a mechanism for the rolling. The catch-slip transition was completely reflected to the tether lifetime and other rolling parameters, such as the mean and fractional stop time. The narrow catch bond regime stabilized the rolling quickly, through steeply increasing fractional stop time to a plateau of about 0.85. Data presented here suggest that the low shear stress threshold serves as a mechanism for most cell rolling events through P-selectin.

  6. Luminol-dependent chemiluminescence of human phagocyte cell lines: comparison between DMSO differentiated PLB 985 and HL 60 cells.

    PubMed

    Ashkenazi, Avraham; Marks, Robert S

    2009-01-01

    The human promyelocytic leukemia HL 60 and PLB 985 cell lines can differentiate into terminally mature neutrophil-like cells via dimethyl sulfoxide (DMSO) induction. In this study the luminol-dependent chemiluminescence (LCL) of both neutrophil-like cells was analayzed and compared in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulants. It was shown that, like human blood neutrophils, both neutrophil-like cells expressed high levels of CD11b, but unlike human blood neutrophils these cells almost lack LCL-detectable intracellular oxidase activity. By studying the pattern of activation to OZ and PMA and priming with GM-CSF, we concluded that there is no difference between the percentage of differentiation and function of DMSO-induced HL 60 and PLB 985. However, the LCL capacity (area under the curve) of DMSO induced PLB 985 cells was higher than that of HL 60 cells in response to both PMA and OZ, which implies a higher capacity to generate reactive oxygen species in PLB 985 cells.

  7. N,N-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells, to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome c and AIF.

    PubMed

    Kim, Byeong Mo; Choi, Yun Jung; Lee, Yong Heon; Joe, Young Ae; Hong, Sung Hee

    2010-08-01

    Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.

  8. Identification of transcription factors expressed during ATRA-induced neutrophil differentiation of HL60 cells.

    PubMed

    Mills, K I; Walsh, V; Gilkes, A F; Woodgate, L J; Brown, G; Burnett, A K

    1998-10-01

    A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in differentiation. In order to understand this block the cascade of molecular events needs to be characterized. Haemopoietic differentiation is ultimately controlled at the level of gene transcription which is mediated by an array of transcription factors. Many transcription factors contain similar structural protein sequences, and we have used an RT-PCR-based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc-finger consensus amino acid sequences and to the POU-homeodomain and POU-specific domain were used to amplify genes on the basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum-independent HL60 cell line was induced towards the neutrophil lineage by treatment with all-trans retinoic acid (ATRA) for 24 h. CD38+ cells committed towards this lineage were enriched and a population of these cells treated with dihydroxyvitamin D3 to induce neutrophil maturation. RNA extracted from uninduced, ATRA-induced CD38+ cells, and vitamin D3 treated maturing cell cultures were amplified using the degenerate primers. PCR fragments were cloned, sequenced, clustered into homologous groups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was identified using the POU family primers. The zinc-finger primers identified three zinc-finger genes. The pattern of gene expression was suggested from the number of clones in each group at neutrophil commitment and maturation. The differential expression of the genes in the zinc finger and POU families will lead to a better understanding of the cascade of gene expression which occurs following ATRA-induced differentiation.

  9. Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model.

    PubMed

    Lin, Jen-Jyh; Hsu, Hui-Ying; Yang, Jai-Sing; Lu, Kung-Wen; Wu, Rick Sai-Chuen; Wu, King-Chuen; Lai, Tung-Yuan; Chen, Po-Yuan; Ma, Chia-Yu; Wood, W Gibson; Chung, Jing-Gung

    2011-09-15

    We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca(2+) levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca(2+) release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo. Copyright © 2011 Elsevier GmbH. All rights reserved.

  10. Induction of apoptosis in human promyelocytic leukemia HL60 cells by an extract from Erythrina suberosa stem bark.

    PubMed

    Agrawal, Satyam Kumar; Agrawal, Madhunika; Sharma, Parduman Raj; Gupta, Bishan Datt; Arora, Saroj; Saxena, Ajit Kumar

    2011-01-01

    In this study, the apoptosis-inducing effect of an alcoholic extract from Erythrina suberosa stem bark (ESB) was investigated using human promyelocytic leukemia HL60 cells. Cell viability was estimated by MTT assay. We found that the ESB inhibited cell proliferation in a dose- and time-dependent manner. A series of well-documented morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and nuclear fragmentation, were observed by fluorescence microscopy. The gold standard scanning electron micrographs showed apoptotic bodies and formation of blebs. Cell cycle analysis showed a significant increase in Sub G(0) population of cells above 50 μg/ml. ESB treatment resulted in a dose-dependent increase in annexin V positive cells. Increase in intracellular ROS production up to sixfold was detected in ESB-treated HL60 cells by DCFH-DA assay. Dissipation of mitochondrial membrane potential of intact cells accompanied by increase in cytosolic cytochrome c was observed, which was followed by activation of caspase-9 and -3 but not caspase-8. DNA fragmentation analysis revealed typical ladders as early as 18 h indicative of caspase-3 role in the apoptotic pathway. The overall results suggest that ESB induces mitochondria-mediated intrinsic apoptotic pathway in HL60 cells and might have therapeutic value against human leukemia.

  11. The roles of bioactive sphingolipids in resveratrol-induced apoptosis in HL60: acute myeloid leukemia cells.

    PubMed

    Cakir, Zeynep; Saydam, Guray; Sahin, Fahri; Baran, Yusuf

    2011-02-01

    Acute promyelocytic leukemia results from a translocation between 15 and 17 chromosomes that produce PML/RARα fusion protein. PML/RARα inhibits differentiation of myeloid precursor cells at stem cell level. Resveratrol is a phytoalexin that exerts cytotoxic effects on cancer cells. Ceramides have crucial roles in cell growth, proliferation, differentiation, drug resistance, and apoptosis. In this study, we examined the possible cytotoxic effects of resveratrol on acute myeloid leukemia cells and determined the roles of ceramide-metabolizing genes in resveratrol-induced apoptosis, in addition to investigating the possibility of increasing the sensitivity of HL60 cells to resveratrol by manipulating sphingolipids. Cytotoxic effects of resveratrol, C8:ceramide, PDMP, and SK-1 inhibitor were determined by XTT cell proliferation assay. Changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured using caspase-3 colorimetric assay and JC-1 MMP detection kit. Expression levels of ceramide-metabolizing genes were examined by RT-PCR. The results revealed that manipulations of ceramide metabolism toward generation or accumulation of apoptotic ceramides increased apoptotic effects of resveratrol in HL60 cells, synergistically. More importantly, gene expression analyses revealed that resveratrol-induced apoptosis via increasing expression levels of ceramide-generating genes and decreasing expression levels of antiapoptotic sphingosine kinase-1 and glucosylceramide synthase genes. These results showed for the first time that increasing intracellular levels of ceramides by biochemical approaches has also increased sensitivity of HL60 cells to resveratrol. We also showed that resveratrol induces apoptosis through manipulating ceramide-metabolizing genes that resulted in the accumulation of ceramides in HL60 cells.

  12. Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

    PubMed

    Stabellini, Giordano; Brugnoli, F; Calastrini, C; Vizzotto, L; Vertemati, M; Baroni, T; Caramelli, E; Marinucci, L; Pellati, A; Bertagnolo, V

    2004-01-01

    Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

  13. Transient formation of DNA strand breaks during the induced differentiation of a human promyelocytic leukaemic cell line, HL-60.

    PubMed Central

    Farzaneh, F; Meldrum, R; Shall, S

    1987-01-01

    During the induced differentiation of the human promyelocytic leukaemic cell line, HL-60, along the myelocytic lineage, DNA strand-breaks are formed. These breaks which are formed in the face of a proficient DNA repair mechanism, are only transiently maintained and subsequently become religated. The ligation of these breaks requires the activity of the nuclear adenosine diphosphoribosyl transferase (ADPRT). Inhibition of nuclear ADPRT, an enzyme totally dependent on the presence of DNA strand-breaks for its activity and required for efficient DNA repair in eukaryotic cells, blocks the religation of these breaks but not their formation. The inhibition of DNA strand ligation in the differentiating HL-60 cells results in loss of viability and cell death. PMID:3106933

  14. Avemar, a nontoxic fermented wheat germ extract, induces apoptosis and inhibits ribonucleotide reductase in human HL-60 promyelocytic leukemia cells.

    PubMed

    Saiko, Philipp; Ozsvar-Kozma, Maria; Madlener, Sibylle; Bernhaus, Astrid; Lackner, Andreas; Grusch, Michael; Horvath, Zsuzsanna; Krupitza, Georg; Jaeger, Walter; Ammer, Kirsten; Fritzer-Szekeres, Monika; Szekeres, Thomas

    2007-06-08

    Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in human HL-60 promyelocytic leukemia cells. After 24, 48, and 72 h of incubation, Avemar inhibited the growth of HL-60 cells with IC50 values of 400, 190, and 160 microg/ml, respectively. Incubation with MSC caused dose-dependent induction of apoptosis in up to 85% of tumor cells. In addition, Avemar attenuated the progression from G2-M to G0-G1 phase of the cell cycle and was also found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of de novo DNA synthesis. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.

  15. TRIM32 promotes retinoic acid receptor {alpha}-mediated differentiation in human promyelogenous leukemic cell line HL60

    SciTech Connect

    Sato, Tomonobu; Okumura, Fumihiko; Iguchi, Akihiro; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer TRIM32 enhanced RAR{alpha}-mediated transcriptional activity even in the absence of RA. Black-Right-Pointing-Pointer TRIM32 stabilized RAR{alpha} in the human promyelogenous leukemic cell line HL60. Black-Right-Pointing-Pointer Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. Black-Right-Pointing-Pointer TRIM32 may function as a coactivator for RAR{alpha}-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor {alpha} (RAR{alpha}). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR{alpha} and enhances transcriptional activity of RAR{alpha} in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR{alpha}, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR{alpha}-mediated transcriptional activity even in the absence of RA and stabilizes RAR{alpha} in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR{alpha}-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  16. Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines

    PubMed Central

    Tayarani-Najaran, Zahra; Makki, Farideh-Sadat; Alamolhodaei, Nafiseh-Sadat; Mojarrab, Mahdi; Emami, Seyed Ahmad

    2017-01-01

    Objective(s): Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies. Materials and Methods: In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol: water (1:1 v/v) extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60) and J774 as normal cells. Results: CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells. Conclusion: The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines. PMID:28293393

  17. Chemical modulation of the ultra-weak photon emission from Saccharomyces cerevisiae and differentiated HL-60 cells

    NASA Astrophysics Data System (ADS)

    Červinková, Kateřina; Nerudová, Michaela; Hašek, Jiří; Cifra, Michal

    2015-01-01

    The ultra-weak photon emission (UPE) is a universal phenomenon common to all cells with active oxidative metabolism. Generally accepted mechanism of the origin of the ultra-weak photon emission considers reactions of radical or nonradical reactive oxygen species (ROS) with biomolecules such as lipids and proteins which lead to the formation of electron excited species. During the transition to the ground state the excess energy is released as a photon with a wavelength in the visible range of the electromagnetic spectrum. Since the intensity of the light is very low it is possible to be measured only by highly sensitive devices. We used Hamamatsu Photonics PMT module H7360-01 mounted into a light-tight chamber for the purposes of this work. The goal of our research is to delineate an origin of UPE from two model organisms; differentiated HL-60 cells (human promyelocytic leukemia) and yeast cells Saccharomyces cerevisiae. While the UPE from the yeast cells arises spontaneously during the growth without any external stimuli, UPE from HL-60 is induced by phorbol 12-myristate, 13-acetate (PMA). It is possible to modulate the UPE production by certain antioxidants which scavenge ROS formed during the metabolism (yeast cells) or respiratory burst (HL-60 cells). The experiments are focused on the description of effects caused by antioxidants. Several kinds of antioxidants (ascorbic acid, mannitol, glutathione) with different concentration were used and we studied the changes in the UPE intensities of and the temporal developments of the optical signal.

  18. Regulated expression of the MRP8 and MRP14 genes during terminal differentiation of human promyelocytic leukemic HL-60 cells

    SciTech Connect

    Warner-Bartnicki, A.L.; Murao, S.; Collart, F.R.; Huberman, E.

    1992-02-14

    The calcium-binding proteins MRP8 and MRP14 are induced during monomyelocytic cell maturation and may mediate the growth arrest in differentiating HL-60 cells. We determined the levels of a protein complex (PC) containing MRP8 and MRP14 and investigated the mechanism by which the genes encoding these proteins are regulated in HL-60 cells treated with the differentiation-inducing agent mycophenolic acid. Elevated levels of the PC were found to directly parallel gains in the steady-state levels of MRP8 and MRP14 mRNA. Transcription studies with the use of nuclear run-on experiments revealed increased transcription initiation at the MRP8 and MRP14 promoters after MPA treatment. 1{alpha},25-Dihydroxyvitamin D{sub 3}, which induces HL-60 cell differentiation by another mechanism, was also found to increase transcription initiation at the MRP8 and MRP14 promoters, suggesting that this initiation is the major control of MRP8 and MRP14 gene expression during terminal differentiation of human promyelocytic cells.

  19. Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

    PubMed

    Khan, Saifur R; Baghdasarian, Argishti; Nagar, Prarthna H; Fahlman, Richard; Jurasz, Paul; Michail, Karim; Aljuhani, Naif; Siraki, Arno G

    2015-09-05

    In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

  20. Cytotoxic evaluation and induction of mitochondria-mediated apoptosis in human leukaemia HL-60 cells by Carissa spinarum stem isolate.

    PubMed

    Sehar, Irum; Pal, Harish Chandra; Shukla, Sangeeta; Bhushan, Shashi; Hamid, Abid; Gupta, Bishan Dutt; Saxena, Ajit Kumar

    2011-08-01

    To evaluate Carissa spinarum stem isolate for its anti-cancer therapeutic potential. The n-butanol fraction of aqueous extract from Carissa spinarum stem was assessed for its cytotoxic and pro-apoptotic activity. We report for the first time the anti-cancer potential of C. spinarum stem aqueous extract (CSE) and its n-butanol fraction (CSF). Both inhibited cell proliferation of various human cancer cell lines in which leukaemia HL-60 cells treated with CSF showed maximum growth inhibition having an inhibitory concentration (IC(50) ) value of 34.58±0.91 µg/ml. In addition, CSF induced concentration-dependent apoptosis in HL-60 cells as measured by various end-points (e.g. Annexin V binding, DNA laddering, apoptotic body formation and an increase in hypodiploid subG0 DNA content). Moreover, persistent levels of reactive oxygen species caused translocation of Bax to mitochondria and Bcl-2 degradation, which led to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. These events were associated with significant activation of caspase-3, caspase-6 and caspase-9 leading to poly (ADP-ribose) polymerase cleavage. All the above parameters revealed that CSF induced apoptosis through the mitochondrial dependent pathway in HL-60 cells. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  1. Identification of Polymorphonuclear Leukocyte and HL-60 Cell Receptors for Adhesins of Streptococcus gordonii and Actinomyces naeslundii

    PubMed Central

    Ruhl, Stefan; Cisar, John O.; Sandberg, Ann L.

    2000-01-01

    Interactions of oral streptococci and actinomyces with polymorphonuclear leukocytes (PMNs), mediated by sialic acid- and Gal/GalNAc-reactive adhesins, respectively, result in activation of the PMNs and thereby may contribute to the initiation of oral inflammation. Sialidase treatment of PMNs or HL-60 cells abolished adhesion of Streptococcus gordonii but was required for adhesion of Actinomyces naeslundii. The same effects of sialidase were noted for adhesion of these bacteria to a major 150-kDa surface glycoprotein of either PMNs or undifferentiated HL-60 cells and to a 130-kDa surface glycoprotein of differentiated HL-60 cells. These glycoproteins were both identified as leukosialin (CD43) by immunoprecipitation with a specific monoclonal antibody (MAb). Adhesion of streptococci and actinomyces to a 200-kDa minor PMN surface glycoprotein was also detected by bacterial overlay of untreated and sialidase-treated nitrocellulose transfers, respectively. This glycoprotein was identified as leukocyte common antigen (CD45) by immunoprecipitation with a specific MAb. CD43 and CD45 both possess extracellular mucinlike domains in addition to intracellular domains that are implicated in signal transduction. Consequently, the interactions of streptococci and actinomyces with the mucinlike domains of these mammalian cell surface glycoproteins result not only in adhesion but, in addition, may represent the initial step in PMN activation by these bacteria. PMID:11035744

  2. Oxidative stress-mediated intrinsic apoptosis in human promyelocytic leukemia HL-60 cells induced by organic arsenicals

    PubMed Central

    Fan, Xiao-Yang; Chen, Xin-You; Liu, Yu-Jiao; Zhong, Hui-Min; Jiang, Feng-Lei; Liu, Yi

    2016-01-01

    Arsenic trioxide has shown the excellent therapeutic efficiency for acute promyelocytic leukemia. Nowadays, more and more research focuses on the design of the arsenic drugs, especially organic arsenicals, and on the mechanism of the inducing cell death. Here we have synthesized some organic arsenicals with Schiff base structure, which showed a better antitumor activity for three different kinds of cancer cell lines, namely HL-60, SGC 7901 and MCF-7. Compound 2a (2-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) and 2b (2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) were chosen for further mechanism study due to their best inhibitory activities for HL-60 cells, of which the half inhibitory concentration (IC50) were 0.77 μM and 0.51 μM, respectively. It was illustrated that 2a or 2b primarily induced the elevation of reactive oxygen species, decrease of glutathione level, collapse of mitochondrial membrane potential, release of cytochrome c, activation of Caspase-3 and apoptosis, whereas all of the phenomena can be eliminated by the addition of antioxidants. Therefore, we concluded that compound 2a and 2b can induce the oxidative stress-mediated intrinsic apoptosis in HL-60 cells. Both the simplicity of structure with Schiff base group and the better anticancer efficiency demonstrate that organic arsenicals are worthy of further exploration as a class of potent antitumor drugs. PMID:27432798

  3. Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.

    PubMed Central

    Tonkinson, J L; Stein, C A

    1994-01-01

    We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells. Images PMID:7937155

  4. Gene expression profiling for nitric oxide prodrug JS-K to kill HL-60 myeloid leukemia cells.

    PubMed

    Liu, Jie; Malavya, Swati; Wang, Xueqian; Saavedra, Joseph E; Keefer, Larry K; Tokar, Erik; Qu, Wei; Waalkes, Michael P; Shami, Paul J

    2009-07-01

    The nitric oxide (NO) prodrug JS-K is shown to have anticancer activity. To profile the molecular events associated with the anticancer effects of JS-K, HL-60 leukemia cells were treated with JS-K and subjected to microarray and real-time RT-PCR analysis. JS-K induced concentration- and time-dependent gene expression changes in HL-60 cells corresponding to the cytolethality effects. The apoptotic genes (caspases, Bax, and TNF-alpha) were induced, and differentiation-related genes (CD14, ITGAM, and VIM) were increased. For acute phase protein genes, some were increased (TP53, JUN) while others were suppressed (c-myc, cyclin E). The expression of anti-angiogenesis genes THBS1 and CD36 and genes involved in tumor cell migration such as tissue inhibitors of metalloproteinases, were also increased by JS-K. Confocal analysis confirmed key gene changes at the protein levels. Thus, multiple molecular events are associated with JS-K effects in killing HL-60, which could be molecular targets for this novel anticancer NO prodrug.

  5. In vitro hydroquinone-induced instauration of histone bivalent mark on human retroelements (LINE-1) in HL60 cells.

    PubMed

    Mancini, Monica; Mandruzzato, Martina; Garzia, Alba C; Sahnane, Nora; Magnani, Elena; Macchi, Filippo; Oulad-Abdelghani, Mustapha; Oudet, Pierre; Bollati, Valentina; Fustinoni, Silvia; Furlan, Daniela; Bonapace, Ian M

    2017-04-01

    Benzene is extensively used in industry despite its leukemogenic activity, representing a significant occupational hazard. We investigated if long-term treatment with low-doses hydroquinone (HQ), a benzene metabolite, might be sufficient to alter in vitro the epigenetic signature underlining LINE-1 sequences, a poorly explored step in health risks associated with benzene exposure. In HL-60 cell line, exploring the epigenetic events occurring in chromatin, we found the transient instauration of the distinctive signature combining the repressive H3Lys27 tri-methylation mark and the activating H3Lys4 tri-methylation mark (H3K27me3/H3K4me3), indicating a tendency toward a poised chromatin conformation. These alterations are lost in time after short-term treatments, while the long-term setting, performed using a concentration within the levels of total HQ in peripheral blood of benzene-exposed workers, showed a gradual increase in H3K4me3. We observed the absence of statistically significant variations in DNA methylation and expression levels of LINE-1, despite a decrease in protein levels of UHRF1, DNA methyl-transferases and histone methyl-transferases. In conclusion, in vitro treatment with low-dose HQ determined the instauration of a reversible poised state of chromatin in LINE-1 sequences, suggesting that prolonged exposure could cause persistent epigenetic alterations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction.

    PubMed

    Yu, Mei-Xia; Liu, Xun; Zhou, Yong-Ming; Cheng, Yan-Xiang; Cheng, Jing; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2014-10-01

    This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that

  7. Multidrug transporter proteins and cellular factors involved in free and mAb linked calicheamicin-gamma1 (gentuzumab ozogamicin, GO) resistance and in the selection of GO resistant variants of the HL60 AML cell line.

    PubMed

    Cianfriglia, Maurizio; Mallano, Alessandra; Ascione, Alessandro; Dupuis, Maria Luisa

    2010-06-01

    In this study we elucidated the role of ATP-binding cassette (ABC) multi-drug transporter proteins and cellular factors such as Bcl-2 expression and CD33 down-modulation contributing to free and hP67.6 mAb linked calicheamicin-gamma1 (CalC-gamma1) resistance. We analyzed in a well designed HL60 cell system the relationship between the expression of ABC proteins, Bcl-2 and CD33 modulation with the activity of free and mAb-linked CalC-gamma1. The results herein reported and discussed, strongly suggest that both MDR1-Pgp and MRP1 efflux systems are engaged by CalC-gamma1, but only MDR1-Pgp over-expression efficiently abrogates drug cytotoxicity in MDR cells. Paradoxically, Bcl-2 expression, as observed for other anticancer compounds belonging to the enediyne family of drugs, confers CalC-gamma1 susceptibility rather than resistance in HL60 cells. Further, the isolation of a resistant HL60 subline (HL60AL) that was developed by exposing the parental sensitive cells to sub-effective doses of gemtuzumab ozogamicin (GO) over an extended period of time shows a reduced level of CD33 expression that represents an important escape mechanism of HL60 MDR cells to the cytotoxic effect of GO.

  8. Magnetic force microscopy analysis of apoptosis of HL-60 cells induced by complex of antisense oligonucleotides and magnetic nanoparticles.

    PubMed

    Shen, He-bai; Long, De-hong; Zhu, Long-zhang; Li, Xing-Yu; Dong, Ya-ming; Jia, Neng-qin; Zhou, Hai-qing; Xin, Xi; Sun, Yang

    2006-06-20

    Magnetic force microscopy (MFM) has been employed to observe antisense oligonucleotides (ASOs)-coupled silica-coated magnetic iron oxide nanoparticles (SMNPs) internalized into human leukemia (HL-60) cells. The experiment demonstrated that the ASOs-coupled SMNPs delivery into the cells really occurred. The nanoparticles were internalized into the cells and the apoptotic topography can be directly visualized simultaneously with MFM technology. These present observations offer direct morphology evidence on studying the apoptosis of tumor cells and provide useful information for better design of new diagnostic and therapeutic tools in tumor treatment.

  9. The induction of monocytopoiesis in HL-60 promyelocytic leukemia cells is inhibited by hydroquinone, a toxic metabolite of benzene

    SciTech Connect

    Oliveira, N.L.

    1992-01-01

    Chronic exposure of humans to benzene has been shown to have a cytotoxic effect on hematopoietic progenitor cells in intermediate stages of differentiation which can lead to aplastic anemia and acute myelogenous leukemia. This thesis examined the effect of hydroquinone, a toxic metabolite of benzene found in the bone marrow, on the human promyelocytic leukemia cell line (HL-60) which can be induced to differentiate to both monocyte and myeloid cells, and thus has been used as a surrogate for a granulocyte/macrophage progenitor cell. Exposure of HL-60 cells to noncytotoxic concentrations of hydroquinone for three hours prior to induction with 12-O-tetradecanoyl phorbol-13-acetate caused a dose-dependent inhibition of the acquisition of characteristics of monocytic differentiation. These included adherence, nonspecific esterase activity and phagocytosis. Hydroquinone had no effect on cell proliferation. Hydroquinone appeared to be affecting maturation beyond the monoblast/promonocyte stages. Hydroquinone also prevented differentiation induced by 1, 25-dihydroxy vitamin D[sub 3], however, the block occurred after the acquisition of adherence. Hydroquinone at concentrations that inhibited monocytic differentiation had no effect on differentiation to granulocytes, suggesting that the block in the differentiation of these bipotential cells is at a step unique to the monocytic pathway. Hydroquinone was unable to prevent differentiation induced by the macrophage-derived cytokine interleukin-1, a differentiation factor for cells of the monocytic lineage. These data demonstrate that treatment of Hl-60 cells with hydroquinone prior to induction of differentiation prevents the acquisition of the monocytic phenotype induced by TPA or 1, 25(OH)[sub 2]D[sub 3] by a mechanism which at present is unknown, but which appears to be specific for the monocytic pathway. These results are of considerable significance for benzene hematotoxicity.

  10. Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3) in acute myeloid leukemia HL-60 cells.

    PubMed

    Song, Hairong; Fares, Mona; Maguire, Kim R; Sidén, Ake; Potácová, Zuzana

    2014-01-01

    Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5-100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

  11. Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

    PubMed

    Chien, Chih-Chiang; Wu, Ming-Shun; Shen, Shing-Chuan; Yang, Liang-Yo; Wu, Wen-Shin; Chen, Yen-Chou

    2013-04-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.

  12. The Novel Retinoid, 9cUAB30, Inhibits Telomerase and Induces Apoptosis in HL60 Cells1

    PubMed Central

    Love, William K; DeAngelis, J Tyson; Berletch, Joel B; Phipps, Sharla MO; Andrews, Lucy G; Brouillette, Wayne J; Muccio, Donald D; Tollefsbol, Trygve O

    2008-01-01

    Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells. PMID:18795149

  13. Wogonin induces apoptosis and endoplasmic reticulum stress in HL-60 leukemia cells through inhibition of the PI3K-AKT signaling pathway.

    PubMed

    Hu, Chengjun; Xu, Maozhong; Qin, Rujuan; Chen, Weifeng; Xu, Xin

    2015-06-01

    Wogonin is a flavonoid isolated from Scutellaria baicalensis root and has multiple pharmacological effects, including anticancer effects. Recent studies have shown that wogonin induces cell cycle arrest and reverses multi-drug resistance in the human K562 leukemia cell line. However, its pharmacological function in the apoptosis of leukemia cells remains unknown. Therefore, we hypothesized that wogonin can induce apoptosis in the HL-60 leukemia cell line. In the present study, the HL-60 cells were treated with different doses of wogonin (0-150 µM). Wogonin inhibited the viability of HL-60 cells in a dose-dependent and time-dependent manner. Flow cytometry and analyses of caspase and PARP-1 activation and the Bax/Bcl-2 ratio, demonstrated that the cytotoxic effect of wogonin on HL-60 cells was mediated by caspase-dependent and mitochondrial-dependent apoptosis. Wogonin also induced the expression of certain members of the endoplasmic reticulum (ER) stress pathway (CHOP, GRP94 and GRP78) and the activation of multiple branches of ER stress transducers (IRE1α, PERK-eIF2α and ATF6) in the HL-60 cells. In addition, wogonin reduced the phosphorylation of PI3K and AKT in the HL-60 cells. Furthermore, constitutive activation of AKT induced by adenoviral vectors inhibited the pro-apoptotic effects and ER stress induced by wogonin in the HL-60 cells. In summary, our results indicated that wogonin induced apoptosis and ER stress in HL-60 cells, which was mediated by the inhibition of the PI3K-AKT signaling pathway.

  14. Biosynthesis of platelet activating factor (PAF) via alternate pathways: subcellular distribution of products in HL-60 cells

    SciTech Connect

    Record, M.; Snyder, F.

    1986-05-01

    Final steps in the biosynthesis of PAF can be catalyzed by two different routes: CDP-choline:1-alkyl-2-acetyl-Gro cholinephosphotransferase (dithiothrietol (DTT)-insensitive) or acetyl-CoA:1-alkyl-2-lyso-GroPCho acetyltransferase. The authors have investigated the conversion of tritium-labeled 1-alkyl-2-acetyl-Gro and 1-alkyl-2-lyso-GroPCho (lyso-PAF) to PAF and other lipid products in HL-60 cells and in subcellular organelles isolated by centrifugation in a Percoll gradient. When cells are incubated with the labeled precursors (2 ..mu..M) the total amount of labeled PAF and 1-alkyl-2-acyl-GroPCho formed was similar from both precursors (60 pmol from 1-alkyl-2-acetyl-Gro and 50 pmol from lyso-PAF). However, PAF formed from 1-alkyl-2-acetyl-Gro represented 70% of the total products, whereas with lyso-PAF the major labeled product was 1-alkyl-2-acyl-GroPCho. Formation of PAF from 1-(/sup 3/H)alkyl-2-acetyl-Gro was linear to at least 30 min at 20/sup 0/C. After a 15-min incubation of this neutral lipid with HL-60 cells, the labeled PAF produced was located exclusively in the plasma membrane fraction as opposed to the label in the 1-alkyl-2-acyl-GroPCho, which was found only in the endoplasmic reticulum; none of the labeled PAF product was released to the media. The authors results suggest PAF might be synthesized by the DTT-insensitive cholinephosphotransferase at the site of the plasma membrane in HL-60 cells.

  15. CD14 mediated endogenous TNF-alpha release in HL60 AML cells: a potential model for CD14 mediated endogenous cytokine release in the treatment of AML.

    PubMed

    Treon, S P; Anand, B; Ulevitch, R; Broitman, S A

    1994-01-01

    In previous studies, HL60 AML cells treated with tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and lipopolysaccharides (LPS) displayed decreased growth and viability, enhanced monocytic pathway differentiation and endogenous TNF release. Endogenous TNF release by LPS/TNF/IFN treated HL60 cells was postulated to play a role with the above findings. In these studies, HL60 cells expressed CD14 when treated with TNF, IFN, and LPS. CD14 mediates TNF release in monocytes/macrophages in response to binding of LPS with LPS binding protein (LBP). CD14 was not expressed in either untreated or LPS only treated HL60 cells. CD14 expression was present and greater with HL60 cells cultured with LPS/TNF/IFN vs TNF/IFN (47.47% vs 9.07% positive, respectively) suggesting synergism for LPS in CD14 induction. CD14 expression was associated with endogenous TNF release, and with significantly higher levels by HL60 cells treated with LPS/TNF/IFN vs TNF/IFN (p < 0.001). Addition of anti-CD14 antibody significantly reduced release of TNF in TNF/IFN (p < 0.001) and LPS/TNF/IFN (p = 0.0013) treated cells. KG1 and U937 AML cells treated with LPS, TNF, and IFN did not express CD14, nor release TNF. A model for inducing release of endogenous growth inhibitory cytokines by CD14 bearing AML cells is proposed as an approach to AML therapy.

  16. Effect of highly lipolyzed goat cheese on HL-60 human leukemia cells: antiproliferative activity and induction of apoptotic DNA damage.

    PubMed

    Yasuda, S; Kuwata, H; Kawamoto, K; Shirakawa, J; Atobe, S; Hoshi, Y; Yamasaki, M; Nishiyama, K; Tachibana, H; Yamada, K; Kobayashi, H; Igoshi, K

    2012-05-01

    To establish cheese as a dairy product with health benefits, we embarked on examining the multifunctional role of cheeses, especially in the field of cancer prevention. The current study was designed to investigate whether different types of commercial goat cheeses may possess antiproliferative activity, using an HL-60 human promyelocytic leukemia cell line as a cancer cell model. Among 11 cheese extracts tested at 500μg/mL, 6 (Crottin de Chavignol, Pouligny Saint-Pierre, Chabichou du Poitou, Valencay, Kavli, and Sainte-Maure de Touraine) resulted in a significant decrease of cell viability, which is consistent with a decrease in viable cell number. Compared with the half-maximal inhibitory concentration (IC(50)) value of individual cheeses in cellular proliferation assays, the Pouligny Saint-Pierre extract showed strong inhibition. Incubation of cells in the presence of Pouligny Saint-Pierre extract resulted in induction of cellular morphological changes and apoptotic DNA fragmentation as well as expression of the active form of caspase-3 protein. Based on the quantification of the ratio of free fatty acids to triglycerides in different cheese samples, a significant correlation was detected between lipolytic ripeness and IC(50) values for antiproliferative capacity tested in HL-60 cells. Collectively, these results support a potential role of highly lipolyzed goat cheeses in the prevention of leukemic cell proliferation.

  17. Cytotoxic and Apoptotic Effects of Different Extracts of Artemisia turanica Krasch. on K562 and HL-60 Cell Lines

    PubMed Central

    Tayarani-Najaran, Zahra; Sareban, Mahla; Gholami, Atefeh; Emami, Seyed Ahmad; Mojarrab, Mahdi

    2013-01-01

    Artemisia is an important genus of Iranian flora. Cytotoxic activities for some species of the genus have already been reported. In this study, we have investigated the cytotoxic effects of n-hexane, CH2Cl2, EtOAc, EtOH, and EtOH/H2O (1 : 1) extracts of A. turanica Krasch. on two human leukemic cancer cell lines (K562 and HL-60) and J774 as normal cells using alamarBlue (resazurin) assay. PI staining of the fragmented DNA and western blot analysis were used to evaluate the possible apoptotic effect of the extract. The CH2Cl2 extract of A. turanica showed the most antiproliferative effect on cancer cells among all tested extracts with IC50 values of 69 and 104 μg/mL on K562 and HL-60 cells, respectively, whereas the normal cells were not affected significantly by this extract. Sub-G1 peak in the flow cytometry histogram of the cells treated with CH2Cl2 extract of A. turanica and cleavage of PARP protein confirmed the induction of apoptosis with CH2Cl2 extract. Taken together, the findings of the present work suggest the anticancer potential of CH2Cl2 extract of A. turanica on human leukemic cancer cell lines. PMID:24288497

  18. [Synthetic peptides -- analogs of biologically active fragment of the differentiation factor from HL-60 cells show radioprotective and adaptogenic activities].

    PubMed

    Goncharenko, E N; Deev, L I; Kostanian, I A; Astapova, M V; Akhalaia, M Ia; Kudriashova, N Iu; Surina, E A

    2002-01-01

    It was shown that the addition of synthetic six-membered peptide (HLDF-6) and its Tyr-analog (HLDF-Y) to cultural medium significantly increased the survival of cells HL-60, treated by cold shock. The prophylactic administration of HDLF-Y (1 mg/kg, 4 hours prior to applied actions) decreased the response of hypothalamushypophysis-adrenal glands system and sympathicoadrenal system of rat males on supercooling and also increased the resistance of mouse males to supercooling and X-irradiation. In the experiences with females HDLF-Y did not show the similar biological activity.

  19. Inhibition of mitochondrial function in HL60 cells is associated with an increased apoptosis and expression of CD14.

    PubMed

    Mills, K I; Woodgate, L J; Gilkes, A F; Walsh, V; Sweeney, M C; Brown, G; Burnett, A K

    1999-09-24

    The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population. Copyright 1999 Academic Press.

  20. Induction of growth inhibition and differentiation of human leukemia HL-60 cells by a Tunisian gerboui olive leaf extract.

    PubMed

    Abaza, Leila; Talorete, Terence P N; Yamada, Parida; Kurita, Yui; Zarrouk, Mokhtar; Isoda, Hiroko

    2007-05-01

    Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.

  1. Antrodia camphorata induces G(1) cell-cycle arrest in human premyelocytic leukemia (HL-60) cells and suppresses tumor growth in athymic nude mice.

    PubMed

    Yang, Hsin-Ling; Kumar, K J Senthil; Kuo, Ya-Ting; Chang, Hebron C; Liao, Jiunn-Wang; Hsu, Li-Sung; Hseu, You-Cheng

    2014-09-01

    Antrodia camphorata is a well-known medicinal mushroom in Taiwan. The broth from a fermented culture of Antrodia camphorata (AC) has been shown to induce apoptosis in cultured human premyelocytic leukemia (HL-60) cells. In the present study, we examined the effects of AC on cell cycle arrest in vitro in HL-60 cells and on tumor regression in vivo using an athymic nude mouse model. We found that AC (20-80 μg mL(-1)) treatment significantly induced G1 cell-cycle arrest in HL-60 cells by reducing the levels of cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and phosphorylation of retinoblastoma protein (p-Rb). Moreover, AC treatment led to significantly increased protein expression levels of CDK inhibitors, including p21(WAF1) and p15(NIK4B). Additionally, AC treatment markedly induced intracellular ROS generation and mitochondrial dysfunction in HL-60 cells. Furthermore, the in vivo study results revealed that AC treatment was effective in terms of delaying the tumor incidence in nude mice that had been inoculated with HL-60 cells as well as in reducing the tumor burden. Histological analysis confirmed that AC treatment significantly modulated the xenografted tumor progression as demonstrated by a reduction in mitotic cells. Our data strongly suggest that Antrodia camphorata could be an anti-cancer agent for human leukemia.

  2. Effects of portulacerebroside a on apoptosis of human leukemia HL60 cells and p38/JNK signaling pathway

    PubMed Central

    Ye, Qidong; Zhang, Na; Chen, Kai; Zhu, Jiashi; Jiang, Hui

    2015-01-01

    Acute myeloid leukemia is known as one of the most malignant diseases. We aimed at exploring the effect of portulacerebroside A (PCA) on the apoptosis in human leukemia HL60 cells and clarify the possible mechanisms involved in. By MTT analysis, we found that PCA (1-100 μM) inhibited the cell viability in a time- and dose-dependent manner, and cell cycle was arrested at G0/G1 period. PCA treatment from 5 to 50 μM dose-dependently induced apoptosis from 12.7 ± 1.56% to 52.7 ± 6.214% of HL60 cells. Mitochondrial membrane potential (MMP) was decreased and reactive oxygen species (ROS) accumulated obviously. mRNA expressions and protein levels of Bax/Bcl-2, caspase-3 and caspase-9 were elevated significantly. ERK1/2, JNK1/2 and p38 MAPK pathway were blocked detected by western blot analysis. In conclusion, PCA can act as a new agent for leucocythemia treatment. PMID:26823708

  3. Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells.

    PubMed

    Hsing, Chung-Hsi; Chen, Chia-Ling; Lin, Wei-Chieh; Lin, Chiou-Feng

    2015-01-01

    Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.

  4. Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

    PubMed Central

    Hsing, Chung-Hsi; Chen, Chia-Ling; Lin, Wei-Chieh; Lin, Chiou-Feng

    2015-01-01

    Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells. PMID:26061531

  5. The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

    SciTech Connect

    Reich, Charles F.; Pisetsky, David S.

    2009-03-10

    Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for {beta}-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

  6. In vitro cytotoxicity of Mokko lactone in human leukemia HL-60 cells: induction of apoptotic cell death by mitochondrial membrane potential collapse.

    PubMed

    Yun, Y G; Oh, H; Oh, G S; Pae, H O; Choi, B M; Kwon, J W; Kwon, T O; Jang, S I; Chung, Hun-Taeg

    2004-08-01

    We studied the effect of mokko lactone (ML) isolated from the roots of Saussurea lappa (Compositae), a plant that is used for medicinal purposes in Korea, on the induction of apoptosis in human leukemia HL-60 cells. ML was cytotoxic to HL-60 cells, and this cytotoxic effect of ML appears to be attributable to its induction of apoptotic cell death, as ML induced nuclear morphologic changes and internucleosomal DNA fragmentation and increased the proportion of Annexin V-positive cells and the activity of caspase-3. Further studies revealed that the induction of apoptosis by ML was associated with the loss of mitochondrial membrane potential. Collectively, our results suggest that apoptosis induced by ML in HL-60 cells was executed by a collapse of mitochondrial membrane potential followed by the activation of caspase-3. This is the first report on the mechanism of apoptosis-inducing effect of ML.

  7. Antitumor effects with apoptotic death in human promyelocytic leukemia HL-60 cells and suppression of leukemia xenograft tumor growth by irinotecan HCl.

    PubMed

    Chen, Yung-Liang; Chueh, Fu-Shin; Yang, Jai-Sing; Hsueh, Shu-Ching; Lu, Chi-Cheng; Chiang, Jo-Hua; Lee, Ching-Sung; Lu, Hsu-Feng; Chung, Jing-Gung

    2015-07-01

    Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation.

  8. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells

    PubMed Central

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells. PMID:25194051

  9. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells.

    PubMed

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells.

  10. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

    PubMed Central

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  11. Enhancement of 1α,25-dihydroxyvitamin D3-induced differentiation of human leukaemia HL-60 cells into monocytes by parthenolide via inhibition of NF-κB activity

    PubMed Central

    Kang, S N; Kim, S H; Chung, S W; Lee, M H; Kim, H J; Kim, T S

    2002-01-01

    Transcription factors such as NF-κB provide powerful targets for drugs to use in the treatment of cancer. In this report parthenolide (PT), a sesquiterpene lactone of herbal remedies such as feverfew (Tanacetum parthenium) with NF-κB inhibitory activity, markedly increased the degree of human leukaemia HL-60 cell differentiation when simultaneously combined with 5 nM 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3). PT by itself did not induce HL-60 cell differentiation. Cytofluorometric analysis indicated that PT stimulated 1,25-(OH)2D3-induced differentiation of HL-60 cells predominantly into monocytes. Pretreatment of HL-60 cells with PT before the 1,25-(OH)2D3 addition also potentiated the 1,25-(OH)2D3-induced HL-60 cell differentiation in both a dose- and a time-dependent manner, in which the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-κB binding activity by PT. In contrast, santonin, a sesquiterpene lactone without an inhibitory activity of NF-κB binding to the κB sites, did not enhance the 1,25-(OH)2D3-induced HL-60 cell differentiation. In transfection experiments, PT enhanced 1,25-(OH)2D3-induced VDRE-dependent promoter activity. Furthermore, PT restored 1,25-(OH)2D3-induced VDRE-dependent promoter activity inhibited by TNF-α, an activator of NF-κB signalling pathway. These results indicate that PT strongly potentiates the 1,25-(OH)2D3-induced HL-60 cell differentiation into monocytes via the inhibition of NF-κB activity and provide evidence that inhibition of NF-κB activation can be a pre-requisite to the efficient entry of promyelocytic leukaemia cells into a differentiation pathway. PMID:11877332

  12. Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Amor, M Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-03-01

    Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

  13. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    PubMed

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  14. Double minute chromatin bodies and other chromosome alterations in human myeloid HL-60 leukemia cells susceptible or resistant to induction of differentiation by phorbol-12-myristate-13-acetate

    SciTech Connect

    Au, W.W.; Callaham, M.F.; Workman, M.L.; Huberman, E.

    1983-12-01

    An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was conducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes (9p- and t(10;13)), which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13-acetate-induced cell differentiation contained double minute chromatine bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of double minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cell differed also in a number of other chromosomal alteration, including gains or losses involving chromosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes.

  15. Ant 4,4, a polyamine-anthracene conjugate, induces cell death and recovery in human promyelogenous leukemia cells (HL-60).

    PubMed

    Traquete, Rui; Ghani, Radiah A; Phanstiel, Otto; Wallace, Heather M

    2013-04-01

    One of the major problems in cancer therapy is the lack of specificity of chemotherapeutic agents towards cancer cells, resulting in adverse side effects. One means to counter this is to selectively deliver the drug to the cancer cell. Cancer cells accumulate increased concentrations of polyamines compared to normal cells, mainly through an increased uptake of preformed polyamines via the polyamine transport system (PTS). Furthermore, the non-stringent structural requirements of the PTS enable the transport of a range of polyamine-based molecules. Thus, the PTS can be used to transport compounds linked to polyamines selectively to cancer cells. In our laboratory, polyamine-anthracene conjugates have shown potent anti-tumour activity towards HL-60 cells. The aim of this study was to determine the cytotoxicity of Ant-4,4, a homospermidine-anthracene conjugate, and assess the long-term effects by determining whether cancer cells were able to recover from treatment. During exposure, Ant-4,4 was an effective growth-inhibitory agent in HL-60 cells decreasing viable cell number, protein and polyamine content. Evidence indicates concomitant cell-cycle arrest and increased apoptosis. Once the drug was removed, HL-60 cells recovered gradually over time. Increasing cell number, protein content and polyamine content, as well as diminished effects on cell-cycle and apoptotic stimuli were observed over time. These data suggest that, despite being an effective way of delivering anthracene, these polyamine conjugates do not exert long-lasting effects on HL-60 cells.

  16. In vitro and in vivo apoptosis-inducing antileukemic effects of Mucuna macrocarpa stem extract on HL-60 human leukemia cells.

    PubMed

    Lu, Kuan-Hung; Chang, Yuh-Fang; Yin, Pen-Hui; Chen, Ting-Ting; Ho, Yu-Ling; Chang, Yuan-Shiun; Chi, Chin-Wen

    2010-09-01

    Mucuna macrocarpa Wallich (Leguminosae) is believed to hold blood circulation activating effects, and has been used as a folk remedy in Southeast Asia for the treatment of various hematologic and circulatory-related ailments. The objective of this study was to investigate whether crude methanolic extract of M macrocarpa (CMEMM) possessed antileukemic effects on HL-60, human leukemia cells. CMEMM was prepared from dried stems of this plant, and its apoptosis-inducing effects were investigated using HL-60 cells in vitro and in vivo. With treatment of 25 to 75 μg/mL CMEMM, the in vitro antiproliferative effect on HL-60 cells increased in a dose- and time-dependent manner during the 72-hour treatment period. The concentration of CMEMM that exhibited a 50% growth inhibition (IC(50)) for 72-hour exposure was 36.4 μg/mL. Apoptosis triggered by CMEMM in HL-60 cells was confirmed by the following observations: ( a) characteristic apoptotic nuclear fragmentation, (b) dose-dependent accumulation of sub-G(1) phase in cell cycle analyses, (c) increased percentages of annexin V-positive apoptotic cells, and (d) dose-dependent elevation of active caspase-3. Furthermore, an in vivo tumor growth suppression effect by CMEMM (500 mg/kg/d intraperitoneally) was observed in mouse xenografts. The results suggest that CMEMM exerts antileukemic effects via an apoptotic pathway in HL-60 cells, and could be a candidate for developing antileukemic agents in the future.

  17. JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

    PubMed

    Udupi, Vidya; Yu, Margaret; Malaviya, Swati; Saavedra, Joseph E; Shami, Paul J

    2006-10-01

    Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

  18. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique.

    PubMed

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-07-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R(2) = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R(2) ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining.

  19. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique

    PubMed Central

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-01-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R2 = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R2 ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining. PMID:25071957

  20. Patterns of alpha-L-fucosidase in acute myeloid leukemia cells. Comparison with promyelocytic HL-60 cell line.

    PubMed

    Dosbaa, I; Bernard, M; Foglietti, M J; Percheron, F; Emiliani, C

    1992-12-15

    Changes were observed in alpha-L-fucosidase forms in cells from acute myelocytic leukemias (AML). Total alpha-L-fucosidase activity was not significantly different for normal granulocytes and leukemic cells, but enzymic profiles obtained by chromatofocusing are quite different. In granulocyte profile, two main peaks are present (B and more acidic A) which were eluted at pH 5.2 with a shoulder at pH 4.6. In AMLs the B form is present but weakly expressed, whereas the more acidic forms are the major ones. This pattern may be related either to the malignancy character or to the stage at which the differentiation is stopped. Experiments on an HL-60 cell line (promyelocytic cells corresponding to the AML 3 type) showed that differentiation induced by dimethyl sulfoxide leads to the appearance of the B form present in normal mature cells. Thus the repartition of the enzyme forms seems to be related to the stage of differentiation of the myelocytic cells.

  1. PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells.

    PubMed

    Barque, J P; Lagaye, S; Ladoux, A; Della Valle, V; Abita, J P; Larsen, C J

    1987-09-30

    PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.

  2. Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

    PubMed Central

    Xu, Dawei; Popov, Nikita; Hou, Mi; Wang, Qian; Björkholm, Magnus; Gruber, Astrid; Menkel, Annette R.; Henriksson, Marie

    2001-01-01

    Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression. PMID:11274400

  3. Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells.

    PubMed

    Santos-Beneit, A M; Mollinedo, F

    2000-05-01

    Neutrophils possess a very short lifespan, dying by apoptosis. HL-60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil-differentiating HL-60 cells correlates with the achievement of an apoptosis-related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase-polymerase chain reaction, cloning, and sequencing techniques, we have found that HL-60 cells express bak, bik, bax, bad, bcl-2, bcl-xL, bcl-w, bfl-1, fas, and caspases 1-4 and 7-10. After DMSO treatment, bak, bcl-w, bfl-1, fas, and caspases 1 and 9 were up-regulated, whereas bik, bcl-2, and caspases 2, 3, and 10 were down-regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase-2 mRNA and protein expression was drastically reduced after HL-60 cell differentiation, being absent in both HL-60-differentiated neutrophils and mature neutrophils, whereas caspase-3 and -10 mRNA and protein expression were diminished upon HL-60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl-1 mRNA levels were largely increased during DMSO-induced differentiation of HL-60 cells, and these genes were the bcl-2 family members that were expressed most abundantly in mature neutrophils. Bcl-2 overexpression or caspase inhibition prevented differentiation-induced apoptosis in HL-60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl-w, bfl-1, fas, and caspases 1, 3, 4, and 7-10, but hardly expressed bcl-2, bcl-xL, bik, bax, and caspase-2. These results suggest that the above gene expression changes in neutrophil-differentiating HL-60 cells may play a role in the acquisition of the neutrophil

  4. A polysaccharide from the fruiting bodies of Agaricus blazei Murill induces caspase-dependent apoptosis in human leukemia HL-60 cells.

    PubMed

    Li, Xiaohui; Zhao, Xin; Wang, Hongmin; Han, Junqing; Liu, Li

    2014-09-01

    Polysaccharides are the major active ingredients of fungus Agaricus blazei for treating and preventing cancer. However, there are no reports showing anti-tumor activity of A. blazei polysaccharides (ABP) on human leukemia (HL)-60 cells in vitro and in vivo. In this study, we demonstrated that ABP efficiently inhibited proliferation of cultured HL-60 cells, which was associated with the induction of apoptosis. The increase in ABP-induced apoptosis was accompanied by loss of mitochondria membrane potential (∆Ψm), cytochrome c release from the mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase (PARP), and the elevated ratio of Bcl-2-associated X (Bax)/B-cell lymphoma 2 (Bcl-2). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, reversed the cytotoxic effects and apoptotic characteristics induced by ABP in HL-60 cells. Furthermore, we confirmed that ABP could obviously inhibit the solid cancer growth of leukemia HL-60 in tumor xenograft model. These data demonstrated that ABP effectively induced the apoptosis of HL-60 cells via a signaling cascade of mitochondrial caspase-3-dependent pathway.

  5. Portulacerebroside A inhibits adhesion, migration, and invasion of human leukemia HL60 cells and U937 cells through the regulation of p38/JNK signaling pathway

    PubMed Central

    Ye, Qidong; Liao, Xuelian; Fu, Pan; Dou, Jiaying; Chen, Kai; Jiang, Hui

    2016-01-01

    Acute myeloid leukemia (AML) is a highly malignant hematopoietic tumor. This study aimed to explore the effect of portulacerebroside A (PCA) on the adhesion, migration, and invasion in human leukemia HL60 cells and U937 cells and clarify the possible mechanisms involved, which could provide potential strategies for the treatment of AML. By methyl thiazolyl tetrazolium analysis, it was found that PCA (1–10 μM) suppressed the cell viability in a time- and dose-dependent manner. A total of 1, 2, and 5 μM of PCA dramatically inhibited the adhesion, migration, and invasion of HL60 cells and U937 cells in a dose-dependent manner. Phosphorylation level of JNK and P38 protein level was measured by Western blot. After the real-time quantification polymerase chain reaction and Western blot detection of the total RNA and protein, messenger RNA, and protein expression levels of Ras homologous C (RhoC), metastasis-associated gene 1 (MTA1) and matrix metalloproteinase-2/9 (MMP-2/9) were decreased significantly in a dose-dependent manner. The phosphorylation level of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) was decreased dramatically in HL60 cells and U937 cells after PCA treatment. In conclusion, PCA significantly inhibits the adhesion, migration, and invasion of HL60 cells and U937 cells by suppressing the p38/JNK pathway and regulating the expressions of related genes. PMID:27956839

  6. Upregulation of CD54 and downregulation of HLA‑ABC contribute to the novel enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by ATRA plus VPA.

    PubMed

    Zou, Huijuan; Li, Lianlian; Han, Yang; Ma, Ruiping; Liao, Qiong; Tian, Jing; Zhang, Xiaoyu; Ren, Xia; Song, Guanhua; Guo, Qiang; Li, Xia; Ding, Huifang; Jiang, Guosheng

    2017-01-01

    Enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by all-trans-retinoic acid (ATRA) plus valproate (VPA) was evaluated. In addition to the synergistic effect of ATRA plus VPA on HL-60 cells, the optimal concentration of 1 mM VPA plus 0.5 µM ATRA increased the cytotoxic sensitivity of HL-60 cells to NK cells. The expression of the activated receptors NKp30 and NKG2D on NK-92 cells was higher compared with the levels noted for the other receptors, and the expression of NKG2D ligands MICA/B on HL-60 cells was not significantly upregulated in the ATRA plus VPA goup compared with the control. Moreover, it was observed that the ligands of NKp30 on HL-60 cells presented the same variation trend. As to the co-stimulatory and adhesion molecules on NK-92 and their ligands on HL-60 cells post exposure to ATRA and VPA alone or their combination, there was no obvious change in the expression of CD112, CD48 and CD70 on the HL-60 cells. However, the expression of CD54 on HL-60 cells was significantly upregulated. In contrast, the expression of NKG2A ligands HLA-ABC on HL-60 cells was obviously downregulated. In addition, the expression of HLA-E on the HL-60 cells in the group treated with ATRA plus VPA was not significantly increased. In conclusion, the combination of VPA and ATRA not only induced the differentiation of HL-60 cells, but also induced enhancement of the sensitivity of HL-60 cells to NK cells by downregulating the expression of HLA-ABC and upregulating the expression of CD54, but not MICA/MICB. The results provide experimental and theoretical basis for the clinical combination of a low-dose of ATRA plus VPA for the treatment of leukemia.

  7. Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells.

    PubMed

    Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake; Ozaki, Norio; Noda, Yukihiro

    2016-09-01

    Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100μM) and doxorubicin (0.2µM) decreased the cell survival rate, but olanzapine (1-100µM) did not. Under granulocytic differentiation for 5days, clozapine, even at a concentration of 25μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation.

  8. INDUCTION OF CELL PROLIFERATION AND APOPTOSIS IN HL60 AND HACAT CELLS BY ARSENIC, ARSENATE, AND ARSENIC-CONTAMINATED DRINKING WATER

    EPA Science Inventory

    Induction of cell proliferation and apoptosis in HL-60 and HaCaT cells by arsenite, arsenate and arsenic-contaminated drinking water. T-C. Zhang, M. Schmitt, J. L. Mumford National Research Council, Washington DC and U.S. Environmental Protection Agency, NHEERL, Research Triangle...

  9. INDUCTION OF CELL PROLIFERATION AND APOPTOSIS IN HL60 AND HACAT CELLS BY ARSENIC, ARSENATE, AND ARSENIC-CONTAMINATED DRINKING WATER

    EPA Science Inventory

    Induction of cell proliferation and apoptosis in HL-60 and HaCaT cells by arsenite, arsenate and arsenic-contaminated drinking water. T-C. Zhang, M. Schmitt, J. L. Mumford National Research Council, Washington DC and U.S. Environmental Protection Agency, NHEERL, Research Triangle...

  10. Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in HL-60 cells are not reproducible.

    PubMed

    Speit, Günter; Gminski, Richard; Tauber, Rudolf

    2013-08-15

    Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Various results indicating a genotoxic potential of RF-EMF were reported by the collaborative EU-funded REFLEX (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) project. There has been a long-lasting scientific debate about the reliability of the reported results and an attempt to reproduce parts of the results obtained with human fibroblasts failed. Another part of the REFLEX study was performed in Berlin with the human lymphoblastoid cell line HL-60; genotoxic effects of RF-EMF were measured by means of the comet assay and the micronucleus test. The plausibility and reliability of these results were also questioned. In order to contribute to a clarification of the biological significance of the reported findings, a repeat study was performed, involving scientists of the original study. Comet-assay experiments and micronucleus tests were performed under the same experimental conditions that had led to genotoxic effects in the REFLEX study. Here we report that the attempts to reproduce the induction of genotoxic effects by RF-EMF in HL-60 cells failed. No genotoxic effects of RF-EMF were measured in the repeat experiments. We could not find an explanation for the conflicting results. However, the negative repeat experiments suggest that the biological significance of genotoxic effects of RF-EMF reported by the REFLEX study should be re-assessed. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D/sub 3/ and phorbol-12-myristate-13-acetate

    SciTech Connect

    Murao, S.; Gemmell, M.A.; Callaham, M.F.; Anderson, N.L.; Huberman, E.

    1983-10-01

    Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 x 10/sup -10/ to 10/sup -7/ M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D/sub 3/ and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/. Te resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D/sub 3/ was unable to compete for the phorbol diester binding sites as measured by (/sup 3/H)phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. These results indicate that 1,25-dihydroxyvitamin D/sub 3/ induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages. 40 references, 3 figures, 1 table.

  12. Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in Ca

  13. c-myc but not Hif-1α-dependent downregulation of VEGF influences the proliferation and differentiation of HL-60 cells induced by ATRA.

    PubMed

    Song, Guanhua; Li, Yanmei; Zhang, Zhiyong; Ren, Xia; Li, Hongjiang; Zhang, Wen; Wei, Ruoying; Pan, Sufei; Shi, Lulu; Bi, Kehong; Jiang, Guosheng

    2013-06-01

    Vascular endothelial growth factor (VEGF) plays an important role in solid tumor growth, progression and metastasis as well as in the proliferation and differentiation of hematological malignancies. However, the molecular mechanism that modulates VEGF expression and secretion in leukemia cells has not yet to be elucidated. The purpose of the present study was to investigate the role of the signal pathway in modulating the expression of VEGF in HL-60 cells. Specific siRNAs targeting VEGF were transfected into HL-60 cells and the VEGF expression was measured with reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. The cell proliferation of HL-60 cells was detected by the cell counting kit-8 (CCK-8) assay and the differentiation of HL-60 cells induced by all-trans-retinoic acid (ATRA) was detected by the RT-PCR assay and flow cytometry assay for CD11b. The upstream transcription factors that were related to VEGF expression such as P53, SP-1, c-jun, VHL, cox-2, c-myc and stat3 were detected by RT-PCR assay. In addition, the chromatin immunoprecipitation (ChIP) assay was used to reveal the role of c-myc by binding the target gene VEGF. The results demonstrated the hypoxia-inducible factor 1α-related signaling pathway, not the same as in solid tumors, might not play a key role in modulating VEGF expression. c-myc contributes to the modulation of VEGF expression by targeting the promoter of VEGF, which was indicated by the ChIP assay. In conclusion, our data demonstrate that VEGF plays an important role in the differentiation and proliferation of HL-60 cells; c-myc-dependent downregulation of VEGF induced by ATRA contributes to the differentiation of HL-60 cells.

  14. Eugenol isolated from the essential oil of Eugenia caryophyllata induces a reactive oxygen species-mediated apoptosis in HL-60 human promyelocytic leukemia cells.

    PubMed

    Yoo, Chae-Bin; Han, Ki-Tae; Cho, Kyu-Seok; Ha, Joohun; Park, Hee-Juhn; Nam, Jung-Hwan; Kil, Uk-Hyun; Lee, Kyung-Tae

    2005-07-08

    Eugenol is a major component of essential oil isolated from the Eugenia caryophyllata (Myrtaceae), which has been widely used as a herbal drug. In this study, we investigated the effects of eugenol on the cytotoxicity, induction of apoptosis, and the putative pathways of its actions in human promyelocytic leukemia cells (HL-60) under the standard laboratory illumination. Eugenol-treated HL-60 cells displayed features of apoptosis including DNA fragmentation and formation of DNA ladders in agarose gel electrophoresis. We observed that eugenol transduced the apoptotic signal via ROS generation, thereby inducing mitochondrial permeability transition (MPT), reducing anti-apoptotic protein bcl-2 level, inducing cytochrome c release to the cytosol, and subsequent apoptotic cell death. Taken together, the present study demonstrated that ROS plays a critical role in eugenol-induced apoptosis in HL-60, and this is the first report on the mechanism of the anticancer effect of eugenol.

  15. Differential proteomic analysis of HL60 cells treated with secalonic acid F reveals caspase 3-induced cleavage of Rho GDP dissociation inhibitor 2.

    PubMed

    Li, Ning; Yi, Zhiwei; Wang, Yuqiao; Zhang, Qin; Zhong, Tianhua; Qiu, Yinkun; Wu, Zhen; Tang, Xixiang

    2012-12-01

    Secalonic acid F (SAF) has been previously identified, however, little is known about its cytotoxic activity and related cytotoxic mechanism. The aim of this study was to evaluate the cytotoxic activity of SAF isolated from a deep sea originated fungus Penicillium sp. F11 in HL60 cells and to analyze the differences in protein expression of HL60 cells treated with SAF. The CCK-8 assay and Annexin V-FLUOS/PI assay indicated that SAF displayed dose- and time-dependent inhibition of HL60 cell proliferation and induced apoptosis. Two-dimensional gel electrophoresis (2-DE) analysis of HL60 cells treated with SAF (4 µg/ml) revealed 10 differentially expressed protein spots (P<0.05), 5 upregulated and 5 downregulated. Three spots (1 downregulated and 2 upregulated) were identified as Rho GDP dissociation inhibitor 2 (RhoGDI 2) proteins by MALDI-TOF MS. Western blotting further demonstrated the decreased abundance of full-length RhoGDI 2 together with the increased abundance of caspase 3-cleaved product of RhoGDI 2. The caspase 3 inhibitor Ac-DEVD-CHO could suppress the cytotoxic effect of SAF and significantly block the cleavage of RhoGDI 2. RhoGDI 2 is a cytosolic regulator of Rho GTPase and the caspase 3-cleaved product of RhoGDI 2 can advance progression of the apoptotic process. Our data showed that SAF may modulate RhoGDI 2 levels in HL60 cells, thereby potentially disrupting cell signaling pathways important for HL60 cell function.

  16. 6-C-methyl flavonoids isolated from Pinus densata inhibit the proliferation and promote the apoptosis of the HL-60 human promyelocytic leukaemia cell line.

    PubMed

    Yue, Rongcai; Li, Bo; Shen, Yunheng; Zeng, Huawu; Li, Bo; Yuan, Hu; He, Yiren; Shan, Lei; Zhang, Weidong

    2013-08-01

    Three structurally related 6-C-methyl flavonoids isolated from Pinus densata, including 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone (PD1), 5,7,4'-trihydroxy-3,8-dimethoxy-6-C-methylflavone (PD2), and 5,7,4'-trihydroxy-3-methoxy-6-C-methylflavone (PD3), were tested for their ability to inhibit the proliferation and promote the apoptosis of the HL-60 human leukaemia cell line. Cytotoxicity assays in the HL-60 human cancer cell line demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone exhibited the most potent cytotoxicity of the three structurally related 6-C-methyl flavonoids. 5,4'-Dihydroxy-3,7,8-trimethoxy-6-C-methylflavone inhibited the proliferation of HL-60 cells in a dose-dependent manner with an IC₅₀ of 7.91 µM (48 h treatment). Furthermore, 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-induced apoptosis was associated with mitochondrial membrane disruption and cytochome c release. Flow cytometry analyses revealed an increase in the hypodiploid population in 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-treated HL-60 cells. Treatment with a concentration of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone that induced apoptosis activated caspase-3 but did not activate caspase-1. A caspase-3 inhibitor (Ac-DEVD-CHO), but not a caspase-1 inhibitor (Ac-YVAD-CHO), reversed the cytotoxic effects of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone in HL-60 cells. These data demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone effectively induced the apoptosis of HL-60 cells and exhibited significant anticancer activity via the mitochondrial caspase-3-dependent apoptosis pathway.

  17. Changes in the gene expression of C-myc and CD38 in HL-60 cells during differentiation induced by nicotinic acid-related compounds.

    PubMed

    Ida, Chieri; Ogata, Shin; Okumura, Katsuzumi; Taguchi, Hiroshi

    2008-03-01

    Changes in gene expression levels of c-myc and CD38 were examined during the differentiation of HL-60 cells to granulocytes due to three nicotinic acid-related compounds. CD38 expression was increased by isonicotinic acid and all-trans-retinoic acid (ATRA). Nicotinamide and nicotinamide N-oxide drastically decreased c-myc expression, but isonicotinic acid had no effect, suggesting that these compounds differentiate HL-60 to granulocytes through different pathways. These results should provide useful information as to the mechanisms of cell differentiation.

  18. Differentially expressed nuclear proteins in human CCRF-CEM, HL-60, MEC-1 and Raji cells correlate with cellular properties.

    PubMed

    Henrich, Silke; Crossett, Ben; Christopherson, Richard I

    2007-10-01

    The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.

  19. BCL-x{sub L}/MCL-1 inhibition and RARγ antagonism work cooperatively in human HL60 leukemia cells

    SciTech Connect

    Perri, Mariarita; Yap, Jeremy L.; Yu, Jianshi; Cione, Erika; Fletcher, Steven; Kane, Maureen A.

    2014-10-01

    The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia–retinoic acid receptor, alpha fusion protein (PML–RARα). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates >80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RARα). Whereas combination therapy with As{sub 2}O{sub 3} has increased survival further, patients that experience relapse and are refractory to atRA and/or As{sub 2}O{sub 3} is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-x{sub L}) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RARα agonist), and SR11253 (RARγ antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-x{sub L}/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment. - Highlights: • Novel Bcl-x{sub L}/Mcl-1 inhibitor JY-1-106 reduces HL60 cell viability. • JY-1-106 is investigated in combination with retinoic acid, AM580, and SR11253. • AM580 is an RARα agonist; SR11253 is an RARγ antagonist. • Combined use of JY-1-106/SR11253 exhibited the greatest cell viability reduction. • JY-1-106 alone or in combination with retinoids induces apoptosis.

  20. The nanoparticulate Quillaja saponin KGI exerts anti-proliferative effects by down-regulation of cell cycle molecules in U937 and HL-60 human leukemia cells.

    PubMed

    Berenjian, Saideh; Hu, Kefei; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Bashir Hassan, Sadia; Morein, Bror

    2014-07-01

    Cancer cells are characterized by uncontrolled replication involving loss of control of cyclin dependent kinases (CDKs) and cyclins, and by abolished differentiation. In this study we introduce KGI, which is a nanoparticle with a Quillaja saponin as an active molecule. By the use of RNA array analysis and confirmation at the protein level, we show that KGI affects myeloid leukemia cells (in particular, the U937 monoblast cancer cell) by the following mechanisms: (A) ceasing cell replication via proteasome degradation, (B) down-regulation of key molecules at check points between G1/S and G2/M phases, (C) reduction of thymidine kinase activity, followed by (D) exit to differentiation and production of interleukin-8 (IL-8), eventually leading to apoptosis. Leukemia cell lines (U937 and HL-60 cells) were exposed to KGI for 8 h, after which the drug was removed. The cancer cells did not revert to replication over the following 10 days. Thus our findings suggest that the nanoparticle KGI inhibits proliferation and promotes differentiation in leukemic cells by interfering with the cell cycle process.

  1. Inhibition of DNA topoisomerases I and II and growth inhibition of HL-60 cells by novel acridine-based compounds.

    PubMed

    Janočková, Jana; Plšíková, Jana; Kašpárková, Jana; Brabec, Viktor; Jendželovský, Rastislav; Mikeš, Jaromír; Kovaľ, Ján; Hamuľaková, Slávka; Fedoročko, Peter; Kuča, Kamil; Kožurková, Mária

    2015-08-30

    HL-60 cancer cells were treated with a series of novel acridine derivatives (derivatives 1-4) in order to test the compounds' ability to inhibit both cancer cell growth and topoisomerase I and II activity. Binding studies of derivatives 1-4 with calf thymus DNA were also performed using a number of techniques (UV-Vis and fluorescence spectroscopy, thermal denaturation, linear dichroism and viscometry) to determine the nature of the interaction between the compounds and ctDNA. The binding constants for the complexes of the studied acridine derivatives with DNA were calculated from UV-Vis spectroscopic titrations (K=3.1×10(4)-2.0×10(3)M(-1)). Some of the compounds showed a strong inhibitory effect against Topo II at the relatively low concentration of 5μM. Topo I/II inhibition mode assays were also performed and verified that the novel compounds are topoisomerase suppressors rather than poisons. The biological activities of derivatives were studied using MTT assay and flow cytometric methods (detection of mitochondrial membrane potential, measurement of cell viability) after 24 and 48h incubation. The ability of derivatives to impair cell proliferation was tested by an analysis of cell cycle distribution.

  2. Antiproliferative activity of O4-benzo[c]phenanthridine alkaloids against HCT-116 and HL-60 tumor cells.

    PubMed

    Hatae, Noriyuki; Fujita, Erina; Shigenobu, Saori; Shimoyama, Sayumi; Ishihara, Yuhsuke; Kurata, Yuhki; Choshi, Tominari; Nishiyama, Takashi; Okada, Chiaki; Hibino, Satoshi

    2015-07-15

    The O4-benzo[c]phenanthridine alkaloids exhibit potent antiproliferative activity against cancer cells, which is derived from their ability to inhibit of topoisomerase I and II. It has been reported that in the alkaloids a cationic quaternary ammonium atom, which results in resonance effects between ring A and B, is necessary for increased antiproliferative activity. These findings indicate the role of their substituents at ring A on inhibition of tumor cell proliferation. In the present study, we systematically assessed the cytotoxic activities of naturally occurring alkaloids and their derivatives containing various ring A substituents against two tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. Among the cationic iminium alkaloids, which displayed more potent activity than the corresponding neutral derivatives, and the 7,8-oxygenated benzo[c]phenanthridine alkaloids, chelerythrine and NK109, exhibited stronger antiproliferative activity than the 8,9- and 9,10-oxygenated alkaloids. The activity of cationic iminium alkaloids could be correlated with the bond lengths of their ring A substituents and the electrostatic potentials of their ammonium molecules by DFT calculation.

  3. Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells

    PubMed Central

    Murphy, Mark P.; Caraher, Emma

    2015-01-01

    Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics. PMID:26371179

  4. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.

    PubMed Central

    Wolf, D; Rotter, V

    1985-01-01

    The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein. Images PMID:2858093

  5. Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells.

    PubMed

    Murphy, Mark P; Caraher, Emma

    2015-11-01

    Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

  6. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    PubMed

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  7. Preferential involvement of both ROS and ceramide in fenretinide-induced apoptosis of HL60 rather than NB4 and U937 cells.

    PubMed

    Jiang, Lulu; Pan, Xiaoling; Chen, Ying; Wang, Kankan; Du, Yanzhi; Zhang, Ji

    2011-02-11

    Leukemic cells responding to apoptosis-inducing drugs can be varied in terms of the mechanisms of action. Fenretinide, a synthetic retinoid, is worth of study as a promising candidate for apoptosis-based therapy of leukemia. Yet, it remains unclear whether this drug exerts the similar mechanisms on different leukemic cells. Here, we report a comparative analysis of fenretinide-induced apoptosis in three acute myeloid leukemic (AML) cell lines including HL60, NB4 and U937. Through a series of antagonist assays, we revealed similarities and differences of mechanisms involved in these three cell lines. Antioxidant vitamin C completely abrogated fenretinide-induced apoptosis in all cell lines, demonstrating that ROS is an essential and common mediator. However, the apoptotic effects of fenretinide could be blocked by ceramide synthase inhibitor fumonisin B1 only in HL60 rather than the other two. Moreover, fumonisin B1 was unable to inhibit the generation of ROS in fenretinide-treated HL60 cells, indicating that ROS may function as upstream stimulus of ceramide-mediated apoptosis. These comparative results strongly suggest that the apoptotic response induced by fenretinide in HL60 involves both ROS and ceramide, whereas drug-induced apoptosis in NB4 and U937 requires ROS but is independent of ceramide. Differentiated modes of action exerting on AML may guide the use of this apoptosis-inducing drug, and hence advance our knowledge about the nature of cancer-specific responses to this drug. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Quercus Suber L. Cork Extracts Induce Apoptosis in Human Myeloid Leukaemia HL-60 Cells.

    PubMed

    Bejarano, Ignacio; Godoy-Cancho, Belén; Franco, Lourdes; Martínez-Cañas, Manuel A; Tormo, María A

    2015-08-01

    Quercus suber L. cork contains a diversity of phenolic compounds, mostly low molecular weight phenols. A rising number of reports support with convergent findings that polyphenols evoke pro-apoptotic events in cancerous cells. However, the literature related to the anti-cancer bioactivity of Q. suber L. cork extractives (QSE) is still limited. Herein, we aim to describe the antitumor potential displayed by cork extractives obtained by different extraction methods in the human promyelocytic leukaemia cells. In order to quantify the effects of QSE on cancer cells viability, phosphatidylserine exposure, caspase-3 activity, mitochondrial membrane potential and cell cycle were evaluated. The results indicated that the QSE present a time-dependent and dose-dependent cytotoxicity in the human promyelocytic leukaemia cells. Such a noxious effect leads these leukaemia cells to their death through apoptotic processes by altering the mitochondrial outer membrane potential, activating caspase-3 and externalizing phosphatidylserine. However, cells cycle progression was not affected by the treatments. This study contributes to open a new way to use this natural resource by exploiting its anti-cancer properties. Moreover, it opens new possibilities of application of cork by-products, being more efficient in the sector of cork-based agriculture. Copyright © 2015 John Wiley & Sons, Ltd.

  9. α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells.

    PubMed

    Huang, Huarong; Chen, Shaohua; Van Doren, Jeremiah; Li, Dongli; Farichon, Chelsea; He, Yan; Zhang, Qiuyan; Zhang, Kun; Conney, Allan H; Goodin, Susan; Du, Zhiyun; Zheng, Xi

    2015-06-01

    α‑Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α‑tomatine on human myeloid leukemia HL‑60 cells were investigated. Treatment of HL‑60 cells with α‑tomatine resulted in growth inhibition and apoptosis in a concentration‑dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL‑60 cells. Growth inhibition and apoptosis induced by α‑tomatine in HL‑60 cells was partially abrogated by addition of cholesterol indicating that interactions between α‑tomatine and cell membrane‑associated cholesterol may be important in mediating the effect of α‑tomatine. Activation of nuclear factor‑κB by the phorbol ester, 12‑O‑tetradecanoylphorbol‑13‑acetate failed to prevent apoptosis in HL‑60 cells treated with α‑tomatine. In animal experiments, it was found that treatment of mice with α‑tomatine inhibited the growth of HL‑60 xenografts in vivo. Results from the present study indicated that α‑tomatine may have useful anti‑leukemia activities.

  10. Induction of apoptosis in the human promyelocytic leukemia cell line HL60 by falconensone A and its derivatives, new polyenes.

    PubMed

    Takahashi, N; Kubo, Y; Iwahori, A; Kawai, K I; Fukui, T

    2000-06-01

    Falconensones A and B are a new type of yellow pigment with structural similarity to retinoic acid isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis or Emericella fruticulosa. In the present study we show that falconensone A alone induced apoptosis of HL60 human leukemia cells, while falconensone B, the 4'-nor-methyl derivative of falconensone A, had much lower activity. The synthetic derivatives of falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime, were more potent than natural falconensone A and B as far as the induction of apoptosis was concerned. The induction of apoptosis by the falconensones correlated with their inhibition of cell growth. In addition, falconensones A and B, and falconensone A dioxime, increased the generation of intracellular reactive oxygen species, while falconensone A p-bromophenylhydrazone was inactive. These results suggest that falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime are potential new apoptosis-inducing agents. The enhanced generation of reactive oxygen species in cells may be involved in apoptosis induced by falconensone A and falconensone A dioxime, but not by falconensone A p-bromophenylhydrazone. It is also suggested that the methyl residue at the 4' position of the falconensone A cyclopentenone ring may be essential for the induction of apoptosis. Based on these results, falconensone A and its derivatives may be clinically useful in the treatment of some leukemias.

  11. 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies.

    PubMed

    Liu, Jun-Jen; Wu, Hsueh-Hsia; Chen, Tzu-Ho; Leung, Wan; Liang, Yu-Chih

    2015-08-17

    15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

  12. 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies

    PubMed Central

    Liu, Jun-Jen; Wu, Hsueh-Hsia; Chen, Tzu-Ho; Leung, Wan; Liang, Yu-Chih

    2015-01-01

    15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways. PMID:26287183

  13. Quercetin induces mitochondrial-derived apoptosis via reactive oxygen species-mediated ERK activation in HL-60 leukemia cells and xenograft.

    PubMed

    Lee, Wei-Jiunn; Hsiao, Michael; Chang, Junn-Liang; Yang, Shun-Fa; Tseng, Tsui-Hwa; Cheng, Chao-Wen; Chow, Jyh-Ming; Lin, Ke-Hsun; Lin, Yung-Wei; Liu, Chung-Chi; Lee, Liang-Ming; Chien, Ming-Hsien

    2015-07-01

    Quercetin is a plant-derived bioflavonoid that was recently shown to have multiple anticancer activities in various solid tumors. Here, novel molecular mechanisms through which quercetin exerts its anticancer effects in acute myeloid leukemia (AML) cells were investigated. Results from Western blot and flow cytometric assays revealed that quercetin significantly induced caspase-8, caspase-9, and caspase-3 activation, poly ADP-ribose polymerase (PARP) cleavage, and mitochondrial membrane depolarization in HL-60 AML cells. The induction of PARP cleavage by quercetin was also observed in other AML cell lines: THP-1, MV4-11, and U937. Moreover, treatment of HL-60 cells with quercetin induced sustained activation of extracellular signal-regulated kinase (ERK), and inhibition of ERK by an ERK inhibitor significantly abolished quercetin-induced cell apoptosis. MitoSOX red and 2',7'-dichlorofluorescin fluorescence, respectively, showed that mitochondrial superoxide and intracellular peroxide levels were higher in quercetin-treated HL-60 cells compared with the control group. Moreover, both N-acetylcysteine and the superoxide dismutase mimetic, MnTBAP, reversed quercetin-induced intracellular reactive oxygen species production, ERK activation, and subsequent cell death. The in vivo xenograft mice experiments revealed that quercetin significantly reduced tumor growth through inducing intratumoral oxidative stress while activating the ERK pathway and subsequent cell apoptosis in mice with HL-60 tumor xenografts. In conclusions, our results indicated that quercetin induced cell death of HL-60 cells in vitro and in vivo through induction of intracellular oxidative stress following activation of an ERK-mediated apoptosis pathway.

  14. Quercetin simultaneously induces G0 /G1 -phase arrest and caspase-mediated crosstalk between apoptosis and autophagy in human leukemia HL-60 cells.

    PubMed

    Chang, Junn-Liang; Chow, Jyh-Ming; Chang, Jer-Hwa; Wen, Yu-Ching; Lin, Yung-Wei; Yang, Shun-Fa; Lee, Wei-Jiunn; Chien, Ming-Hsien

    2017-07-01

    Quercetin is a plant-derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL-60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL-60 cell line in vitro and in vivo. Quercetin-induced G0 /G1 -phase arrest occurred when expressions of cyclin-dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)-1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)-II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3-methyladenine, significantly enhanced quercetin-mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL-60 cells with the pan-caspase inhibitor, Z-VAD-fmk, dramatically reversed quercetin-mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin-mediated HL-60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL-60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML. © 2017 Wiley Periodicals, Inc.

  15. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

    PubMed

    Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  16. [Effect of 5-aza-CdR demethylation on expression of SHP-1 and C-kit genes in leukemia HL-60 cells].

    PubMed

    Meng, Zhen; Li, Ying-Hua; Wang, Dong-Mei; Luo, Jian-Min

    2014-12-01

    This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of 5-aza-CdR demethylation on expression of SHP-1 and C-kit genes. RT-PCR was used to detect the mRNA expression level of SHP-1 and C-kit mRNA in HL-60 cells of the drug-treated group and control group.The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 and C-kit genes in HL-60 cells.The results showed that after being treated with 5-aza-CdR, the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally was not expressed. Meanwhile, the high expression level of C-kit mRNA in HL-60 cells was decreased. When HL-60 cells were treated with 0, 0.5, 1.0, 2.0 µmol/L 5-aza-CdR, the demethylation effect was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and the expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05) . It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with 5-aza-CdR suggest that the hypermethylation of SHP-1 gene relates with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of 5-aza-CdR on expression of SHP-1 and C-kit shows dose-dependency, the higher the 5-aza-CdR concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of 5-aza-CdR shows time-dependency in specific concentration.The SHP-1 mRNA expression negatively correlates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.

  17. Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells.

    PubMed

    Kikuchi-Ueda, Takane; Ubagai, Tsuneyuki; Ono, Yasuo

    2013-12-01

    Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.

  18. Subcellular localization of DJ-1 in human HL-60 leukemia cells in response to diallyl disulfide treatment.

    PubMed

    Li, Qingye; Tang, Yuxian; Qin, Jing; Yi, Lan; Yang, Yening; Wang, Juan; He, Jie; Su, Qi; Tan, Hui

    2016-11-01

    Diallyl disulfide (DADS) has been demonstrated to exert potent anticancer effects in vitro and in vivo. Previous studies indicate that DADS may induce the differentiation and/or apoptosis of human leukemia cells in vitro. However, the mechanisms underlying these anticancer effects remain elusive. The aim of the present study was to investigate alterations in the subcellular localization of protein deglycase DJ‑1 (also known as Parkinsonism associated deglycase-7, PARK-7) in the cytoplasm, nucleus and mitochondria of human leukemia HL‑60 cells induced by DADS, in order to provide novel experimental evidence for the molecular mechanisms underlying the anticancer mechanisms of DADS in leukemia cells. HL‑60 cells induced by DADS were collected at different time points, and proteins from the cytoplasm, nucleus and mitochondria of the cells were isolated using specific cellular component isolation kits. The protein expression levels of DJ‑1 in these subcellular fractions of HL60 cells following exposure to DADS for varying lengths of time, were determined using western blotting, immunocytochemistry and immunofluorescence techniques. Following exposure of HL‑60 cells to 1.25 mg/l DADS for 8 h, the protein expression levels of DJ‑1 were significantly decreased in the cytoplasm, while nuclear fractions exhibited a significant increase in DJ‑1 expression when compared with untreated controls. The protein expression levels of DJ‑1 in mitochondria of HL‑60 cells were significantly decreased following treatment with 5 and 10 mg/l DADS. These results demonstrate that exposure of HL‑60 cells to low concentrations of DADS may promote DJ‑1 protein translocation from the cytoplasm to the nucleus, which suggests that DJ‑1 may function as a transcription factor or cofactor binding protein in the process of cell differentiation. The expression of DJ‑1 in mitochondria may be associated with induction of apoptosis in HL‑60 cells treated with moderate

  19. The Effects of Purified Artemia Extract Proteins on Proliferation, Differentiation and Apoptosis of Human Leukemic HL-60 Cells

    PubMed Central

    Deezagi, Abdolkhaleg; Chashnidel, Azadeh; Hagh, Neda Vaseli; Shahraki, Mahvash Khodabandeh

    2016-01-01

    There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 µg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 µg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 µg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells. PMID:28122447

  20. Electrokinetic measurements of dielectric properties of membrane for apoptotic HL-60 cells on chip-based device.

    PubMed

    Huang, Chengjun; Chen, Ailiang; Wang, Lei; Guo, Min; Yu, Jun

    2007-06-01

    The specific membrane capacitance and conductance of mammalian cells reflect the surface morphological complexities and barrier functions of cell membrane, respectively, and could potentially respond to cell physiological and pathological changes in a measurable manner. In this study, an electrokinetic system was developed by using negative dielectrophoretic force (nDEP force) assisted positioning and electroroation (ROT) measurement. Numerical simulations regarding the geometric model of the electrode were performed primarily for the electric field analysis. The dielectric responses of membrane for apoptotic HL-60 cells induced by bufalin were detected. The membrane capacitance of the cells was found to fall from an initial value of 15.6 +/- 0.9 mF/cm(2) to 6.4 +/- 0.6 mF/cm(2) after a 48 h treatment with 10 nM bufalin. However, the membrane conductance remained almost constant at (2.25 +/- 1.1) x 10(3) S/m(2) during the first 12 h of bufalin treatment and then increased distinctly to (4.2 +/- 1.3) x 10(3) S/m(2) thereafter. Scan electron microscopy (SEM) studies of the cells revealed a decreased complexity in cell membrane morphology following bufalin treatments, suggesting that the observed changes in the membrane capacitance was dominated by the alterations of cell surface structures. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents.

  1. The Effects of Purified Artemia Extract Proteins on Proliferation, Differentiation and Apoptosis of Human Leukemic HL-60 Cells

    PubMed

    Deezagi, Abdolkhaleg; Chashnidel, Azadeh; Vaseli Hagh, Neda; Khodabandeh Shahraki, Mahvash

    2016-12-01

    There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 μg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 μg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 μg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells

  2. Steroidal saponins from the aerial parts of Dracaena draco and their cytostatic activity on HL-60 cells.

    PubMed

    Mimaki, Y; Kuroda, M; Ide, A; Kameyama, A; Yokosuka, A; Sashida, Y

    1999-03-01

    Chemical examination of the aerial parts of Dracaena draco has led to the isolation of a total of nine steroidal saponins, including five new ones. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L -arabinopyranosyl} 24-O-beta-D-fucopyranoside, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta, 23,24-tetrol 1-O-{O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L -arabinopyranoside}, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(4-O- acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyransoide} , (23S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-alpha-L- rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyranoside} and (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-(4-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L- arabinopyranoside}. The isolated saponins were evaluated for their cytostatic activity on leukemia HL-60 cells.

  3. Structure-activity relationship studies of 5,7-dihydroxyflavones as naturally occurring inhibitors of cell proliferation in human leukemia HL-60 cells.

    PubMed

    Ninomiya, Masayuki; Nishida, Kyohei; Tanaka, Kaori; Watanabe, Kunitomo; Koketsu, Mamoru

    2013-07-01

    Flavonoids are widely occurring polyphenols that are found in plants. The aim of this study was to investigate the structure-activity relationships of 5,7-dihydroxyflavones, with a focus on the effect of B ring structure substitution on the antiproliferative effects of the compounds in human leukemia HL-60 cells. We prepared a series of 5,7-dihydroxyflavones and evaluated their ability to inhibit the proliferation of HL-60 cells by using the MTT assay. The apoptosis- and cell differentiation-inducing ability of the most potent flavones were investigated using staining and morphological analyses. This study explored the antileukemic and chemopreventive potency of 5,7-dihydroxyflavones, particularly diosmetin and chrysoeriol, which have both hydroxy and methoxy groups on the B ring.

  4. Uvangoletin induces mitochondria-mediated apoptosis in HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances.

    PubMed

    Zheng, Zhuanzhen; Qiao, Zhenhua; Gong, Rong; Wang, Yalin; Zhang, Yiqun; Ma, Yanping; Zhang, Li; Lu, Yujin; Jiang, Bo; Li, Guoxia; Dong, Chunxia; Chen, Wenliang

    2016-02-01

    This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

  5. RRD-251 enhances all-trans retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells

    PubMed Central

    Bunaciu, Rodica P.; Yen, Andrew

    2016-01-01

    All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy. PMID:27331409

  6. RRD-251 enhances all-trans retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.

    PubMed

    Wallace, Aaron S; Supnick, Harrison T; Bunaciu, Rodica P; Yen, Andrew

    2016-07-19

    All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy.

  7. Down-regulation of Mcl-1 by small interference RNA induces apoptosis and sensitizes HL-60 leukemia cells to etoposide.

    PubMed

    Karami, Hadi; Baradaran, Behzad; Esfehani, Ali; Sakhinia, Masoud; Sakhinia, Ebrahim

    2014-01-01

    Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time- dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

  8. Fractionation of the Hypericum perforatum L. extract: PMF, and PDT effects of the fractions against HL-60 leukemic cells

    NASA Astrophysics Data System (ADS)

    Tsontou, M.; Dimitriou, H.; Filippidis, G.; Tsimaris, I.; Kalmanti, M.; Skalkos, D.

    2007-02-01

    In the last three years we have prepared and studied the polar methanolic extract PMF, of the herb Hypericum perforatum L, and studied as a new, alternative photosensitizing substance for PDT. Hypericum perforatum L., as well as PMF, contains a number of naphthodianthrone derivatives (hypericins), such as hypericin and pseudohypericin, as its main photosensitizing constituents. PMF has been tested as a PDT agent in vitro in bladder cancer cells, leukemia cells, and in vivo in rat tumor bearing urinary bladder. In order to evaluate the contribution of the hypericins in the overall PDT action, and prepare a better photosensitizing extract than PMF, we have separated the extract in four main fractions (1,2,3,4), and tested their PDT effects against the HL-60 leukemic cells. The concentration of hypericins in the extracts was found 0.08% for fraction 1, 0.09% for fraction 2, 0.8% for fraction 3, and 2,8% for fraction 4. The PDT activity observed among the fractions was proportional to their hypericins concentration, thus increasing in the order of increasing number: fraction 4 > fraction 3 > fraction 2 > fraction 1. Fraction 4 proved to be the most powerful fraction. However, despite its relatively high hypericins concentration (2.8%), compared with the total extract PMF (1.37%), fraction 4 proved to be less active in the cell line tested. This result indicates that there are other photosensitizing constituents within the PMF extract which contribute significantly in the overall PDT action, and therefore the extract should be used as it is for further PDT studies, without any further purification.

  9. A comparison of apoptosis and necrosis induced by ent-kaurene-type diterpenoids in HL-60 cells.

    PubMed

    Suzuki, Ikue; Kondoh, Masuo; Nagashima, Fumihiro; Fujii, Makiko; Asakawa, Yoshinori; Watanabe, Yoshiteru

    2004-05-01

    We previously reported that ent-11alpha-hydroxy-16-kauren-15-one (KD) induces apoptosis through a caspase-dependent pathway and the induction of apoptosis is dependent on its enone group in human leukemia cells. Here we investigated the abilities of some KD-related compounds with enone group (Fig. 1) to induce apoptosis and to activate some caspases. The IC50 values of ent-kaurene-related compounds possessing the enone group, ent-1beta-hydroxy-9(11),16-kauradien-15-one (1), ent-9(11),16-kauradiene-12,15-dione (2) and the rearranged ent-kaurane-type diterpene (3), against HL-60 cells after 12 h of treatment were 40 microM, 1.8 microM and 5.5 microM, respectively. Although treatment with 3 induced apoptosis, DNA ladder formation was not observed after treatment with 1 or 2. Induction of necrosis, as assayed by trypan blue staining, was observed after treatment with 1 or 2. Treatment with compound 1, 2 or 3 induced proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, and processing of caspase-3. Activation of caspase-8 and processing of Bid, a typical substrate of caspase-8, were also observed on treatment with these compounds. Pretreatment with a broad-spectrum inhibitor of caspases attenuated apoptosis induced by 3 but not necrosis induced by 1 and 2. In summary, KD-related compounds are a unique family of diterpenes that cause either caspase-dependent apoptotic or necrotic cell death.

  10. Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells.

    PubMed

    Fabiani, Roberto; Fuccelli, Raffaela; Pieravanti, Federica; De Bartolomeo, Angelo; Morozzi, Guido

    2009-07-01

    Hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth-suppressive and pro-apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro-apoptotic activity of 3,4-DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4-DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V-FITC kit. The concentration of H(2)O(2) in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. The pro-apoptotic effect of 3,4-DHPEA (100 muM) was prevented by N-acetyl-cysteine, ascorbate, and alpha-tocopherol. Catalase suppressed the 3,4-DHPEA-induced apoptosis, while the Fe(II)-chelating reagent o-phenantroline showed no effect, suggesting the involvement of H(2)O(2 )but not of OH(*). Indeed, 3,4-DHPEA caused accumulation of H(2)O(2) in the culture medium. Tyrosol (p-hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4-DHPEA but not able to generate H(2)O(2), did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4-DHPEA is mediated by the extracellular production of H(2)O(2).

  11. Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

    PubMed Central

    Molnár, Judit; Szebeni, Gábor J.; Csupor-Löffler, Boglárka; Hajdú, Zsuzsanna; Szekeres, Thomas; Saiko, Philipp; Ocsovszki, Imre; Puskás, László G.; Hohmann, Judit; Zupkó, István

    2016-01-01

    Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4β,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents. PMID:26901188

  12. [Demethylation effect of inhibitor As2O3 on expression of SHP-1 and C-kit genes in leukemia HL-60 cells].

    PubMed

    Meng, Zhen; Wang, Dong-Mei; Li, Ying-Hua; Liu, Xiao; Guo, Su-Qing; Luo, Jian-Min

    2013-06-01

    This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of inhibitor As2O3 demethylation on SHP-1 and C-kit genes expression. RT-PCR was used to detect the expression level of SHP-1 and C-kit mRNA in drug-treated cell group and control group. The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 gene in HL-60 cells. The results showed that after being treated with As2O3 the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally did not expressed, meanwhile the expression level of C-kit mRNA in HL-60 cells with high expression decreased. When HL-60 cells were treated with As2O3 of 1.0, 2.5, 5.0 µmol/L, the demethylation effects was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05). It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with As2O3 suggest the hypermethylation of SHP-1 gene related with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of As2O3 on expression of SHP-1 and C-kit shows dose-dependency, the higher the As2O3 concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of As2O3 shows time-dependency in specific concentration. The SHP-1 mRNA expression negatively relates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.

  13. Beta-escin, a natural triterpenoid saponin from Chinese horse chestnut seeds, depresses HL-60 human leukaemia cell proliferation and induces apoptosis.

    PubMed

    Niu, Yang P; Wu, Li M; Jiang, Yan L; Wang, Wen X; Li, Lian D

    2008-09-01

    Beta-escin, a natural triterpenoid saponin isolated from the seed of the horse chestnut, is known to generate a wide variety of biochemical and pharmacological effects. The purpose of the present study was to examine the apoptotic and antiproliferative activity of beta-escin in HL-60 human acute myeloid leukaemia cells. Antiproliferative activity was examined by soft agar colony assay and the trypan blue exclusion method. Apoptotic activity was evaluated by morphological analysis, annexin V analysis, DNA fragmentation analysis and flow cytometry cell cycle analysis. The results showed that beta-escin caused a significant inhibition of HL-60 cell proliferation in a dose- and time-dependent manner. Morphological evidence of apoptosis, including vacuolization, apoptotic nuclei fragmentation and apoptotic body formation, was observed in cells treated with 30 microg mL(-1) of beta-escin for 24, 48 and 72 h. A significant increase in the population of annexin V+ and PI- cells (early apoptotic) among the total cells was observed in cells treated with beta-escin (30-50 microg mL(-1)) for 24 h (P<0.001). Typical DNA ladders, DNA with a unit length of about 180 bp, were detected in cells treated with beta-escin (30-50 microg mL(-1)) for 48 h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin (30-50 microg mL(-1)) induced G1-S arrest and led to a significant accumulation of the sub-G1 population in HL-60 cells (P<0.05). Taken together, the results demonstrate that beta-escin is a potent natural inhibitor of cell proliferation and inducer of apoptosis in HL-60 acute myeloid leukaemia cells. The results indicate that beta-escin may be a useful candidate agent for exploring potential antileukaemic drugs.

  14. Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin.

    PubMed

    Marcinkowska, E; Chrobak, A; Wiedłocha, A

    2001-10-15

    Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells. Copyright 2001 Academic Press.

  15. Tricetin Induces Apoptosis of Human Leukemic HL-60 Cells through a Reactive Oxygen Species-Mediated c-Jun N-Terminal Kinase Activation Pathway

    PubMed Central

    Chien, Ming-Hsien; Chow, Jyh-Ming; Lee, Wei-Jiunn; Chen, Hui-Yu; Tan, Peng; Wen, Yu-Ching; Lin, Yung-Wei; Hsiao, Pei-Ching

    2017-01-01

    Tricetin is a dietary flavonoid with cytostatic properties and antimetastatic activities in various solid tumors. The anticancer effect of tricetin in nonsolid tumors remains unclear. Herein, the molecular mechanisms by which tricetin exerts its anticancer effects on acute myeloid leukemia (AML) cells were investigated. Results showed that tricetin inhibited cell viability in various types of AML cell lines. Tricetin induced morphological features of apoptosis such as chromatin condensation and phosphatidylserine (PS) externalization, and significantly activated proapoptotic signaling including caspase-8, -9, and -3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in HL-60 AML cells. Of note, tricetin-induced cell growth inhibition was dramatically reversed by a pan caspase and caspase-8- and -9-specific inhibitors, suggesting that this compound mainly acts through a caspase-dependent pathway. Moreover, treatment of HL-60 cells with tricetin induced sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and inhibition of ERK and JNK by their specific inhibitors respectively promoted and abolished tricetin-induced cell apoptosis. Dichlorofluorescein (DCF) staining showed that intracellular reactive oxygen species (ROS) levels were higher in tricetin-treated HL-60 cells compared to the control group. Moreover, an ROS scavenger, N-acetylcysteine (NAC), reversed tricetin-induced JNK activation and subsequent cell apoptosis. In conclusion, our results indicated that tricetin induced cell death of leukemic HL-60 cells through induction of intracellular oxidative stress following activation of a JNK-mediated apoptosis pathway. A combination of tricetin and an ERK inhibitor may be a better strategy to enhance the anticancer activities of tricetin in AML. PMID:28758971

  16. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed Central

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-01

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells. Images PMID:1988936

  17. The Synthetic Tryptanthrin Analogue Suppresses STAT3 Signaling and Induces Caspase Dependent Apoptosis via ERK Up Regulation in Human Leukemia HL-60 Cells

    PubMed Central

    Pathania, Anup S.; Kumar, Suresh; Guru, Santosh K.; Bhushan, Shashi; Sharma, Parduman R.; Aithagani, Sravan K.; Singh, Parvinder P.; Vishwakarma, Ram A.; Kumar, Ajay; Malik, Fayaz

    2014-01-01

    Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways. PMID:25383546

  18. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-15

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells.

  19. Retinoic acid-induced differentiation of retrovirus-infected HL-60 cells is associated with enhanced transcription from the viral long terminal repeat

    SciTech Connect

    Collins, S.J.

    1988-11-01

    The author infected different human leukemic cell lines with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. In retrovirus-infected G418-resistant HL-60 cells, induction of granulocyte differentiation by retinoic acid was invariably accompanied by a marked increase (5- to 10-fold) in the transcriptional activity of the integrated retroviral long terminal repeat.

  20. Ethanolic extract of fermented Thunb induces human leukemic HL-60 and Molt-4 cell apoptosis via oxidative stress and a mitochondrial pathway.

    PubMed

    Banjerdpongchai, Ratana; Kongtawelert, Prachya

    2011-01-01

    Houttuynia cordata Thunb (HCT) is a medicinal plant of the Saururaceae family which features antimutagenic and antiviral properties. For extraction, the whole plants were fermented or non-fermented with yeast and ethanol then the whole plants were dried, ground and extracted with 95% ethanol or water. The aims of this study were to compare cytotoxic effects, apoptosis induction, and mechanism(s) with the ethanolic and water extracts of fermented and non-fermented HCT. Cytotoxicity was assessed using the MTT assay in human leukemic HL-60, Molt-4 and peripheral blood mononuclear cells (PBMCs). Apoptotic death was characterized by staining with propidium iodide and examined under a fluorescence microscope. Peroxide radical production and reduction of mitochondrial transmembrane potential (MTP) were determined using 2',7'-dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. The expression of caspase-9 was identified by immunoblotting. The ethanolic extract of fermented HCT was cytotoxic to HL-60 >Molt- 4 > PBMCs, to a greater extent than the non-fermented preparation and the number of apoptotic cells was higher. The alcoholic (fermented) extract produced more radicals than the non-fermented in HL-60 cells but the converse was observed in Molt-4 cells. Reduction of MTP was found in HL-60 and Molt-4 cells treated with the alcoholic (fermented) extract and caspase-9 was cleaved dose-dependently in both cells. In conclusion, the alcoholic extract of fermented HCT was more toxic to human leukemic cells than the non-fermented and both cell lines underwent apoptosis via oxidative stress and a mitochondrial pathway.

  1. Arsenic Trioxide (ATO) cooperates with All Trans Retinoic Acid (ATRA) to enhance MAPK activation and differentiation in Human Myeloblastic Leukemia (HL-60) cells

    PubMed Central

    Nayak, Satyaprakash; Shen, Miaoqing; Varner, Jeffrey D.; Yen, Andrew

    2016-01-01

    Arsenic trioxide (ATO) synergistically promotes retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells, a PML-RARα negative cell line. In PML-RARα positive myeloid leukemia cells, ATO is known to cause degradation of PML-RARα with subsequent induced myeloid differentiation. We find now that ATO by itself does not cause differentiation of the PML-RARα negative HL-60 cells, but enhances RA’s capability to cause differentiation. RA-induced differentiation of HL-60 cells is known to be propelled by an induced hyperactive/persistent MAPK signal. ATO augmented RA induced RAF/MEK/ERK axis signaling and expression of CD11b, an integrin receptor that is a myeloid differentiation marker. p47PHOX, a component of the respiratory burst machinery and inducible oxidative metabolism, functional differentiation marker were also enhanced. However, ATO did not enhance RA-induced CD38 expression, an early cell surface differentiation marker. ATO enhanced RA-induced population growth retardation without evidence of apoptosis or an enhanced G1/0 growth arrest. But compared to RA, ATO plus RA showed reduced pAKT, suggesting that an overall biosynthetic/metabolic retardation was seminal to the apparent enhanced growth retardation due to ATO. In sum, our results indicate that ATO can augment action of RA in causing differentiation of myeloid leukemia cells through promoting MAPK signaling and independent of PML-RARα. PMID:20615082

  2. C/EBPα and PU.1 are involved in distinct differentiation responses of acute promyelocytic leukemia HL-60 and NB4 cells via chromatin remodeling.

    PubMed

    Savickiene, Jurate; Treigyte, Grazina; Vistartaite, Giedre; Tunaitis, Virginijus; Magnusson, Karl-Eric; Navakauskiene, Ruta

    2011-01-01

    C/EBPα and PU.1 are the basic transcription factors that control differentiation-related genes, including granulocyte- colony-stimulating factor (G-CSFR) and human neutrophil elastase (HNE). Here, we analyzed a role of C/EBPα and PU.1 in human acute leukemia cell lines, HL-60 and NB4, in association with a modified chromatin structure by histone deacetylase inhibitors, FK228, sodium phenyl butyrate and vitamin B3. We found that sodium phenyl butyrate alone and 6h-pretreatment with phenyl butyrate or FK228 before the induction of differentiation with all-trans-retinoic acid in the presence of vitamin B3 effectively accelerated and enhanced differentiation to granulocytes in HL-60 but not in NB4 cells as detected by NBT test and the expression of CD11b and CD114 (G-CSFR) using flow cytometric analysis. HDACIs induced a time- and dose-dependent accumulation of hyper-acetylated histone H4 in both cell lines with the delay in NB4 cells. Time-dependent different induction of HL-60 and NB4 cell differentiation was paralleled by the activation of C/EBPα and PU.1 binding to the G-CSFR and the HNE promoters in electrophoretic mobility shift assay. Chromatin immunoprecipitation analysis revealed histone H4 acetylation in the G-CSF receptor promoter at the C/EBPα binding site in HL-60 but not in NB4 cells under the combined treatment. The results indicate that epigenetic events, such as histone acetylation, are involved in the activity modulation of the key transcription factors responsible for the induction of granulocytic differentiation in promyelocytic leukemia cells.

  3. Inhibitory Effect of Isoflavones from Erythrina poeppigiana on the Growth of HL-60 Human Leukemia Cells through Inhibition of Glyoxalase I.

    PubMed

    Hikita, Kiyomi; Yamada, Saori; Shibata, Rina; Katoh, Miyako; Murata, Tomiyasu; Kato, Kuniki; Tanaka, Hitoshi; Kaneda, Norio

    2015-09-01

    It has been reported that many malignant human tissues, including breast, colon, and lung cancers, may show an elevated expression of glyoxalase I (GLO I). GLO I catalyzes the reaction to transform hemimercaptal, a compound formed from methylglyoxal (MG) and reduced glutathione, into S-D-lactoylglutathione, which is then converted to D-lactic acid by glyoxalase II. GLO I inhibitors are expected to be useful for inhibiting tumorigenesis through the accumulation of apoptosis-inducible MG in tumor cells. Here, we investigated the anti-proliferative activity of eight kinds of isoflavone isolated from Erythrina poeppigiana against the growth of HL-60 human leukemia cells from the viewpoint of GLO I inhibition. Of the compounds tested, the diprenyl isoflavone, isolupalbigenin, was shown to exhibit the highest anti-proliferative activity against HL-60 cells. Upon the treatment of HL-60 cells with isolupalbigenin, MG was significantly accumulated in the culture medium, and the caspase 3 activity of the cell lysate was elevated in a time-dependent manner. Thus, it is suggested that isolupalbigenin inhibits the enzyme GLO I, resulting in MG accumulation in the medium, and leading to cell apoptosis. Isolupalbigenin, with two prenyl groups in its A- and B-rings, might be expected to become a potent leading compound for the development of anticancer agents.

  4. Antiproliferative and differentiating effects of polysaccharide fraction from fu-ling (Poria cocos) on human leukemic U937 and HL-60 cells.

    PubMed

    Chen, Yue-Yawn; Chang, Hung-Min

    2004-05-01

    Poria cocos (PC), Fu-Ling, is an oriental fungus and has been widely used as a Chinese traditional herbal medicine for centuries. In the present study, a neutral polysaccharide fraction from PC (PC-PS) was isolated by a series of chromatographies and its effects on antiproliferation and differentiation of human leukemic cells, U937 and HL-60, were investigated in vitro. Results showed that the molecular weight of isolated PC-PS was approximately 160 kDa, as estimated by gel permeation chromatography. The conditioned medium prepared with PC-PS (15 microg/ml)-stimulated human blood mononuclear cells for 5 days (PC-PS-MNC-CM5) exhibited a potent activity in suppressing the proliferation of U937 and HL-60 cells by 87.3 and 74.7%, respectively. Furthermore, PC-PS-MNC-CM5 induced about 66.6% of the U937 cells and 49.4% of the HL-60 cells to differentiate into mature monocytes/macrophages, which also markedly expressed surface antigens of CD11b, CD14, and CD 68. The differentiated U937 and HL-60 cells displayed physiological functions such as respiratory burst and phagocytosis, while PC-PS group and control group showed insignificant effects. Interestingly, the levels of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in PC-PS-MNC-CM were about 41 and 10 times, respectively, higher than that of control group. Antibody neutralization tests of the PC-PS-MNC-CM5 revealed that the growth-inhibitory and differentiation-inducing activities were mainly due to the elevated cytokines of IFN-gamma and TNF-alpha. It suggests that PC-PS is a biological response modifier (BRM), instead of a cytotoxic reagent, and may be a potential alternative in leukemia therapy.

  5. Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with alpha-enolase devoid of plasminogen binding activity.

    PubMed

    Gentile, Fabrizio; Pizzimenti, Stefania; Arcaro, Alessia; Pettazzoni, Piergiorgio; Minelli, Rosalba; D'Angelo, Daniela; Mamone, Gianfranco; Ferranti, Pasquale; Toaldo, Cristina; Cetrangolo, Gianpaolo; Formisano, Silvestro; Dianzani, Mario U; Uchida, Koji; Dianzani, Chiara; Barrera, Giuseppina

    2009-08-13

    HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.

  6. Tempranillo-derived grape seed extract induces apoptotic cell death and cell growth arrest in human promyelocytic leukemia HL-60 cells.

    PubMed

    Espino, Javier; González-Gómez, David; Moreno, Daniel; Fernández-León, María F; Rodríguez, Ana B; Pariente, José A; Delgado-Adámez, Jonathan

    2013-12-01

    Although grape seed extract (GSE) has proven to be effective against various cancers, few studies have investigated the effects of GSE on human leukemia. In this study, we analysed the mechanisms involved in the apoptotic effects induced by GSE on human promyelocytic leukemia HL-60 cells. Thus, GSE treatment succeeded in activating caspase-3 (P < 0.05), the activation being dose-dependent and time-dependent. Activation of caspase-3 induced by GSE was accompanied by mitochondrial membrane depolarization (P < 0.05). Moreover, disruption of mitochondrial integrity caused by GSE treatment subsequently led to activation of caspase-9 (P < 0.05), and also produced a slight increase in ROS levels (P < 0.05). Cytotoxic effects elicited by GSE treatment ultimately resulted in extensive S-phase arrest (P < 0.05) and a substantial increase in the intrinsic rate of apoptosis (P < 0.05). Our findings suggest that the GSE induces apoptotic cell death and cell growth inhibition in human leukemic HL-60 cells, which seems to be dependent on mitochondrial damage. Therefore, the GSE obtained from Tempranillo cultivars could be an effective approach to restrain uncontrolled cell proliferation and survival in leukemia cells.

  7. Effects of the monoamine oxidase inhibitor, tranylcypromine, on induction of HL60 cell differentiation by hexamethylene bisacetamide and N-acetyl-1,6-diaminohexane.

    PubMed

    Snyder, S W; Egorin, M J; Zuhowski, E G; Schimpff, E C; Callery, P S

    1990-01-01

    Hexamethylene bisacetamide (HMBA) is converted by successive deacetylation and oxidation reactions to four major metabolites; in vitro, the initial deacetylated metabolite, N-acetyl-1,6-diaminohexane (NAD-AH), is more potent than HMBA (Synder, S.W.; Egorin, M.J.; Geelhaar, L.A.; Hamburger, A.W.; Callery, P.S. Cancer Res. 48:3613-3616; 1988). We propose that monoamine oxidase (MAO) catalyzed metabolism of NADAH to 6-acetamidohexanoic acid (AcHA) is an inactivation pathway and, therefore, investigated whether blocking such metabolism with the MAO inhibitor, tranylcypromine, would potentiate induction of cell differentiation by HMBA and NADAH. Tranylcypromine, at concentrations up to 30 micrograms/mL, did not inhibit HL60 cell growth and did not induce differentiation of HL60 cells. Tranylcypromine did, however, produce concentration-dependent enhancement of HMBA- and NADAH-induced differentiation. In contrast, 30 micrograms/mL of tranylcypromine did not effect the ability of dimethylsulfoxide, at concentrations between 0.25% and 1.25%, to induce differentiation of HL60 cells. Tranylcypromine, at 30 micrograms/mL, did not change cellular concentrations of HMBA or NADAH but did reduce intracellular concentrations of AcHA, consistent with inhibition of MAO catalyzed conversion of NADAH to AcHA. These results support the hypothesis that MAO catalyzed metabolism of NADH to AcHA is an inactivation pathway and may provide the basis for a clinical trail in which HMBA metabolism is modulated with concurrent tranylcypromine therapy.

  8. Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line.

    PubMed

    Sheng, Xianfu; Zhong, Hua; Wan, Haixia; Zhong, Jihua; Chen, Fangyuan

    2016-07-01

    Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of

  9. Tiron and trolox potentiate the autophagic cell death induced by magnolol analog Ery5 by activation of Bax in HL-60 cells.

    PubMed

    Kumar, Suresh; Kumar, Ajay; Pathania, Anup Singh; Guru, Santosh Kumar; Jada, Srinivas; Sharma, Parduman Raj; Bhushan, Shashi; Saxena, Ajit Kumar; Kumar, H M Sampath; Malik, Fayaz

    2013-05-01

    This study describes the mechanism of trolox and tiron induced potentiation of cytotoxicity caused by Ery5, an analog of magnolol, in human myeloid leukemia HL-60 cells. Ery5 induced cytotoxicity in HL-60 cells by involving activation of bax and cleavage of caspase 3, which contributed towards activation of both apoptotic and autophagic pathways. Trolox and tiron, even at non-toxic concentrations, contributed to the cytotoxicity of Ery5 by activation of autophagic proteins like ATG7, ATG12 and LC3-II. Z-VAD-fmk mediated reduction in the cytotoxicity and expression of autophagic proteins, further suggested that autophagy induced by Ery5 is largely dependent upon caspases. Interestingly, Ery5 induced autophagy was accompanied by the downregulation of PI3K/AKT pathway whereas, trolox and tiron strongly enhanced this effect. In addition to that treatment of cells with Ery5, trolox and tiron individually, displayed a marked upregulation of Bax. The involvement of Bax in trolox and tiron induced enhancement of the cytotoxicity of Ery5 was confirmed, when siRNA induced silencing of Bax led to increased viability of the cells and exerted a strong inhibitory effect on LC3-II accumulation and p62 degradation in case of cells treated by the combination of Ery5 with trolox or tiron. Additionally, an important role of PARP in Ery5 mediated cell death has been suggested by PARP silencing experiments, however, potentiation of autophagic cytotoxicity by trolox and tiron did not seem to be dependent on PARP-1. Therefore, Bax seems to play a vital role in trolox and tiron mediated potentiation of autophagic cell death by Ery5 in HL-60 cells.

  10. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation

    SciTech Connect

    Kauss, M. Ariel; Reiterer, Gudrun; Bunaciu, Rodica P.; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G{sub 1} to S to G{sub 2}/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G{sub 0} cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  11. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation.

    PubMed

    Kauss, M Ariel; Reiterer, Gudrun; Bunaciu, Rodica P; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G(1) to S to G(2)/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G(0) cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  12. [Expression pattern of myeloid differentiation-related transcription factor mRNA in differentiation of NB4 and HL-60 cells induced by all-trans retinoic acid].

    PubMed

    Wu, Yong; Li, Xian-Fang; Yang, Jing-Hui; Liao, Xiao-Ying; Huang, Hui-Fang; Chen, Yuan-Zhong

    2011-08-01

    Hematopoiesis is coordinated by a complex regulatory network of transcription factors that involves proliferation, differentiation and maturation of a very small population of pluripotent hematopoietic stem cells with self-renewing and differentiating into various specialized and distinct blood cell types. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML). The purpose of this study was to investigate the expression pattern of transcription factor mRNA in acute myeloid leukemia (AML) cells during in vitro differentiation. The 2 human leukemic cell lines HL-60 and NB4 had been used as model cell lines. Differentiation of HL-60 and NB4 cells was induced by all-trans retinoic acid (ATRA) for 4 days. Morphological changes were observed by May-Grunwald Giemsa stainings, the CD11b expression level was detected by flow cytometry. Transcription factor mRNA profiles (PU.1, C/EBPα, ε, γ, GATA-1, GATA-2) were determined by real time RT-PCR during in vitro HL-60 and NB4 differentiation; The expression level of transcription factor mRNA was relatively quantitatively analyzed by using 2(-ΔΔCT) and compared with control group. The results showed that the expression levels of PU.1 and C/EBP ε mRNA in NB4 differentiation group were 5.75 and 6.16, respectively, which were significantly higher than those in untreated group; while the expression level of C/EBPα, γ, GATA-1, GATA-2 mRNA in NB4 differentiation group were 62%, 31%, 63% and 8.7% respectively, which were significantly lower than those in untreated group; In HL-60 differentiation group, the expression levels of PU.1, C/EBPα, ε were 1.97, 1.95 and 2.35 respectively, which were significantly higher than those in untreated group; while the expression levels of C/EBPγ, GATA-1, GATA-2 in HL-60 differentiation group were 20%, 21% and 18% respectively, which were significantly lower than those in untreated group. It is concluded that dysregulation of transcription factors is a

  13. Protodioscin, Isolated from the Rhizome of Dioscorea tokoro Collected in Northern Japan is the Major Antiproliferative Compound to HL-60 
Leukemic Cells.

    PubMed

    Oyama, Manami; Tokiwano, Tetsuo; Kawaii, Satoru; Yoshida, Yasunori; Mizuno, Kouichi; Oh, Keimei; Yoshizawa, Yuko

    2017-06-01

    The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.

  14. The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells.

    PubMed

    Cheng, Yung-Yi; Yang, Jai-Sing; Tsai, Shih-Chang; Liaw, Chih-Chuang; Chung, Jing-Gung; Huang, Li-Jiau; Lee, Kuo-Hsiung; Lu, Chi-Cheng; Chien, Hsi-Cheng; Tsuzuki, Minoru; Kuo, Sheng-Chu

    2012-10-01

    The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in vitro.

  15. A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

    PubMed

    Shashi, Bhushan; Jaswant, Singh; Madhusudana, Rao J; Kumar, Saxena A; Nabi, Qazi G

    2006-02-01

    AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.

  16. Euchromatic histone methyltransferase 2 inhibitor, BIX-01294, sensitizes human promyelocytic leukemia HL-60 and NB4 cells to growth inhibition and differentiation.

    PubMed

    Savickiene, Jurate; Treigyte, Grazina; Stirblyte, Ieva; Valiuliene, Giedre; Navakauskiene, Ruta

    2014-07-01

    The involvement of histone lysine methyltransferases (HMT) in carcinogenesis is not well understood. Here, we describe a dose-dependent growth and survival inhibitory effects of BIX-01294, a specific inhibitor of euchromatic HMT2, in promyelocytic leukemia HL-60 and NB4 cells. BIX-01294 combined with all-trans retinoic acid or together with histone deacetylase and DNA methyltransferase inhibitors enhanced cell differentiation to granulocytes and induced cell line-specific changes in the expression of cell cycle-, survival- and differentiation regulating genes and proteins in association with histone modification state. Our results suggest that targeting EHMT2 may be of therapeutical benefits in myeloid leukemia.

  17. Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

    PubMed

    Shang, Hung-Sheng; Shih, Yung-Luen; Lee, Ching-Hsiao; Hsueh, Shu-Ching; Liu, Jia-You; Liao, Nien-Chieh; Chen, Yung-Liang; Huang, Yi-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2017-01-01

    Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca(2+) production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca(2+) production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

  18. miR-181a sensitizes resistant leukaemia HL-60/Ara-C cells to Ara-C by inducing apoptosis.

    PubMed

    Bai, Haitao; Cao, Zhongwei; Deng, Chong; Zhou, Lili; Wang, Chun

    2012-04-01

    Ara-C is one of the most commonly used drugs in the treatment of AML. However, the development of drug resistance always prevented its further use. It has been shown that miR-181a is associated with the clinical outcome of AML patients. Here, we investigated the possible role of miR-181a in AML Ara-C resistance. miR-181a expression was measured by real-time PCR. Cell viability was detected by MTT assay. Protein expressions were measured by western blotting. Caspase activity was examined by fluorescence assay. We found that miR-181a expression was downregulated in the Ara-C-resistant cell line HL-60/Ara-C compared with its parental cell line HL-60. Overexpression of miR-181a in HL-60/Ara-C cells sensitized the cells to Ara-C treatment. Furthermore, Bcl-2 was confirmed as a direct miR-181a target by immunoblot analysis and reporter gene assays. Knockdown of Bcl-2 mimicked the effect of enforced miR-181a expression by reducing cell viability. In addition, the apoptosis pathway was activated by cytochrome C release and caspase 9/caspase 3 activation after miR-181a overexpression. This study for the first time demonstrated that downregulation of miR-181a and upregulation of Bcl-2 in leukaemia cells confer resistance to Ara-C-based therapy. These results suggest that restoration of miR-181a expression might provide a promising therapeutic in drug resistance of leukaemia.

  19. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

    PubMed

    Gabig, T G; Crean, C D; Mantel, P L; Rosli, R

    1995-02-01

    Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates

  20. In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line

    PubMed Central

    2014-01-01

    Background The essential oil (EO) of Artemisia vulgaris L. has been traditionally used worldwide for treating a large number of diseases. Although major components in A. vulgaris EO have been shown to inhibit growth of different cancer cells, as pure compounds or part of other plants extracted oil, no information is known about its anti-proliferative activities. Therefore, the current investigation has evaluated the toxicity of the plant extracted oil from buds (AVO-b) and leaves (AVO-l) and characterized their growth inhibitory effects on cancer cells. Methods AVO-b and AVO-l from A. vulgaris L. were extracted by hydrodistillation, and their effect on the viability of human HL-60 promyelocytic leukemia and various other cancer cell lines was tested using MTT assay. Flow cytometric analysis of apoptosis, DNA fragmentation assay, caspases enzymatic activities and Western blotting were used to determine the apoptotic pathway triggered by their action on HL-60 cells. Results Low concentrations of AVO-b and AVO-l inhibited the growth of HL-60 cells in a dose- and time-dependent manner. Employing flow cytometric, DNA fragmentation and caspase activation analyses, demonstrated that the cytotoxic effect of the oils is mediated by a caspase-dependent apoptosis. Kinetic studies in the presence and absence specific caspase inhibitors showed that activation of caspase-8 was dependent and subsequent to the activation of caspases-9 and -3. In addition, the essential oil caused a disruption of the mitochondrial transmembrane potential (ΔΨm), increased the release of cytochrome c to the cytosol, and altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), Apaf-1 and XIAP. Interestingly, low doses of AVO-b and AVO-1 also induced apoptosis in various cancer cell lines, but not in noncancerous cells. Conclusions The results demonstrate that the EO-induced apoptosis in HL-60 cells is mediated by caspase-dependent pathways, involving caspases-3, -9, and -8

  1. Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells*

    PubMed Central

    Patel, Satyananda; Djerdjouri, Bahia; Raoul-Des-Essarts, Yannick; Dang, Pham My-Chan; El-Benna, Jamel; Périanin, Axel

    2010-01-01

    Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (Km 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67phox and p47phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47phox, p67phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. PMID:20693286

  2. Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy- vitamin D3-induced differentiation of HL-60 cells.

    PubMed Central

    López-Lluch, G; Burón, M I; Alcaín, F J; Quesada, J M; Navas, P

    1998-01-01

    1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1

  3. Synthesis and Biological Activity of Diastereomeric and Geometric Analogs of Calcipotriol, PRI-2202 and PRI-2205, Against Human HL-60 Leukemia and MCF-7 Breast Cancer Cells.

    PubMed

    Milczarek, Magdalena; Chodyński, Michał; Filip-Psurska, Beata; Martowicz, Agnieszka; Krupa, Małgorzata; Krajewski, Krzysztof; Kutner, Andrzej; Wietrzyk, Joanna

    2013-10-31

    Diastereomeric and geometric analogs of calcipotriol, PRI-2202 and PRI-2205, were synthesized as advanced intermediates from vitamin D C-22 benzothiazoyl sulfones and side-chain aldehydes using our convergent strategy. Calcitriol, calcipotriol (PRI-2201) and tacalcitol (PRI-2191) were used as the reference compounds. Among a series of tested analogs the diastereomeric analog PRI-2202 showed the strongest antiproliferative activity on the human breast cancer cell line MCF-7, whereas the geometric analog PRI-2205 was the weakest. Both analogs were less potent in antiproliferative activity against HL-60 cells compared to the reference compounds. The ability to potentiate antiproliferative effect of cisplatin or doxorubicin against HL-60 cells or that of tamoxifen against the MCF-7 cell line was observed at higher doses of PRI-2202 or PRI-2205 than those of the reference compounds. The proapoptotic activity of tamoxifen, expressed as the diminished mitochondrial membrane potential, as well as the increased phosphatidylserine expression, was partially attenuated by calcitriol, PRI-2191, PRI-2201 and PRI-2205. The treatment of the MCF-7 cells with tamoxifen alone resulted in an increase in VDR expression. Moreover, a further increase in VDR expression was observed when the analogs PRI-2201 or PRI-2205, but not PRI-2191, were used in combination with tamoxifen. This observation could partially explain the potentiation of the antiproliferative effect of tamoxifen by vitamin D analogs.

  4. Synthesis and Biological Activity of Diastereomeric and Geometric Analogs of Calcipotriol, PRI-2202 and PRI-2205, Against Human HL-60 Leukemia and MCF-7 Breast Cancer Cells

    PubMed Central

    Milczarek, Magdalena; Chodyński, Michał; Filip-Psurska, Beata; Martowicz, Agnieszka; Krupa, Małgorzata; Krajewski, Krzysztof; Kutner, Andrzej; Wietrzyk, Joanna

    2013-01-01

    Diastereomeric and geometric analogs of calcipotriol, PRI-2202 and PRI-2205, were synthesized as advanced intermediates from vitamin D C-22 benzothiazoyl sulfones and side-chain aldehydes using our convergent strategy. Calcitriol, calcipotriol (PRI-2201) and tacalcitol (PRI-2191) were used as the reference compounds. Among a series of tested analogs the diastereomeric analog PRI-2202 showed the strongest antiproliferative activity on the human breast cancer cell line MCF-7, whereas the geometric analog PRI-2205 was the weakest. Both analogs were less potent in antiproliferative activity against HL-60 cells compared to the reference compounds. The ability to potentiate antiproliferative effect of cisplatin or doxorubicin against HL-60 cells or that of tamoxifen against the MCF-7 cell line was observed at higher doses of PRI-2202 or PRI-2205 than those of the reference compounds. The proapoptotic activity of tamoxifen, expressed as the diminished mitochondrial membrane potential, as well as the increased phosphatidylserine expression, was partially attenuated by calcitriol, PRI-2191, PRI-2201 and PRI-2205. The treatment of the MCF-7 cells with tamoxifen alone resulted in an increase in VDR expression. Moreover, a further increase in VDR expression was observed when the analogs PRI-2201 or PRI-2205, but not PRI-2191, were used in combination with tamoxifen. This observation could partially explain the potentiation of the antiproliferative effect of tamoxifen by vitamin D analogs. PMID:24202449

  5. Volatile isoprenoid constituents of fruits, vegetables and herbs cumulatively suppress the proliferation of murine B16 melanoma and human HL-60 leukemia cells.

    PubMed

    Tatman, Dana; Mo, Huanbiao

    2002-01-25

    Substantial evidence from epidemiological studies supports the inverse association between the intake of fruits, vegetables and other plant products and cancer incidence. Cancer-preventive constituents of fruits and vegetables may inhibit carcinogen activation, enhance carcinogen detoxification, prevent carcinogens from interacting with critical target sites, or impede tumor progression. These activities, however, are achievable only when levels of individual bioactive constituents reach beyond those attainable from a normal balanced diet. Isoprenoids, a broad class of mevalonate-derived phytochemicals ubiquitous in the plant kingdom, suppress the proliferation of tumor cells and the growth of implanted tumors. A search for volatile isoprenoid constituents of food products spanning seven plant families identified 179 isoprenoids. Of these, 41 purchased from commercial sources were screened for efficacy in suppressing the proliferation of murine B16 melanoma cells. Individual isoprenoids suppressed the proliferation of B16 and HL-60 promyelocytic leukemia cells with varying degrees of potency. Cell cycle arrest at the G(0)-G(1) phase and apoptosis account, at least in part, for the suppression. Blends of isoprenoids suppressed B16 and HL-60 cell proliferation with efficacies equal to the sum of the individual impacts. These findings suggest that the cancer-protective property of fruits, vegetables, and related products is partly conferred by the cumulative impact of volatile isoprenoid constituents.

  6. Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

    PubMed

    Husain, Islam; Bala, Kiran; Wani, Abubakar; Makhdoomi, Ubaid; Malik, Fayaz; Sharma, Anjana

    2017-08-25

    Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn(2+) and Mg(2+) ions enhanced arginase activity while, Li(+), Cu(2+), and Al(3+) ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml(-1)) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Oxidative DNA damage is prevented by extracts of olive oil, hydroxytyrosol, and other olive phenolic compounds in human blood mononuclear cells and HL60 cells.

    PubMed

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Servili, Maurizio; Montedoro, Gian Francesco; Morozzi, Guido

    2008-08-01

    Our aim in this study was to provide further support to the hypothesis that phenolic compounds may play an important role in the anticarcinogenic properties of olive oil. We measured the effect of olive oil phenols on hydrogen peroxide (H(2)O(2))-induced DNA damage in human peripheral blood mononuclear cells (PBMC) and promyelocytic leukemia cells (HL60) using single-cell gel electrophoresis (comet assay). Hydroxytyrosol [3,4-dyhydroxyphenyl-ethanol (3,4-DHPEA)] and a complex mixture of phenols extracted from both virgin olive oil (OO-PE) and olive mill wastewater (WW-PE) reduced the DNA damage at concentrations as low as 1 micromol/L when coincubated in the medium with H(2)O(2) (40 micromol/L). At 10 micromol/L 3,4-DHPEA, the protection was 93% in HL60 and 89% in PBMC. A similar protective activity was also shown by the dialdehydic form of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) on both kinds of cells. Other purified compounds such as isomer of oleuropein aglycon (3,4-DHPEA-EA), oleuropein, tyrosol, [p-hydroxyphenyl-ethanol (p-HPEA)] the dialdehydic form of elenoic acid linked to tyrosol, caffeic acid, and verbascoside also protected the cells against H(2)O(2)-induced DNA damage although with a lower efficacy (range of protection, 25-75%). On the other hand, when tested in a model system in which the oxidative stress was induced by phorbole 12-myristate 13-acetate-activated monocytes, p-HPEA was more effective than 3,4-DHPEA in preventing the oxidative DNA damage. Overall, these results suggest that OO-PE and WW-PE may efficiently prevent the initiation step of carcinogenesis in vivo, because the concentrations effective against the oxidative DNA damage could be easily reached with normal intake of olive oil.

  8. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells.

    PubMed

    Lee, Wei-Jiunn; Chen, Yun-Ru; Tseng, Tsui-Hwa

    2011-02-01

    Quercetin, a naturally occurring flavonoid abundant in fruits and vegetables, has been demonstrated as a multipotent bioflavonoid with great potential for the prevention and treatment of cancer. Apoptosis is thought to be an important response to most chemotherapeutic agents in leukemia cells. However, the underlying mechanism of induction of apoptosis by quercetin involving epigenetic regulation is poorly understood. In the present study, by evaluation of fragmentation of DNA, poly (ADP-ribose) polymerase (PARP) and procaspases, we found that quercetin was able to induce apoptosis of human leukemia HL-60 cells in a dose-dependent manner. Quercetin triggered the extrinsic apoptosis pathway through activation of caspase-8 and induction of Bid cleavage, Bax conformation change and cytochrome c release. Furthermore, quercetin induced Fas ligand (FasL) expression involving activation of the extracellular signal-regulated kinase (ERK) and Jun N-terminus kinase (JNK) signaling pathways. In addition to activation of c-Jun, quercetin increased histone H3 acetylation which resulted in the promotion of the expression of FasL. Quercetin exhibited potential for the activation of histone acetyltransferase (HAT) and the inhibition of histone deacetyltransferase (HADC), both of which contributed to histone acetylation. However, only the activation effect on HAT was associated with the ERK and JNK pathway. These results demonstrated that quercetin induced FasL-related apoptosis by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in HL-60 cells.

  9. The putative benzene metabolite 2,3, 5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells.

    PubMed

    Bratton, S B; Lau, S S; Monks, T J

    2000-07-01

    In this study, we show that 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a putative metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Prior to the onset of apoptosis, TGHQ depletes intracellular glutathione (GSH) in a reactive oxygen species (ROS)-independent manner. Neutral, Mg(2+)-dependent sphingomyelinases, which are normally inhibited by GSH, are subsequently activated, as evidenced by increases in intracellular ceramide and depletion of sphingomyelin. As ceramide levels rise, effector caspase (DEVDase) activity steadily increases. Interestingly, while catalase has no effect on TGHQ-mediated depletion of GSH, this hydrogen peroxide (H(2)O(2)) scavenger does inhibit DEVDase activity and apoptosis, provided the enzyme is added to HL-60 cells before an increase in ceramide can be observed. Since ceramide analogues inhibit the mitochondrial respiratory chain, these data imply that ceramide-mediated generation of H(2)O(2) is necessary for the activation of effector caspases-3 and/or -7, and apoptosis. In summary, these studies indicate that TGHQ, and perhaps many quinol-based toxicants and chemotherapeutics, may induce apoptosis in hematopoietic cells by depleting GSH and inducing the proapoptotic ceramide-signaling pathway.

  10. ARF1(2-17) does not specifically interact with ARF1-dependent pathways. Inhibition by peptide of phospholipases C beta, D and exocytosis in HL60 cells.

    PubMed

    Fensome, A; Cunningham, E; Troung, O; Cockcroft, S

    1994-07-25

    The small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N-terminal peptide ARF1(2-17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-depleted HL60 cells was inhibited by ARF1(2-17). In the presence of endogenous cytosol, ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD and exocytosis. GTP-gamma-S-stimulated phospholipase C-beta (PLC-beta) was also inhibited by ARF(2-17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2-17) inhibited the reconstitution of GTP-gamma-S-stimulated PLC-beta activity with exogenously-added PLC-beta 1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2-17) peptide inhibits both ARF-independent (i.e. PLC-beta) and ARF-dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.

  11. Cyclic AMP-dependent protein kinase interferes with GTP. gamma. S stimulated IP sub 3 formation in differentiated HL-60 cell membranes

    SciTech Connect

    Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro )

    1989-01-01

    The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of {sup 3}H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37{degree}C decreased GTP {gamma}S-stimulated inositol trisphosphate (IP{sub 3}) formation by about 30%, but had no influence on Ca{sup 2+}-stimulated IP{sub 3} formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some {sup 32}P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation.

  12. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 and influences the expression of genes involved in the PI3K/AKT/mTOR signaling pathway.

    PubMed

    Han, Shuwen; Zhang, Gang; Li, Maidong; Chen, Dongyun; Wang, Ying; Ye, Wencai; Ji, Zhaoning

    2014-05-01

    The Securinega alkaloids are a class of natural products isolated from plants of the Euphorbiaceae family. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 indicating its potential as an efficient natural antitumor drug with low toxicity. The aim of the present study was to investigate the apoptotic effects of L-securinine on HL-60 cells and to explore its potential underlying molecular mechanism(s) as an antitumor agent. HL-60 cells were cultured with L-securinine. The proliferation and changes in cell morphology were evaluated by cell counting Kit-8 (CCK-8) assay and electron microscopy, respectively. Induction of apoptosis and cell cycle progression were investigated by flow cytometry. The PI3K/AKT/mTOR pathway gene expression was measured by quantitative PCR (qPCR). L-securinine decreased the viability of HL-60 cells in a dose- and time-dependent manner, with IC50 values at 24, 48 and 72 h post-treatment of 47.88, 23.85 and 18.87 µmol/l, respectively. Numerous apoptotic bodies were observed in the HL-60 cells treated with 25 µmol/l L-securinine for 48 h. L-securinine at 12.5, 25 and 50 µmol/l increased the rate of apoptosis in HL-60 cells, and G1/S phase progression was retarded. Furthermore, L-securinine induced downregulation of PI3K, AKT and mTOR gene expression and upregulation of PTEN gene expression in a dose-dependent manner. In conclusion, L-securinine induces apoptosis and inhibition of cell cycle progression in HL-60 cells by modulation of the PI3K/AKT/mTOR pathway gene expression. These observations indicate the potential of L-securinine as an antitumor agent.

  13. Mechanism study of PEGylated polyester and β-cyclodextrin integrated micelles on drug resistance reversal in MRP1-overexpressed HL60/ADR cells.

    PubMed

    Ji, Qian; Qiu, Liyan

    2016-08-01

    Chemotherapy is one of the main strategies for cancer treatment, but its effective application is seriously limited by the development of drug resistance. In this study, we designed micellar vectors for doxorubicin based on amphiphilic copolymers sequentially linking β-cyclodextrin (β-CD), polylacticacid (PLA) or polycaprolactone (PCL) block, and polyethylene glycol (PEG) block to overcome drug resistance in human acute myeloid leukemia cells (HL60/ADR) overexpressing multidrug resistance protein 1 (MRP1). The significant enhancement in cytotoxicity and inhibited HL60/ADR tumor growth in mouse was achieved. More importantly, several analyses were performed to understand the interactions between various polymers and MRP1 at the cellular level. The results showed that the polymers did not show remarkable correlation of MRP1 gene and protein expression, but could decrease intracellular ATP, mitochondrial membrane potential and glutathione levels, which was greatly dependent on the molecular structure of polymers. In conclusion, these novel micelles can be considered as a kind of promising drug delivery system for tumor therapy to reverse drug resistance related to MRP1 overexpression. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Down-regulation of Notch1 expression is involved in HL-60 cell growth inhibition induced by 4-hydroxynonenal, a product of lipid peroxidation.

    PubMed

    Pizzimenti, Stefania; Barrera, Giuseppina; Calzavara, Elisabetta; Mirandola, Leonardo; Toaldo, Cristina; Dianzani, Mario Umberto; Comi, Paola; Chiaramonte, Raffaella

    2008-11-01

    The role of the Notch1 pathway has been well assessed in leukemia. Notch1 mutations are the most common ones in T acute lymphoblastic leukaemia patients which carry either oncogenic Notch1 forms or ineffective ubiquitin ligase implicated in Notch1 turnover. Abnormalities in the Notch1-Jagged1 system have been reported also in acute myelogenous leukaemia (AML) patients where Jagged1 is frequently over-expressed. Moreover, activating Notch1 mutations, as well, can occur in human AML and in leukemia cases with lineage infidelity. As a result, Notch1 signalling inhibition is an attractive goal in leukaemia therapy. Blockage/delay in cell differentiation and/or increase of proliferation are the main results of Notch1 signalling activation in several leukemic cell lines. Moreover, specific genes involved in cell growth control have been identified as Notch1 transcriptional targets, i.e. Cyclin D1 and c-Myc. 4-Hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, is involved in several pathological and physiological conditions, including inflammation; atherosclerosis; and neurodegenerative and chronic liver diseases. Moreover HNE has an antiproliferative/ differentiative effect in several cell lines, by affecting the expression of key genes, such as oncogenes (e.g. c-Myc, c-Myb), cyclins and telomerase. This prompted us to study the effect of HNE on Notch1 expression and its related signalling in HL-60 cells, a leukemic cell line widely used for differentiation studies. RT-PCR as well as Western blot assay showed Notch1down-regulation in HNE-treated HL-60 cells. The expression of Hes1, a Notch1 target gene, was concomitantly down-regulated by HNE treatment, reflecting Notch1 signalling inhibition. DAPT, an inhibitor of Notch activity, when added contemporary to HNE, further increased cell growth inhibition, without affecting apoptosis. Moreover, DAPT treatment reversed the HNE-induced differentiation. Overall these results suggest that Notch1 is a target

  15. Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

    PubMed Central

    Uto, Takuhiro; Tung, Nguyen Huu; Thongjankaew, Pinjutha; Lhieochaiphant, Sorasak; Shoyama, Yukihiro

    2016-01-01

    Background: Mammea siamensis (Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the flower of M. siamensis and demonstrated that kayeassamin A (KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines. Objective: In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by flow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly (ADP-ribose) polymerase (PARP) and caspases were determined by Western blotting. Results: KA exhibited a significant cytotoxic effect in a dose- and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and -8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment. Conclusion: These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and -8 in HL-60 cells. SUMMARY Kayeassamin A (KA) isolated from the flower of Mammea siamensis exhibited a significant cytotoxic effect in HL-60 human leukemia cells. KA triggers apoptotic cell death by activating caspase-3/-8. Abbreviations Used: KA: Kayeassamin A; MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  16. Neoplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibited by mango (Mangifera indica L.) juice and mango juice extracts.

    PubMed

    Percival, Susan S; Talcott, Stephen T; Chin, Sherry T; Mallak, Anne C; Lounds-Singleton, Angela; Pettit-Moore, Jennifer

    2006-05-01

    The mango, Mangifera indica L., is a fruit with high levels of phytochemicals, suggesting that it might have chemopreventative properties. In this study, whole mango juice and juice extracts were screened for antioxidant and anticancer activity. Antioxidant activity of the mango juice and juice extracts was measured by 3 standard in vitro methods. The results of the 3 methods were in general agreement, although different radicals were measured in each. Anticancer activity was measured by examining the effect on cell cycle kinetics and the ability to inhibit chemically induced neoplastic transformation of mammalian cell lines. Incubation of HL-60 cells with whole mango juice and mango juice fractions resulted in an inhibition of the cell cycle in the G(0)/G(1) phase. A fraction of the eluted mango juice with low peroxyl radical scavenging ability was most effective in arresting cells in the G(0)/G(1) phase. Whole mango juice was effective in reducing the number of transformed foci in the neoplastic transformation assay in a dose-dependent manner. These techniques provide valuable screening tools for health benefits derived from mango phytochemicals.

  17. Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells

    PubMed Central

    Husain, Islam; Sharma, Anjana; Kumar, Suresh; Malik, Fayaz

    2016-01-01

    Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7–8 and temperature 35–40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10−3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h) and trypsin (T1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes. PMID:26891220

  18. Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells.

    PubMed

    Husain, Islam; Sharma, Anjana; Kumar, Suresh; Malik, Fayaz

    2016-01-01

    Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7-8 and temperature 35-40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10-3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h) and trypsin (T1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.

  19. Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism.

    PubMed

    Bello, Rosario I; Gómez-Díaz, Consuelo; López-Lluch, Guillermo; Forthoffer, Nathalie; Córdoba-Pedregosa, María C; Navas, Plácido; Villalba, José M

    2005-06-01

    This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G0/1 after serum deprivation. However, when cells were treated with 5 microM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 microM) and copper[II]diisopropyl salicylate (CuDIPS, 10 microM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.

  20. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    PubMed

    Takeda, Yuji; Fu, Junfen; Suzuki, Kichiya; Sendo, Dai; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-06-10

    GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

  1. Effect of modification of the physicochemical properties of ICAM-1-derived peptides on internalization and intracellular distribution in the human leukemic cell line HL-60

    PubMed Central

    Majumdar, Sumit; Tejo, Bimo A.; Badawi, Ahmed; Moore, David; Krise, Jeffrey P.; Siahaan, Teruna J.

    2009-01-01

    The objective of this work is to test the hypothesis that increasing the hydrophilicity of DOX-peptide conjugates may modify their entry mechanisms into HL-60 cells from passive diffusion to receptor-mediated uptake. To test this hypothesis, the entry mechanisms and the intracellular disposition of DOX-cIBR7, DOX-PEGcIBR7, FITC-cIBR, and FITC-cIBR7 were evaluated in HL-60 cells. To increase the hydrophilicity of the peptide, the cIBR peptide (cyclo(1,12)PenPRGGSVLVTGC) was modified to cIBR7 peptide (cyclo(1,8)CPRGGSVC) by removing the hydrophobic residues at the C-terminus. DOX-cIBR7 conjugate, which has higher hydrophilicity than DOX-cIBR, was synthesized. Secondly, a hydrophilic linker (11-amino-3,6,9-trioxaundecanate linker) was incorporated between DOX and cIBR7 to generate DOX-PEGcIBR7 with higher hydrophilicity than DOX-cIBR7. As controls, FITC-cIBR and FITC-cIBR7 were used to check for any endocytic uptake process of the peptide. As previously found with DOX-cIBR, DOX-cIBR7, and DOX-PEGcIBR7, conjugates enter the cells via passive diffusion and not via the energy-dependent endocytic process. This result suggests that an increase in hydrophilicity does not influence the entry mechanism of the DOX-peptide conjugates. In contrast to the DOX-cIBR7 conjugate, the FITC-cIBR7 conjugate showed energy-dependent cellular entry into the cells and followed an endocytic pathway similar to that for dextran. Finally, the entry of DOX-cIBR7 and DOX-PEGcIBR into the cell cytosol was shown to be due to the properties of DOX and not to those of the peptide. PMID:19296614

  2. Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells.

    PubMed

    Ishisaka, R; Kanno, T; Akiyama, J; Yoshioka, T; Utsumi, K; Utsumi, T

    2001-01-01

    We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.

  3. Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells*

    PubMed Central

    Xu, Yong Zhong; Thuraisingam, Thusanth; Marino, Rafael; Radzioch, Danuta

    2011-01-01

    The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)n repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene. PMID:21300803

  4. Five novel naphthylisoquinoline alkaloids with growth inhibitory activities against human leukemia cells HL-60, K562 and U937 from stems and leaves of Ancistrocladus tectorius.

    PubMed

    Jiang, Chao; Li, Zhan-Lin; Gong, Ping; Kang, Sheng-Li; Liu, Ming-Sheng; Pei, Yue-Hu; Jing, Yong-Kui; Hua, Hui-Ming

    2013-12-01

    Two new 7,6'-coupled naphthylisoquinolines, namely ancistrotectorines A (1) and B (2), two new 5,3'-coupled naphthylisoquinolines, namely ancistrotectorines C (3) and D (4), and one new 7,8-coupled naphthylisoquinoline, namely ancistrotectorine E (5), together with 9 known naphthylisoquinoline alkaloids, hamatine (6), ancistrobertsonine B (7), ancistrocladinine (8), hamatinine (9), ancistrotanzanine A (10), ancistrotanzanine B (11), ancistrotectoriline B (12), 7-epi-ancistrobrevine D (13), and ancistrotectorine (14), were isolated from the 70% EtOH extract of Ancistrocladus tectorius. Their structures were elucidated based on the extensive analysis of spectroscopic data (1D, 2D NMR and MS). Compound 5 exhibited inhibitory activities against HL-60, K562 and U937 cell lines with IC50 values of 1.70, 4.18 and 2.56 μM respectively. © 2013.

  5. Effect of NADPH-cytochrome P450 reductase on all-trans-retinoic acid efficacy and cytochrome P450 26A1 expression in human myeloid leukaemia HL-60 cells.

    PubMed

    Hu, Lei; Lv, Jin-Feng; Zhuo, Wei; Zhang, Cong-Min; Zhou, Hong-Hao; Fan, Lan

    2016-09-01

    All-trans-retinoic acid (ATRA), a naturally occurring metabolite of vitamin A, has been shown to have great potential as an antitumorigenic drug to treat acute leukaemia by promoting cancer cell differentiation. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the microsomal cytochrome P450 enzymes including CYP26A1 which is highly specific for ATRA metabolism and efficacy in human myeloid leukaemia cells. In this study, we aimed to investigate the effect of POR on ATRA efficacy and CYP26A1 expression in human myeloid leukaemia HL-60 cells. Stably expressed POR and POR-RNAi HL-60 cell lines were established by transfecting POR overexpression or RNAi (RNA interference) vectors mediated by lentivirus. The protein expression of POR and CYP26A1 was examined by Western blot. The potential roles of POR on ATRA efficacy in HL-60 cells were explored by cell viability assay, cell cycle distribution, cellular differentiation and apoptosis analysis. All-trans-retinoic acid treatment caused the expression of POR upregulation and CYP26A1 downregulation in dose- and time-dependent manners. POR overexpression decreased CYP26A1 expression in HL-60 cells. When POR gene was interfered, the downregulation of CYP26A1 expression by ATRA was abolished. In addition, POR overexpression in HL-60 cells significantly compromised ATRA-induced cell proliferation inhibition, cell cycle arrest, differentiation and apoptosis, whereas downregulation of POR significantly potentiated ATRA effects. Our study therefore suggested that POR played an important role in regulating ATRA efficacy and CYP26A1 expression in HL-60 cells. © 2016 Royal Pharmaceutical Society.

  6. Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

    PubMed

    Tsai, Tsung-Chang; Huang, Hui-Pei; Chang, Kai-Ting; Wang, Chau-Jong; Chang, Yun-Ching

    2017-04-01

    Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL(-1) ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017.

  7. The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells.

    PubMed

    Kumar, Sunil; Pathania, Anup Singh; Saxena, A K; Vishwakarma, R A; Ali, Asif; Bhushan, Shashi

    2013-09-25

    Erythrina suberosa is an ornamental tall tree found in India, Pakistan, Nepal, Bhutan, Burma, Thailand and Vietnam. We have isolated four known distinct metabolites designated as α-Hydroxyerysotrine, 4'-Methoxy licoflavanone (MLF), Alpinumisoflavone (AIF) and Wighteone. Among the four isolated metabolites the two flavonoids, MLF and AIF were found to be the most potent cytotoxic agent with IC50 of ∼20μM in human leukemia HL-60 cells. We are reporting first time the anticancer and apoptotic potential of MLF and AIF in HL-60 cells. Both MLF and AIF inhibited HL-60 cell proliferation and induce apoptosis as measured by several biological endpoints. MLF and AIF induce apoptosis bodies formation, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), release of cytochrome c, Bax, activation of caspase-9, caspase-3 and PARP (poly ADP Ribose polymers) cleavage in HL-60 cells. MLF and AIF also increase the expression of apical death receptor, Fas, with inhibition of anti-apoptotic protein Bid. All the above parameters revealed that these two flavonoids induce apoptosis through both extrinsic and intrinsic apoptotic pathways in HL-60 cells. In spite of apoptosis, these two flavonoids significantly inhibit nuclear transcription factor NF-κB and STAT (Signal Transducer and Activator of Transcription) signaling pathway, which are highly expressed in leukemia. The present study provide an insight of molecular mechanism of cell death induced by MLF and AIF in HL-60 cells which may be useful in managing and treating leukemia.

  8. Ultrastructural localization of F-actin using phalloidin and quantum dots in HL-60 promyelocytic leukemia cell line after cell death induction by arsenic trioxide.

    PubMed

    Izdebska, Magdalena; Gagat, Maciej; Grzanka, Dariusz; Grzanka, Alina

    2013-06-01

    Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD-streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes.

  9. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

    PubMed

    Wu, Qiuling; Lv, Tingting; Chen, Yan; Wen, Lu; Zhang, Junli; Jiang, Xudong; Liu, Fang

    2015-07-01

    The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

  10. Molecular characteristics of cytochrome b558 isolated from human granulocytes, monocytes and HL60 and U937 cells differentiated into monocyte/macrophages.

    PubMed

    Capeillere-Blandin, C; Masson, A; Descamps-Latscha, B

    1991-08-13

    Enriched cytochrome b558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon gamma induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88-91) for purification of cytochrome b558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the gamma band were used and delta epsilon 427-413 was determined to be equal to 158 mM-1 cm-1. An alpha beta type heterodimeric cytochrome b558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide alpha chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the alpha subunit of PMN cytochrome b558. Immunoblotting studies detected the alpha subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b558-enriched preparations, the heme b binding site was shown to be associated with the alpha subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide beta chain from MN cytochrome b was found to have a significantly higher molecular weight than the beta subunit from PMN at 94 +/- 5 kDa instead of 90 +/- 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme

  11. Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells.

    PubMed

    Hatae, Noriyuki; Nakamura, Jun; Okujima, Tetsuo; Ishikura, Minoru; Abe, Takumi; Hibino, Satoshi; Choshi, Tominari; Okada, Chiaki; Yamada, Hiroko; Uno, Hidemitsu; Toyota, Eiko

    2013-08-15

    9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC50 values of these analogs were indicated over 10 μM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity.

  12. 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

    PubMed

    Gokoh, Maiko; Kishimoto, Seishi; Oka, Saori; Mori, Masahiro; Waku, Keizo; Ishima, Yoshio; Sugiura, Takayuki

    2005-03-15

    Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB2 receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that 2-AG (2-arachidonoylglycerol) is the most efficacious agonist. We have proposed that 2-AG is the true natural ligand for both the CB1 and CB2 receptors. Despite the potential physiological importance of 2-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of 2-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB2 receptor. We found that 2-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by 2-AG was abolished when cells were treated with SR144528, a CB2 receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB2 receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that 2-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

  13. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  14. 6-Formylindolo (3,2-b)carbazole (FICZ) enhances retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells.

    PubMed

    Bunaciu, Rodica P; Yen, Andrew

    2013-05-09

    The aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts. Using flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots. We demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more

  15. Decursinol angelate inhibits PGE2-induced survival of the human leukemia HL-60 cell line via regulation of the EP2 receptor and NFκB pathway

    PubMed Central

    Shehzad, Adeeb; Islam, Salman Ul; Ahn, Eun-Mi; Lee, You Mie; Lee, Young Sup

    2016-01-01

    ABSTRACT Decursinol angelate (DA), an active pyranocoumarin compound from the roots of Angelica gigas, has been reported to possess anti-inflammatory and anti-cancer activities. In a previous study, we demonstrated that prostaglandin E2 (PGE2) plays a survival role in HL-60 cells by protecting them from the induction of apoptosis via oxidative stress. Flow cytometry and Hoechst staining revealed that PGE2 suppresses menadione-induced apoptosis, cell shrinkage, and chromatin condensation, by blocking the generation of reactive oxygen species. Treatment of DA was found to reverse the survival effect of PGE2 as well as restoring the menadione-mediated cleavage of caspase-3, lamin B, and PARP. DA blocked PGE2-induced activation of the EP2 receptor signaling pathway, including the activation of PKA and the phosphorylation of CREB. DA also inhibited PGE2-induced expression of cyclooxygenase-2 and the activation of the Ras/Raf/ Erk pathway, which activates downstream targets for cell survival. Finally, DA greatly reduced the PGE2-induced activation of NF-κB p50 and p65 subunits. These results elucidate a novel mechanism for the regulation of cell survival and apoptosis, and open a gateway for further development and combinatory treatments that can inhibit PGE2 in cancer cells. PMID:27414656

  16. Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

    PubMed

    Xu, Bo; Monsarrat, Bernard; Gairin, Jean Edouard; Girbal-Neuhauser, Elisabeth

    2004-04-01

    The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

  17. 2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-{kappa}B

    SciTech Connect

    Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E. . E-mail: ganey@msu.edu

    2007-06-15

    Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A{sub 2} with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of {sup 3}H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-{kappa}B and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A{sub 2}, reduced {sup 3}H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter {sup 3}H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-{kappa}B. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-{kappa}B prevented the 2244-TCB

  18. Ethyl acetate fraction of Garcina epunctata induces apoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: a bioassay-guided approach.

    PubMed

    Constant Anatole, Pieme; Guru, Santoh Kumar; Bathelemy, Ngamegni; Jeanne, Ngogang; Bhushan, Shashi; Murayama, Tetsuya; Saxena, Ajit Kumar

    2013-11-01

    Number of deaths due to cancer diseases is increasing in the world. There is an urgent need to develop alternative therapeutic measures against the disease. Our study reports the cytotoxicity activity of Garcina epunctata (gutifferae) in human promyelocytic leukemia cells (HL-60) and prostate cancer cells (PC-3) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and morphological changes associated with apoptosis were examined by flow cytometry and Hoescht staining respectively. The results of in vitro antiproliferative screening of fractions and extract from G. epunctata indicated that three fractions inhibited the viability of PC-3 cells with IC₅₀ varied from 50 to 88 μ/ml while two fractions inhibited the proliferation of HL-60 cells with IC₅₀ range between 47.5 and 12 μg/ml. Among the entire fraction tested, Hex-EtOAc (75:25) showed cytotoxic effects on the two cell lines and EtOAc fraction was most active only HL-60 cells (12 μg/ml). Treatment of HL-60 cells with G. epunctata (20, 50, 100 μg/ml) for 24 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a number of apoptotic bodies containing nuclear fragments were observed in cells treated with 100 μg/ml. The EtOAc fraction of G. epunctata treatment significantly arrested HL-60 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated as well as the loss of membrane mitochondrial potential in a concentration dependant manner. The results demonstrated that the EtOAc fraction of G. epunctata inhibited the proliferation of HL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway.

  19. Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells.

    PubMed

    Ka, Hyeon; Park, Hee-Juhn; Jung, Hyun-Ju; Choi, Jong-Won; Cho, Kyu-Seok; Ha, Joohun; Lee, Kyung-Tae

    2003-07-10

    Cinnamaldehyde is an active compound isolated from the stem bark of Cinnamomum cassia, a traditional oriental medicinal herb, which has been shown to inhibit tumor cell proliferation. In this study, we investigated the effects of cinnamaldehyde on the cytotoxicity, induction of apoptosis and the putative pathways of its actions in human promyelocytic leukemia cells. Using apoptosis analysis, measurement of reactive oxygen species (ROS), and assessment of mitochondrial membrane potentials (DeltaPsim), we show that cinnamaldehyde is a potent inducer of apoptosis and that it transduces the apoptotic signal via ROS generation, thereby inducing mitochondrial permeability transition (MPT) and cytochrome c release to the cytosol. ROS production, mitochondrial alteration, and subsequent apoptotic cell death in cinnamaldehyde-treated cells were blocked by the antioxidant N-acetylcystein. Taken together, our data indicate that cinnamaldehyde induces the ROS-mediated mitochondrial permeability transition and resultant cytochrome c release. This is the first report on the mechanism of the anticancer effect of cinnamaldehyde.

  20. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    SciTech Connect

    Gulliya, K.S.; Pervaiz, S.

    1989-03-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested.

  1. Cytotoxic effects of novel phytosphingosine derivatives, including N,N-dimethylphytosphingosine and N-monomethylphytosphingosine, in human leukemia cell line HL60.

    PubMed

    Park, Sook Ryun; Cho, Hyo Jin; Moon, Kyung Jin; Chun, Kyung-Hee; Kong, Sun-Young; Yoon, Sung-Soo; Lee, Jong Seok; Park, Seonyang

    2010-01-01

    Novel phytosphingosine derivatives have been developed based on the inhibition of sphingosine kinase, which has been implicated in cell growth and inhibition of ceramide-mediated apoptosis. This study evaluated the cytotoxic effects and underlying mechanisms of action of novel phytosphingosine derivatives, including N-monomethylphytosphingosine (MMPH) and N,N-dimethylphytosphingosine (DMPH) and the pegylated forms MMPH-PEG and DMPH-PEG, in human leukemia HL60 cells. In viability and proliferation assays using WST-1, all four drugs induced suppression of cell growth and viability in a concentration-dependent manner. Among them, DMPH had the highest antileukemic activity and induced apoptosis via caspase-8, caspase-3, and caspase-9 activation. The apoptotic effect was also associated with Fas/FasL upregulation, Bid cleavage, Bcl-2 downregulation, Bax upregulation, mitochondrial membrane depolarization, and cytochrome c release. DMPH decreased the phosphorylation of ERK and inhibited daunorubicin-induced ERK activation. Furthermore, DMPH displayed synergistic cytotoxicity with daunorubicin in a sequence-dependent manner. Our findings indicate that DMPH has potential as an effective cytotoxic agent for leukemia.

  2. An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells.

    PubMed

    Kumar, Ajay; Malik, Fayaz; Bhushan, Shashi; Sethi, Vijay K; Shahi, Ashok K; Kaur, Jagdeep; Taneja, Subhash C; Qazi, Ghulam N; Singh, Jaswant

    2008-02-15

    An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.

  3. 2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

    SciTech Connect

    Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min; Chen, Yen-Jung; Chen, Chun-Jen; Lin, Yu-Fu; Huang, Li-Jiau; Lee, Kuo-Hsiung; Kuo, Sheng-Chu

    2012-03-01

    2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.

  4. Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors.

    PubMed

    McDowell, C L; Carver, R T; Papoutsakis, E T

    1998-10-20

    Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.

  5. Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

    SciTech Connect

    Kuo, H.-C.; Kuo, W.-H.; Lee, Y.-J.; Wang, C.-J.; Tseng, T.-H. . E-mail: tht@csmu.edu.tw

    2006-10-01

    All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest at the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RAR{alpha} assessed by EMSA assay and enhanced the expression of target genes including RAR{alpha}, C/EBP{epsilon}, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL.

  6. Active extracts of wild fruiting bodies of Antrodia camphorata (EEAC) induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A.

    PubMed

    Lu, Mei-Chin; Du, Ying-Chi; Chuu, Jiunn-Jye; Hwang, Shiuh-Lin; Hsieh, Pao-Chuan; Hung, Chih-Sheng; Chang, Fang-Rong; Wu, Yang-Chang

    2009-02-01

    The endemic species of Antrodia camphorate (AC) is a promising chemotherapeutic drug for cancer. We found that the ethanol extract from wild fruiting bodies of Antrodia camphorata (EEAC) could induce HL 60 cells apoptosis via histone hypoacetylation, up-regulation of histone deacetyltransferase 1 (HDAC 1), and down-regulation of histone acetyltransferase activities including GCN 5, CBP and PCAF in dose-dependent manner. In combination with histone deacetylase inhibitor, trichostatin A (TSA), did not block EEAC-induced apoptosis. Interestingly, combined treatment (100 nM of TSA and 100 microg/ml EEAC) caused synergistic inhibition of cell growth and increase of apoptotic induction. EEAC could effectively increase the cytotoxic sensitivity of TSA through the up-regulation of DR5 and NFkappaB activation. In this present study, bioassay-guided fractionation of EEAC led to a major active compound, zhankuic acid A, as the bioactive marker. Moreover, our findings may represent an experimental basis for developing EEAC as a potential chemotherapeutic adjuvant.

  7. Asterosaponins from the Starfish Astropecten monacanthus suppress growth and induce apoptosis in HL-60, PC-3, and SNU-C5 human cancer cell lines.

    PubMed

    Thao, Nguyen Phuong; Luyen, Bui Thi Thuy; Kim, Eun-Ji; Kang, Hee-Kyoung; Kim, Sohyun; Cuong, Nguyen Xuan; Nam, Nguyen Hoai; Kiem, Phan Van; Minh, Chau Van; Kim, Young Ho

    2014-01-01

    Using various chromatographic experiments, six asterosaponins (1-6) were isolated from the MeOH extract of the Vietnamese starfish Astropecten monacanthus. The cytotoxic activities of the MeOH extract and six asterosaponins were evaluated on three human cancer cell lines, HL-60 (promyelocytic leukemia), PC-3 (prostate cancer), and SNU-C5 (colorectal cancer). Relative to the effects of the postitive control mitoxantrone, the MeOH extract (with IC50 values ranging from 0.84±0.03 to 3.96±0.14 µg/mL) and astrosterioside D (5) (with IC50 values ranging from 4.31±0.07 to 5.21±0.15 µM) exhibited potent cytotoxic effects against all three tested human cancer cell lines. In addition, the MeOH extract and astrosterioside D (5) have an effect on leading to apoptosis. Interestingly, the apoptosis of induction was accompanied by down-regulation of phosphatidyl inositol 3-kinase (PI3K)/AKT signaling and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) signaling, and decrease of c-myc expression. Further studies are required to establish use of the asterosaponins from A. monacanthus as remedial and/or nutraceutical purposes.

  8. Toxicological effects of three types of silver nanoparticles and their salt precursors acting on human U-937 and HL-60 cells.

    PubMed

    Barbasz, Anna; Oćwieja, Magdalena; Walas, Stanisław

    2017-01-01

    The growing popularity of nanomaterials requires a systematic study of their effects on the human body. Silver nanoparticles (AgNPs), due to their antiseptic properties, are used in almost every area of life. The purpose of the study was to examine whether the precursor used for the synthesis of nanoparticles affects their bio-influence and modifies their impact on cells of the human immune system. To compare the effects of precursor silver salts (AgNO3, CH3COOAg and AgClO4) and corresponding nanoparticles (TAN TAA and TAC) cytotoxicity study was conducted on two cell lines U-937 and HL-60. For both cell lines, silver salts are more toxic than the corresponding nanoparticles. Cell viability after treatment with the two forms of silver (salt/particle) is dependent on silver dose and degree of cells differentiation. Addition of the silver salt of doses greater than 5 mg/L results in decreased cell viability by over 60%, whereas nanoparticles' addition reduces cell viability on average by 30%. On the basis of the determined LD50 values it can be stated that for the tested cells the most toxic are AgClO4 and TAC. Production of nitric oxide, which is a mediator of inflammation, is the greatest after treatment of the cells by TAC. Different interactions of studied nanoparticles with albumin has been found and it was shown that addition of albumin to the cells treated by nanoparticles reduces their toxic effects. Obtained by us highly purified, mono-disperse AgNPs exhibit diverse effects relative to the biological systems, depending on the precursor salt used.

  9. All-trans retinoic acid and a novel synthetic retinoid tamibarotene (Am80) differentially regulate CD38 expression in human leukemia HL-60 cells: possible involvement of protein kinase C-delta.

    PubMed

    Uruno, Akira; Noguchi, Naoya; Matsuda, Ken; Nata, Koji; Yoshikawa, Takeo; Chikamatsu, Youichiro; Kagechika, Hiroyuki; Harigae, Hideo; Ito, Sadayoshi; Okamoto, Hiroshi; Sugawara, Akira

    2011-08-01

    ATRA and a synthetic RAR agonist tamibarotene (Am80) induce granulocytic differentiation of human acute leukemia HL-60 cells and have been used in antineoplastic therapy. ATRA induces CD38 antigen during HL-60 cell differentiation, which interacts with CD31 antigen on the vascular EC surface and may induce disadvantages in the therapy. We here examined the mechanisms of the ATRA-mediated CD38 induction and compared the difference between ATRA- and tamibarotene-mediated induction. Tamibarotene-induced HL-60 cell adhesion to ECs was 38% lower than ATRA, and NB4 cell adhesion to ECs by tamibarotene was equivalent to ATRA, which induced CD38 gene transcription biphasically in HL-60 cells, the early-phase induction via DR-RARE containing intron 1, and the delayed-phase induction via RARE lacking the 5'-flanking region. In contrast to ATRA, tamibarotene induced only the early-phase induction, resulting in its lower CD38 induction than ATRA. A PKCδ inhibitor, rottlerin, and siRNA-mediated PKCδ knockdown suppressed the ATRA-induced CD38 promoter activity of the 5'-flanking region, whereas a RAR antagonist, LE540, or RAR knockdown did not affect it. Cycloheximide and rottlerin suppressed the delayed-phase induction of CD38 expression by ATRA but did not affect the early-phase induction. Moreover, ATRA, but not tamibarotene, induced PKCδ expression without affecting its mRNA stability. The diminished effect of tamibarotene on CD38-mediated HL-60 cell adhesion to ECs compared with ATRA is likely a result of the lack of its delayed-phase induction of CD38 expression, which may be advantageous in antineoplastic therapy.

  10. Cytotoxic and DNA-damaging effects of methyl tert-butyl ether and its metabolites on HL-60 cells in vitro

    SciTech Connect

    Tang, G.H.; Shen, Y.; Shen, H.M.

    1996-12-31

    Methyl tert-butyl ether (MTBE) is a widely used oxygenate in unleaded gasoline; however, few studies have been conducted on the toxicity of this compound. This study evaluates the cytotoxic and DNA-damaging effects of MTBE and its metabolites in a human haemopoietic cell line, HL-60. The metabolites of MTBE studied include tertiary butyl alcohol (TBA), {alpha}-hydroxyisobutyric acid (HIBA), and formaldehyde. Comet assay is used to assess DNA damage, and the cytotoxicity is investigated by lactate dehydrogenease (LDH) release. The results show no significant cytotoxic effects of MTBE, TBA, and HIBA over a concentration ranging from 1 to 30 mM. Formaldehyde, in contrast, causes a substantial LDH release at a concentration of 5 {mu}M. Hydrogen peroxide, a known oxidative agent, at concentrations ranging from 10 to 100 {mu}M, produces a significant dose-related increase in DNA damage, whereas a much higher concentration of MTBE (1 to 30 mM) is required to produce a similar observation. The genotoxic effects of TBA and HIBA appear to be identical to that of MTBE. Conversely, DNA damage is observed for formaldehyde at a relatively low concentration range (5 to 100 {mu}M). These findings suggest that MTBE and its metabolites, except formaldehyde, have relatively low cytotoxic and genotoxic effects. 16 refs., 4 figs.

  11. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    PubMed Central

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-01-01

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively. PMID:25262027

  12. An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

    PubMed

    Emiliani, C; Beccari, T; Tabilio, A; Orlacchio, A; Hosseini, R; Stirling, J L

    1990-04-01

    Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.

  13. An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

    PubMed Central

    Emiliani, C; Beccari, T; Tabilio, A; Orlacchio, A; Hosseini, R; Stirling, J L

    1990-01-01

    Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease. Images Fig. 5. Fig. 6. PMID:2139328

  14. Four new dammarane-type triterpene saponins from the stems and leaves of Panax ginseng and their cytotoxicity on HL-60 cells.

    PubMed

    Li, Ke-Ke; Yao, Chun-Mei; Yang, Xiu-Wei

    2012-01-01

    Four new triterpene saponins, ginsenosides Rh(14)-Rh(17)(1- 4), along with two known compounds, 20(S)-ginsenoside Rg₂ and dammar-(E)-20(22),24-diene-3 β,6 α,12 β-triol, were isolated from the stems and leaves of Panax ginseng. The structures of the new compounds were elucidated as 3 β,6 α,12 β,24 ξ-tetrahydroxy-dammar-(E)-20(22),25-diene 6- O- α- L-rhamnopyranosyl-(1 → 2)- β-D-glucopyranoside (1), 3 β,12 β,24 ξ-trihydroxy-dammar-(E)-20(22),25-diene 3- O- β- D-glucopyranosyl-(1 → 2)- β-D-glucopyranoside (2), 3 β,6 α,12 β-trihydroxy-dammar-(E)-20(22),24-diene 3-O-β-D-glucopyranoside (3), and 3-oxo-6 α,12 β,20(S)-trihydroxy-dammar-24-ene 6-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside (4) by means of extensive spectroscopic and chemical methods, respectively. The isolated compounds were tested for IN VITRO cytotoxicity against HL-60 cells. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells

    SciTech Connect

    Sahyoun, N.; Wolf, M.; Besterman, J.; Hsieh, T.S.; Sander, M.; LeVine H. III; Chang, K.J.; Cuatrecasas, P.

    1986-03-01

    DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a K/sub m/ of about 100 nM, the reaction was rapid, occurring at 4/sup 0/C as well as at 30/sup 0/C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca/sup 2 +/ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), but not by the inactive analog o-AMSA.

  16. Evidence for involvement of multiple iron species in DNA single-strand scission by H2O2 in HL-60 cells.

    PubMed

    Byrnes, R W

    1996-01-01

    Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsulfonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elution of DNA of cells treated at 4 degrees to minimize repair during treatment. Strand breakage in the presence of 10% serum was only 40% of that in the absence of serum. This effect was traced to reaction of H2O2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me2SO) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me2SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me2SO-inhibitable strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extracellular iron in serum was taken into account, 60 microM orthophenanthroline (OP) inhibited a maximum of 85% of strand breaks. In cells pretreated with 60 microM OP, the Me2SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decreased to 4.1 nm. These data indicate the existence of a number of different iron species, as characterized by overlapping but not coincidental inhibition by OP and Me2SO, and by differing diffusion distances.

  17. Seed dormancy breaking diterpenoids from the liverwort Plagiochila sciophila and their differentiation inducing activity in human promyelocytic leukemia HL-60 cells.

    PubMed

    Kenmoku, Hiromichi; Tada, Hiroyuki; Oogushi, Megumi; Esumi, Tomoyuki; Takahashi, Hironobu; Noji, Masaaki; Sassa, Takeshi; Toyota, Masao; Asakawa, Yoshinori

    2014-07-01

    To obtain the structural diversity of bioactive compounds similar to cotylenins and fusicoccins that modulate 14-3-3 protein-protein interactions in eukaryotes, screening tests were carried out using the lettuce seed dormancy breaking-assay. An acetone extract of the liverwort Plagiochila sciophila exhibited significant activity against the seeds in the presence of the plant hormone abscisic acid. Activity-guided fractionation of the extract afforded the isolation of seven novel fusicoccane-type diterpenoids, named fusicosciophins A-E (1-5), 8-deacetyl (6) and 9-deacetyl fusicosciophin E (7). Their structures were determined by spectroscopic methods and X-ray crystallographic analyses. All the pure isolated compounds (1-7) exhibited moderate lettuce seed dormancy breaking activity. In addition, the differentiation-inducing activity and cytotoxicity of these isolates, together with fusicoccin A (FC-A) and all-trans retinoic acid (ATRA), were evaluated in human promyelocytic leukemia HL-60 cells and human mouth epidermal carcinoma KB cells, respectively. Fusicosciophins (2 and 4) and FC-A exhibited moderate differentiation-inducing activity (EC50 31.2-59.1 microM) compared with ATRA (EC50 0.3 microM), while 2, 4 and ATRA exhibited higher selectivity indices (IC50/EC50 >3.38-667) than FC-A (IC50/EC50 1.05). This is the first report on the isolation of fusicoccane-type diterpenoids from liverworts having seed dormancy breaking activity and differentiation-inducing activity in mammal cells.

  18. 6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells.

    PubMed

    Bunaciu, Rodica P; Jensen, Holly A; MacDonald, Robert J; LaTocha, Dorian H; Varner, Jeffrey D; Yen, Andrew

    2015-01-01

    6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.

  19. 6-Formylindolo(3,2-b)Carbazole (FICZ) Modulates the Signalsome Responsible for RA-Induced Differentiation of HL-60 Myeloblastic Leukemia Cells

    PubMed Central

    Bunaciu, Rodica P.; LaTocha, Dorian H.; Varner, Jeffrey D.; Yen, Andrew

    2015-01-01

    6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association. PMID:26287494

  20. ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary.

    PubMed

    Congleton, Johanna; Jiang, Hong; Malavasi, Fabio; Lin, Hening; Yen, Andrew

    2011-04-15

    Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11-20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11-20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11-20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function. Copyright © 2010. Published by Elsevier Inc.

  1. ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary

    PubMed Central

    Congleton, Johanna; Jiang, Hong; Malavasi, Fabio; Lin, Hening; Yen, Andrew

    2013-01-01

    Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11–20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11–20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11–20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function. PMID:21156171

  2. Role of Ras-related C3 botulinum toxin substrate 2 (Rac2), NADPH oxidase and reactive oxygen species in diallyl disulphide-induced apoptosis of human leukaemia HL-60 cells.

    PubMed

    Yi, Lan; Ji, Xiao-Xia; Tan, Hui; Lin, Min; Tang, Yi; Wen, Ling; Ma, Yan-Hua; Su, Qi

    2010-12-01

    1. Diallyl disulphide (DADS) has potential as a chemopreventive and therapeutic agent. Previous studies have reported that Ras-related C3 botulinum toxin substrate 2 (Rac2), a regulatory subunit of the NADPH oxidase complex, is upregulated in DADS-induced apoptosis in human leukaemia HL-60 cells. The aim of the present study was to investigate the role of Rac2, NADPH oxidase and reactive oxygen species (ROS) in DADS-induced apoptosis. 2. Expression of the Rac2 gene along with that of five other genes of NADPH oxidase subunits were in HL-60 cells measured by Sybergreen quantitative real-time polymerase chain reaction. RNA interference was used to test the effect of Rac2. Protein expression was evaluated using western blot analysis and ROS levels were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence. DNA fragmentation and flow cytometry analysis were used to detect apoptotic cells. 3. Levels of Rac2 gene and protein were significantly upregulated and NADPH oxidase was activated in DADS-induced apoptosis. Pretreatment of HL-60 cells with small interfering (si) RNAs to inhibit Rac2 blocked DADS-induced apoptosis. Diallyl disulphide-induced intracellular ROS production was increased in phorbol myristate acetate-stimulated cells, but decreased in Rac2 siRNA-treated cells. In Rac2 siRNA-treated cells, activator protein-1 and caspase 3 levels decreased, c-myc protein levels were increased and p38 protein levels were unchanged compared with Rac2-competent, DADS-treated cells. 4. These results demonstrate that NADPH oxidase is the main source of DADS-induced ROS. In addition, Rac2 selectively activates the c-Jun N-terminal kinase pathway, but not the p38 pathway, in DADS-induced apoptosis. So, Rac2, NADPH oxidase and ROS have a critical role in DADS-induced apoptosis in human leukaemia HL-60 cells.

  3. Vitamin K2 and cotylenin A synergistically induce monocytic differentiation and growth arrest along with the suppression of c-MYC expression and induction of cyclin G2 expression in human leukemia HL-60 cells.

    PubMed

    Maniwa, Yasuhisa; Kasukabe, Takashi; Kumakura, Shunichi

    2015-08-01

    Although all-trans retinoic acid (ATRA) is a standard and effective drug used for differentiation therapy in acute promyelocytic leukemia, ATRA-resistant leukemia cells ultimately emerge during this treatment. Therefore, the development of new drugs or effective combination therapy is urgently needed. We demonstrate that the combined treatment of vitamin K2 and cotylenin A synergistically induced monocytic differentiation in HL-60 cells. This combined treatment also synergistically induced NBT-reducing activity and non-specific esterase-positive cells as well as morphological changes to monocyte/macrophage-like cells. Vitamin K2 and cotylenin A cooperatively inhibited the proliferation of HL-60 cells in short-term and long-term cultures. This treatment also induced growth arrest at the G1 phase. Although 5 µg/ml cotylenin A or 5 µM vitamin K2 alone reduced c-MYC gene expression in HL-60 cells to approximately 45% or 80% that of control cells, respectively, the combined treatment almost completely suppressed c-MYC gene expression. We also demonstrated that the combined treatment of vitamin K2 and cotylenin A synergistically induced the expression of cyclin G2, which had a positive effect on the promotion and maintenance of cell cycle arrest. These results suggest that the combination of vitamin K2 and cotylenin A has therapeutic value in the treatment of acute myeloid leukemia.

  4. Regulated expression of the MRP8 and MRP14 genes in human promyelocytic leukemic HL-60 cell treated with the differentiation-inducing agents mycophenolic acid and 1{alpha},25-Dihydroxyvitamin D{sub 3}

    SciTech Connect

    Warner-Bartnicki, A.L.; Murao, S.; Collart, F.R.; Huberman, E.

    1992-12-31

    The calcium-binding proteins MRP8 and MEP14 are present in mature monomyelocytic cells and are induced during differentiation. Previous studies have demonstrated that the proteins may mediate the growth arrest in differentiating HL-60 cells. We determined the levels of a protein complex (PC) containing MRP8 and MRP14 and investigated the mechanism by which the genes encoding these proteins are regulated in HL-60 cells treated with the differentiation-inducing agent mycophenorc acid (MPA)While the PC was barely detectable in untreated cells, MPA treatment resulted in elevated levels of the PC which were maximal at 3-4 d, and were found to directly parallel gains in the steady-state levels of MRP8 and MRP14 MRNA. Transcription studies with the use of nuclear run-on experiments revealed increased transcription initiation at the MRP8 and MRP14 promoters after MPA treatment. 1{alpha},25-Dihydroxyvitamin D{sub 3}, which induces HL-60 cell differentiation by another mechanism, was also found to increase transcription initiation at the MRP8 and MRP14 promoters. Our results suggest that this initiation is the major control of maturation agent-mediated increases in MRP8 and MRPl4 gene expression, and support a role for the PC in terminal differentiation of human monomyelocytic cells.

  5. Cell cycle phase-dependent effect of retinoic acid on the induction of granulocytic differentiation in HL-60 promyelocytic leukemia cells. Evidence for sphinganine potentiation of retinoic acid-induced differentiation.

    PubMed

    Hui, E K; Yung, B Y

    1993-03-01

    The efficiency of retinoic acid (RA)-induced differentiation was dependent on the position of HL-60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA-induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA-induced differentiation.

  6. Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells

    PubMed Central

    Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

    2016-01-01

    Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin – another DPP-4 inhibitor – induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans. PMID:27759084

  7. Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells.

    PubMed

    Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

    2016-10-19

    Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin - another DPP-4 inhibitor - induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans.

  8. Libraries of 2β-(N-substituted piperazino)-5α-androstane-3α, 17β-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

    PubMed

    Roy, Jenny; Maltais, René; Jegham, Hajer; Poirier, Donald

    2011-05-01

    Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 μM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

  9. Single pre-treatment with hypericin, a St. John's wort secondary metabolite, attenuates cisplatin- and mitoxantrone-induced cell death in A2780, A2780cis and HL-60 cells.

    PubMed

    Jendželovská, Zuzana; Jendželovský, Rastislav; Hiľovská, Lucia; Kovaľ, Ján; Mikeš, Jaromír; Fedoročko, Peter

    2014-10-01

    St. John's wort (SJW, Hypericum perforatum L.) is a commonly used natural antidepressant responsible for the altered toxicity of some anticancer agents. These interactions have been primarily attributed to the hyperforin-mediated induction of some pharmacokinetic mechanisms. However, as previously demonstrated by our group, hypericin induces the expression of two ABC transporters: multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Because cisplatin (CDDP) and mitoxantrone (MTX) are potential substrates of ABC transporters, we investigated the effect of 24h hypericin pre-treatment on the cytotoxicity of CDDP and MTX in human cancer cell lines. CDDP-sensitive and -resistant ovarian adenocarcinoma cell lines A2780/A2780cis, together with HL-60 promyelocytic leukemia cells and ABCG2-over-expressing cBCRP subclone, were used in our experiments. We present CDDP cytotoxicity attenuated by hypericin pre-treatment in both A2780 and A2780cis cells and MTX cytotoxicity in HL-60 cells. In contrast, hypericin potentiated MTX-induced death in cBCRP cells. Interestingly, hypericin did not restore cell proliferation in rescued cells. Nevertheless, hypericin did increase the expression of MRP1 transporter in A2780 and A2780cis cells indicating the impact of hypericin on certain resistance mechanisms. Additionally, our results indicate that hypericin may be the potential substrate of BCRP transporter. In conclusion, for the first time, we report the ability of hypericin to affect the onset and/or progress of CDDP- and MTX-induced cell death, despite strong cell cycle arrest. Thus, hypericin represents another SJW metabolite that might be able to affect the effectiveness of anti-cancer drugs and that could interact with ABC transporters, particularly with BCRP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells.

    PubMed

    Sugiura, T; Kondo, S; Kishimoto, S; Miyashita, T; Nakane, S; Kodaka, T; Suhara, Y; Takayama, H; Waku, K

    2000-01-07

    We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

  11. Expression of human kinase suppressor of Ras 2 (hKSR-2) gene in HL60 leukemia cells is directly upregulated by 1,25-dihydroxyvitamin D{sub 3} and is required for optimal cell differentiation

    SciTech Connect

    Wang Xuening; Wang, T.-T.; White, John H.; Studzinski, George P. . E-mail: studzins@umdnj.edu

    2007-08-15

    Induction of terminal differentiation of neoplastic cells offers potential for a novel approach to cancer therapy. One of the agents being investigated for this purpose in preclinical studies is 1,25-dihydroxyvitamin D{sub 3} (1,25D), which can convert myeloid leukemia cells into normal monocyte-like cells, but the molecular mechanisms underlying this process are not fully understood. Here, we report that 1,25D upregulates the expression of hKSR-2, a new member of a small family of proteins that exhibit evolutionarily conserved function of potentiating ras signaling. The upregulation of hKSR-2 is direct, as it occurs in the presence of cycloheximide, and occurs primarily at the transcriptional level, via activation of vitamin D receptor, which acts as a ligand-activated transcription factor. Two VDRE-type motifs identified in the hKSR-2 gene bind VDR-RXR alpha heterodimers present in nuclear extracts of 1,25D-treated HL60 cells, and chromatin immunoprecipitation assays show that these VDRE motifs bind VDR in 1,25D-dependent manner in intact cells, coincident with the recruitment of RNA polymerase II to these motifs. Treatment of the cells with siRNA to hKSR-2 reduced the proportion of the most highly differentiated cells in 1,25D-treated cultures. These results demonstrate that hKSR-2 is a direct target of 1,25D in HL60 cells, and is required for optimal monocytic differentiation.

  12. Differentiation and apoptosis induction by lovastatin and γ-tocotrienol in HL-60 cells via Ras/ERK/NF-κB and Ras/Akt/NF-κB signaling dependent down-regulation of glyoxalase 1 and HMG-CoA reductase.

    PubMed

    Chen, Chun-Chia; Liu, Tzu-Yu; Huang, Shih-Pin; Ho, Chi-Tang; Huang, Tzou-Chi

    2015-11-01

    Glyoxalase 1 (GLO1) and HMG-CoA reductase (HMGCR) are highly expressed in most tumor cells and little in normal cells. In this study, treatment of HL-60 cells with lovastatin induced characteristic apoptosis in a dose-dependent manner. We demonstrated that lovastatin treatment inhibited Ras and Raf protein translocation to cell membrane and eliminated the phosphorylation of the downstream effectors Akt and ERK, and the subsequent NF-κB translocation into nucleus. Specific inhibitors and γ-tocotrienol confirmed the Ras/Raf/ERK/NF-κB/GLO1 and Ras/Akt/NF-κB/GLO1 pathways. Data revealed that lovastatin induced HL-60 cell death was attenuated by mevalonate treatment. We demonstrated also that γ-tocotrienol showed its apoptotic effect on the HL-60 cell through the same pathway. γ-Tocotrienol enhanced the apoptotic effect of lovastatin through the down-regulation of GLO1 and HMGCR resulting in an increase of methylglyoxal and a decrease of cholesterol and led to the apoptosis of HL-60 cells. Data also revealed that both lovastatin and gamma-tocotrienol induced significant HL-60 cell differentiation. These results suggest that both lovastatin and gamma-tocotrienol could induce differentiation and followed by apoptosis.

  13. An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells

    PubMed Central

    Naegelen, Isabelle; Plançon, Sébastien; Nicot, Nathalie; Kaoma, Tony; Muller, Arnaud; Vallar, Laurent; Tschirhart, Eric J.; Bréchard, Sabrina

    2015-01-01

    Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. PMID:25548252

  14. Enhanced antileukemic activity of the novel complex 2,5-dihydroxybenzoate molybdenum(VI) against 2,5-dihydroxybenzoate, polyoxometalate of Mo(VI), and tetraphenylphosphonium in the human HL-60 and K562 leukemic cell lines.

    PubMed

    Thomadaki, Hellinida; Karaliota, Alexandra; Litos, Charalambos; Scorilas, Andreas

    2007-03-22

    We synthesized and studied the antitumor properties of the novel complex compound 2,5-dihydroxybenzoate molybdenum(VI) with tetraphenylphosphonium as counterion, which also acts as cancer cell growth inhibitor. A novel complex compound, the 2,5-dihydroxybenzoate molybdenum(VI) complex, (PPh4)2[Mo3O6(mu-O)2(2,5-DHBA)2] was synthesized. 1H NMR, 13C NMR, IR, and UV-Vis analyses were used for its molecular characterization. The human leukemia cell lines HL-60 and K562 were tested for their viability by assessing the metabolic activity of cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT), the structural integrity of the cell membrane (Trypan blue assay), and cell proliferation ability (growth curves). We showed that both leukemia cell lines are induced to decreased proliferation efficiency after treatment with the novel complex, compared to 2,5-dihydroxybenzoate, tetraphenyl-phosphonium polyoxomolybdate, or tetraphenylphosphonium chloride as individual entities, in a time- and concentration-dependent manner. Our results suggest that the new 2,5-dihydroxybenzoate molybdenum(VI) complex may provide a valuable tool in cancer chemotherapy.

  15. Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

    PubMed Central

    Daga, Martina; Cetrangolo, Giovanni Paolo; Ciamporcero, Eric Stefano; Petrella, Claudia; Graf, Maria; Uchida, Koji; Mamone, Gianfranco; Ferranti, Pasquale; Ames, Paul R. J.

    2015-01-01

    Heat shock 60 kDa protein 1 (HSP60) is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM) of 4-hydroxy-2-nonenal (HNE) yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL) or by copper-catalyzed oxidation (oxLDL), but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed. PMID:26078803

  16. DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C2 C12 mouse and HL-60 human leukemic cells in vitro.

    PubMed

    Alakhras, Raghda S; Stephanou, Georgia; Demopoulos, Nikos A; Grintzalis, Konstantinos; Georgiou, Christos D; Nikolaropoulos, Sotirios S

    2014-08-01

    We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.

  17. (4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

    SciTech Connect

    Magalhães, Hemerson I.F.; Wilke, Diego V.; Bezerra, Daniel P.; Cavalcanti, Bruno C.; Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson; Moraes, Manoel O.; Diniz-Filho, Jairo; Pessoa, Claudia

    2013-10-01

    (4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.

  18. 2,2′,4,4′-Tetrachlorobiphenyl Upregulates Cyclooxygenase-2 in HL-60 cells via p38 Mitogen-Activated Protein Kinase and NF-κB

    PubMed Central

    Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E.

    2007-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2′,4,4′,-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A2 with subsequent release of arachidonic acid (AA), and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 minutes. The increase in COX-2 mRNA was associated with release of 3H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A2, reduced 3H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB

  19. Exposure to p,p'-DDE Induces Morphological Changes and Activation of the PKCα-p38-C/EBPβ Pathway in Human Promyelocytic HL-60 Cells.

    PubMed

    Torres-Avilés, Nallely A; Albores-García, Damaris; Luna, Ana L; Moreno-Galván, Monica; Salgado-Bustamante, Mariana; Portales-Pérez, Diana Patricia; Calderón-Aranda, Emma S

    2016-01-01

    Dichlorodiphenyldichloroethylene (p,p'-DDE), the most persistent metabolite of dichlorodiphenyltrichloroethane (DDT), is still present in the human population. Both are present in the bone marrow of patients with bone marrow disorders, but thus far there are no studies that assess the capability of p,p'-DDE to affect myeloid cells. The aim of this study was to determine the effect of p,p'-DDE on promyelocytic cell differentiation and intracellular pathways related to this event. p,p'-DDE induced morphological changes compatible with promyelocytic differentiation in a concentration-dependent manner. The p,p'-DDE effect on [Ca(2+)]i, C/EBPβ protein levels, PKCα and p38 activation, and the role of oxidative stress or PLA2 was assayed. Exposure to 1.9 μg/mL of p,p'-DDE increased [Ca(2+)]i, PKCα, p38, and C/EBPβ protein levels; the increase of nuclear C/EBPβ protein was dependent on p38. PKCα phosphorylation was dependent on PLA2 and p,p'-DDE-induced oxidative stress. p38 phosphorylation induced by p,p'-DDE was dependent on PLA2, PKC activation, and oxidative stress. These effects of p,p'-DDE at concentrations found in human bone marrow may induce alterations in immature myeloid cells and could affect their cellular homeostasis. In order to establish the risk from exposure to p,p'-DDE on the development of bone marrow disorders in humans, these effects deserve further study.

  20. New Sesquiterpene Lactone Dimer, Uvedafolin, Extracted from Eight Yacon Leaf Varieties (Smallanthus sonchifolius): Cytotoxicity in HeLa, HL-60, and Murine B16-F10 Melanoma Cell Lines.

    PubMed

    Kitai, Yurika; Hayashi, Kana; Otsuka, Moe; Nishiwaki, Hisashi; Senoo, Tatsuya; Ishii, Tomohiko; Sakane, Genta; Sugiura, Makoto; Tamura, Hirotoshi

    2015-12-23

    Uvedafolin, 1, a new sesquiterpene lactone dimer, was isolated from the leaves of Smallanthus sonchifolius with five related compounds, 2-6, and their cytotoxicity was assessed against three tumor cell lines (HeLa, HL-60, B16-F10 melanoma). The stereostructure of 1 was newly elucidated by ESI-TOF-MS, 1D/2D NMR, and single-crystal X-ray diffraction. Dimers 1 and 2 had the most effective IC50 values, 0.2-1.9 μM, against the three tumor cell lines when compared with monomers 3-6 (IC50 values 0.7-9.9 μM) and etoposide (IC50 values 0.8-114 μM). The ester linkages of two sets of monomers, uvedalin, 5, and sonchifolin, 6, for 1, and enhydrin, 4, and sonchifolin, 6, for 2, as well as the acetyl group at the C-9 position, were essential for the high cytotoxicity. Dimers 1 and 2 would have potential as anticancer agents.

  1. Exposure to p,p′-DDE Induces Morphological Changes and Activation of the PKCα-p38-C/EBPβ Pathway in Human Promyelocytic HL-60 Cells

    PubMed Central

    Torres-Avilés, Nallely A.; Albores-García, Damaris; Luna, Ana L.; Moreno-Galván, Monica; Salgado-Bustamante, Mariana; Portales-Pérez, Diana Patricia

    2016-01-01

    Dichlorodiphenyldichloroethylene (p,p′-DDE), the most persistent metabolite of dichlorodiphenyltrichloroethane (DDT), is still present in the human population. Both are present in the bone marrow of patients with bone marrow disorders, but thus far there are no studies that assess the capability of p,p′-DDE to affect myeloid cells. The aim of this study was to determine the effect of p,p′-DDE on promyelocytic cell differentiation and intracellular pathways related to this event. p,p′-DDE induced morphological changes compatible with promyelocytic differentiation in a concentration-dependent manner. The p,p′-DDE effect on [Ca2+]i, C/EBPβ protein levels, PKCα and p38 activation, and the role of oxidative stress or PLA2 was assayed. Exposure to 1.9 μg/mL of p,p′-DDE increased [Ca2+]i, PKCα, p38, and C/EBPβ protein levels; the increase of nuclear C/EBPβ protein was dependent on p38. PKCα phosphorylation was dependent on PLA2 and p,p′-DDE-induced oxidative stress. p38 phosphorylation induced by p,p′-DDE was dependent on PLA2, PKC activation, and oxidative stress. These effects of p,p′-DDE at concentrations found in human bone marrow may induce alterations in immature myeloid cells and could affect their cellular homeostasis. In order to establish the risk from exposure to p,p′-DDE on the development of bone marrow disorders in humans, these effects deserve further study. PMID:27833915

  2. Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells

    PubMed Central

    Boly, Rainatou; Franck, Thierry; Kohnen, Stephan; Lompo, Marius; Guissou, Innocent Pierre; Dubois, Jacques; Serteyn, Didier; Mouithys-Mickalad, Ange

    2015-01-01

    The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds. PMID:25821497

  3. Pisiferdiol and pisiferic acid isolated from Chamaecyparis pisifera activate protein phosphatase 2C in vitro and induce caspase-3/7-dependent apoptosis via dephosphorylation of Bad in HL60 cells.

    PubMed

    Aburai, N; Yoshida, M; Ohnishi, M; Kimura, K

    2010-08-01

    Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates and regulates apoptosis, stress response and growth-related pathways. In the course of screening for PP2C activators from natural sources, we isolated abietane-type diterpenes, pisiferdiol and pisiferic acid from Chamaecyparis pisifera. Pisiferdiol having a unique seven-membered ring showed more specific PP2C activation activity (1.3-fold at 100 microM) than pisiferic acid having a normal six-membered ring and oleic acid, which is known to activate PP2C. Pisiferdiol and pisiferic acid showed mixed-type activation with respect to alpha-casein, and this differed from the non-competitive activation of oleic acid in vitro. In vivo, the cytotoxicity of pisiferdiol toward human promyelocytic leukemia cell line HL60 with an IC(50) value of 18.3 microM was 2-fold and 7-fold stronger than those of pisiferic acid and oleic acid, and pisiferdiol induced apoptosis through a caspase 3/7-dependent mechanism involving the dephosphorylation of Bad(1), which is a PP2C substrate. We thus conclude that pisiferdiol and pisiferic acid are novel PP2C activators, and the more specific activator, pisiferdiol, may be a useful chemical probe to study PP2C-mediated signaling pathways, and a lead compound for pharmaceutical agents.

  4. Rubratoxin-B-induced secretion of chemokine ligands of cysteine-cysteine motif chemokine receptor 5 (CCR5) and its dependence on heat shock protein 90 in HL60 cells.

    PubMed

    Nagashima, Hitoshi

    2015-11-01

    To elucidate the mechanism underlying rubratoxin B toxicity, the effects of rubratoxin B on the secretion of CCR5 chemokines, CCL3, CCL4, and CCL5, in a human promyelocytic leukemia cell line, HL60, were investigated. In addition, to examine whether the molecular chaperone 90-kDa heat shock protein (Hsp90) contributes to rubratoxin B toxicity, the effects of Hsp90-specific inhibitors, radicicol and geldanamycin, were investigated. Exposure to rubratoxin B for 24h induced secretion of each CCR5 chemokine, although the effect on CCL5 secretion was modest, and it enhanced secretion of proinflammatory cytokines tumor necrosis factor-α, CXCL8, and CCL2. Concomitant treatment with radicicol abolished the rubratoxin-induced secretion of all cytokines investigated. Geldanamycin antagonized the rubratoxin B-induced effects on CCL3 and CCL5, but not CCL4; the effects of geldanamycin were less than that of radicicol. Taken together, the results suggest that rubratoxin B, with the contribution of Hsp90, induces secretion of CCR5 chemokines.

  5. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    PubMed

    Jensen, Holly A; Bunaciu, Rodica P; Ibabao, Christopher N; Myers, Rebecca; Varner, Jeffrey D; Yen, Andrew

    2014-01-01

    Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10-20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or "precommitment" phase) or subsequently (the late or "lineage-commitment" phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage

  6. Retinoic Acid Therapy Resistance Progresses from Unilineage to Bilineage in HL-60 Leukemic Blasts

    PubMed Central

    Jensen, Holly A.; Bunaciu, Rodica P.; Ibabao, Christopher N.; Myers, Rebecca; Varner, Jeffrey D.; Yen, Andrew

    2014-01-01

    Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10–20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or “precommitment” phase) or subsequently (the late or “lineage-commitment” phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness

  7. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

    SciTech Connect

    Pai, J.K.; Siegel, M.I.; Egan, R.W.; Billah, M.M.

    1988-09-05

    There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-(32P) lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-(32P)PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-(32P)phosphatidic acid (alkyl-(32P)PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-(32P)PA must be formed via PLD-catalyzed hydrolysis of alkyl-(32P)PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-(32P)phosphatidylethanol (alkyl-(32P)PEt). Formation of alkyl-(32P)PEt parallels that of alkyl-(32P)PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration.

  8. Methylglyoxal-bis(guanylhydrazone), a polyamine analogue, sensitized γ-radiation-induced cell death in HL-60 leukemia cells Sensitizing effect of MGBG on γ-radiation-induced cell death.

    PubMed

    Kim, Jin Sik; Lee, Jin; Chung, Hai Won; Choi, Han; Paik, Sang Gi; Kim, In Gyu

    2006-09-01

    Methylglyoxal-bis(guanylhydrazone) (MGBG), a polyamine analogue, has been known to inhibit the biosynthesis of polyamines, which are important in cell proliferation. We showed that MGBG treatment significantly affected γ-radiation-induced cell cycle transition (G(1)/G(0)→S→G(2)/M) and thus γ-radiation-induced cell death. As determined by micronuclei and comet assay, we showed that it sensitized the cytotoxic effect induced by γ-radiation. One of the reasons is that polyamine depletion by MGBG treatment did not effectively protect against the chemical (OH) or physical damage to DNA caused by γ-radiation. Through in vitro experiment, we confirmed that DNA strand breaks induced by γ-radiation was prevented more effectively in the presence of polyamines (spermine and spermidine) than in the absence of polyamines. MGBG also blocks the cell cycle transition caused by γ-radiation (G(2) arrest), which helps protect cells by allowing time for DNA repair before entry into mitosis or apoptosis, via the down regulation of cyclin D1, which mediates the transition from G(1) to S phase of cell cycle, and ataxia telangiectasia mutated, which is involved in the DNA sensing, repair and cell cycle check point. Therefore, the abrogation of G(2) arrest sensitizes cells to the effect of γ-radiation. As a result, γ-radiation-induced cell death increased by about 2.5-3.0-fold in cells treated with MGBG. However, exogenous spermidine supplement partially relieved this γ-radiation-induced cytotoxicity and cell death. These findings suggest a potentially therapeutic strategy for increasing the cytotoxic efficacy of γ-radiation.

  9. CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway.

    PubMed

    Lin, Chin-Fen; Yang, Jai-Sing; Lin, Chingju; Tsai, Fuu-Jen; Lu, Chi-Cheng; Lee, Miau-Rong

    2016-09-01

    Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti‑leukemic activity in vivo. However, the molecular mechanism of action of CCY‑1a‑E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY‑1a‑E2 suppressed cell viability in multiple leukemia cell lines (HL‑60, K562, KG‑1 and KG‑1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY‑1a‑E2 exhibited a marked toxic effect on HL‑60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY‑1a‑E2 promoted G2/M phase arrest and promoted apoptosis in the HL‑60 cells. CCY‑1a‑E2 treatment upregulated cyclin B, cyclin‑dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY‑1a‑E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6‑diamidino‑2‑phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase‑8, ‑9 and ‑3 were observed during CCY‑1a‑E2‑induced cell apoptosis; their specific inhibitors were found to block CCY‑1a‑E2‑induced apoptosis, respectively. Moreover, CCY‑1a‑E2 time‑dependently disrupted the mitochondrial membrane potential (ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl‑2 expression in the HL‑60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

  10. Sparse-grid-based adaptive model predictive control of HL60 cellular differentiation.

    PubMed

    Noble, Sarah L; Wendel, Lindsay E; Donahue, Maia M; Buzzard, Gregery T; Rundell, Ann E

    2012-02-01

    Quantitative methods such as model-based predictive control are known to facilitate the design of strategies to manipulate biological systems. This study develops a sparse-grid-based adaptive model predictive control (MPC) strategy to direct HL60 cellular differentiation. Sparse-grid sampling and interpolation support a computationally efficient adaptive MPC scheme in which multiple data-consistent regions of the model parameter space are identified and used to calculate a control compromise. The algorithm is evaluated in silico with structural model mismatch. Simulations demonstrate how the multiscenario control strategy more effectively manages the mismatch compared to a single scenario approach. Furthermore, the controller is evaluated in vitro to differentiate HL60 cells in both normal and perturbed environments. The controller-derived input sequence successfully achieves and sustains the specified target level of granulocytes when implemented in the laboratory. The results and analysis given here imply that adoption of this experiment planning technique to direct cell differentiation within more complex tissue engineered constructs will require the use of a reasonably accurate mathematical model and an extension of this algorithm to multiobjective controller design. © 2011 IEEE

  11. RNA INTERFERENCE AGAINST CFTR AFFECTS HL60-DERIVED NEUTROPHIL MICROBICIDAL FUNCTION

    PubMed Central

    Bonvillain, Ryan W.; Painter, Richard G.; Adams, Daniel E.; Viswanathan, Anand; Lanson, Nicholas A.; Wang, Guoshun

    2010-01-01

    Biosynthesis of hypochlorous acid (HOCl), a potent anti-microbial oxidant, in phagosomes is one of the chief mechanisms employed by polymorphonuclear neutrophils (PMNs) to combat infections. This reaction, catalyzed by myeloperoxidase, requires chloride anion (Cl−) as a substrate. Thus, Cl− availability is a rate-limiting factor that affects neutrophil microbicidal function. Our previous research demonstrated that defective CFTR, a cAMP-activated chloride channel, present in cystic fibrosis (CF) patients leads to deficient chloride transport to neutrophil phagosomes and impaired bacterial killing (Painter et al., 2008 & 2010). To confirm this finding, here we used RNA interference against this chloride channel to abate CFTR expression in the neutrophil-like cells derived from HL60 cells, a promyelocytic leukemia cell line, with DMSO. The resultant CFTR deficiency in the phagocytes compromised their bactericidal capability, thereby recapitulating the phenotype seen in CF patient cells. The results provide further evidence suggesting that CFTR plays an important role in phagocytic host defense. PMID:20870018

  12. Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation agents.

    PubMed

    Stoica, Sonia; Magoulas, George E; Antoniou, Antonia I; Suleiman, Sherif; Cassar, Analisse; Gatt, Lucienne; Papaioannou, Dionissios; Athanassopoulos, Constantinos M; Schembri-Wismayer, Pierre

    2016-02-15

    Activation of minoxidil (MNX) with N,N'-carbonyldiimidazole and coupling with natural polyamines (PAs) and commercially available aliphatic or aromatic amines provided a series of new conjugates which were evaluated for their ability to induce differentiation to HL-60 acute myeloid leukemia cancer cells, using a modified NBTZ reduction test. Although neither MNX nor 4,4'-methylenedianiline (MDA) or 2,7-diaminofluorene (DAF), alone or in combination, had any effect, the MNX-spermine (SPM) conjugate (11) and the conjugates 7 and 8 of MNX with MDA and DAF exhibited a differentiation-inducing effect at a concentration of 10 μM without being toxic on proliferating human peripheral blood mononuclear cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Isolation and reconstitution of the N-formylpeptide receptor from HL-60 derived neutrophils.

    PubMed

    Hoyle, P C; Freer, R J

    1984-02-27

    A multifunctional receptor for N-formylpeptides exists on the membranes of neutrophils. This receptor has now been isolated from neutrophils derived from HL-60 promyelocytic leukemia cells. After solubilization by Nonidet-P40 and purification by affinity chromatography and HPLC the isolated receptor was reconstituted into egg phosphatidylcholine vesicles by SM-2 Bio-Bead removal of the Nonidet-P40. Analysis of the affinity and selectivity of the receptor was done by direct binding of two high-affinity ligands, formyl-Met-Leu-[3H]Phe-OH and formyl-Nle-Leu-Phe-[3H]Tyr-OH. The data suggest that the receptor can be isolated and reconstituted without apparent alteration of its binding affinity and selectivity, and that there appear to be no co-factors or subunits upon which these binding characteristics are dependent.

  14. Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

    SciTech Connect

    Macfarlane, D.E.

    1986-05-25

    Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with (/sup 32/P)orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of /sup 32/P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less /sup 32/P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A.

  15. 4H-Chromene-based anticancer agents towards multi-drug resistant HL60/MX2 human leukemia: SAR at the 4th and 6th positions.

    PubMed

    Puppala, Manohar; Zhao, Xinghua; Casemore, Denise; Zhou, Bo; Aridoss, Gopalakrishnan; Narayanapillai, Sreekanth; Xing, Chengguo

    2016-03-15

    4H-Chromene-based compounds, for example, CXL017, CXL035, and CXL055, have a unique anticancer potential that they selectively kill multi-drug resistant cancer cells. Reported herein is the extended structure-activity relationship (SAR) study, focusing on the ester functional group at the 4th position and the conformation at the 6th position. Sharp SARs were observed at both positions with respect to cellular cytotoxic potency and selectivity between the parental HL60 and the multi-drug resistant HL60/MX2 cells. These results provide critical guidance for future medicinal optimization. Copyright © 2016. Published by Elsevier Ltd.

  16. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

    PubMed

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    1999-10-01

    The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

  17. Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production.

    PubMed

    Krautwurst, D; Seifert, R; Hescheler, J; Schultz, G

    1992-12-15

    In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and

  18. All-trans retinoic acid induces different immunophenotypic changes on human HL60 and NB4 myeloid leukaemias.

    PubMed

    Barber, Nicole; Belov, Larissa; Christopherson, Richard I

    2008-02-01

    All-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukaemia (APL), inducing APL cells to differentiate into abnormal neutrophils. To investigate the possible relationship between the chromosome translocation t(15;17) found in APL and ATRA treatment, the human myeloid leukaemia cell lines HL60 and NB4, that are PML-RARalpha negative and positive, respectively, were treated with ATRA and immunophenotyped using a CD antibody microarray. For HL60 cells, ATRA induced major increases in descending order of CD38, CD11b, CD45RO, CD11c, CD54 and CD36 with repression of CD117 and CD44. For NB4 cells, ATRA induced major increases in descending order of CD11c, CD54, CD11a, CD11b, CD53, CD65, CD138, CD66c and T-cell receptor alpha/beta (TCRalpha/beta), with repression of CD38 and CD9. The induction of a number of these CD antigens is consistent with the known differentiation of these leukaemias to abnormal neutrophils. Approximately half of the antigens up-regulated by ATRA on NB4 cells were adhesion molecules, including CD11a, CD11b, CD11c, CD54, CD66c and CD138, consistent with the increased adhesiveness of leukaemia cells observed for APL patients treated with ATRA. On HL60 cells, ATRA induced expression of CD38, CD43 and CD45RO and repressed CD117, while the converse was true on NB4 cells that contain chimeric PML-RARalpha. For NB4 cells, ATRA induced some remarkable increases in CD antigens not seen for HL60: CD14 (16.6-fold), CD32 (27.8), CD53 (20.5), CD65 (139), CD66c (79.7), CD126 (15.1), and CD138 (57.6). The expression of these antigens may be regulated by PML-RARalpha in the presence of ATRA. Such CD antigens could be targets for synergistic treatment of APL with therapeutic antibodies following ATRA treatment.

  19. Rotenoids and flavonoids with anti-invasion of HT1080, anti-proliferation of U937, and differentiation-inducing activity in HL-60 from Erycibe expansa.

    PubMed

    Matsuda, Hisashi; Yoshida, Kazutoshi; Miyagawa, Katsutoshi; Asao, Yasunobu; Takayama, Saya; Nakashima, Souichi; Xu, Fengming; Yoshikawa, Masayuki

    2007-02-01

    Principal rotenoids (deguelin, tephrosin, rotenone, and 12a-hydroxyrotenone) (3-30microM) isolated from the stems of Erycibe expansa significantly inhibited invasion of human fibrosarcoma HT1080 cells through Matrigel-coated filters and release of proMMPs-2 and 9. In addition, deguelin and tephrosin showed differentiation-inducing activity in human promyelocytic leukemia HL-60 cells. Furthermore, effects of various constituents isolated from the ethyl acetate-soluble fraction on proliferation of human leukemia U937 cells were examined. As a result, most of isoflavones and several flavans as well as rotenoids showed moderate or substantial anti-proliferative activities.

  20. Enhanced cellular respiration in cells exposed to doxorubicin.

    PubMed

    Souid, Abdul-Kader; Penefsky, Harvey S; Sadowitz, Peter D; Toms, Bonnie

    2006-01-01

    Doxorubicin executes topoisomerase II mediated apoptosis, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores (PTP). To further define the effects of doxorubicin on cell metabolism, we measured cellular respiration, cellular ATP, DNA fragmentation, and cytochrome c leakage in Jurkat (supersensitive), human leukemia-60 (HL-60, sensitive), and HL-60/MX2 (resistant) cells following exposure to 1.0 microM doxorubicin for 30 min. The measurements were made after 24 h of exposure to the drug. In Jurkat and HL-60 cells, doxorubicin treatment increased cellular mitochondrial oxygen consumption and ATP content by 2-3-fold. The increment in oxygen consumption was blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk) and by the PTP inhibitor cyclosporin A. In HL-60/MX2 cells, which are resistant because of a reduced topoisomerase II activity, doxorubicin treatment was without effect on either respiration or ATP content, suggesting that topoisomerase II was essential for induction of apoptosis and stimulation of respiration and ATP content. The conclusion that both of the latter processes were products of oxidations in the mitochondrial respiratory chain was supported by the further observation that rotenone and sodium cyanide inhibited oxygen consumption and substantially lowered ATP content in the treated and untreated cells. Thus, oxidative phosphorylation is enhanced in cells briefly incubated with doxorubicin for as long as 24 h post drug exposure despite apoptosis-associated mitochondrial insults caused by the drug.

  1. Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

    PubMed

    Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

    2016-01-01

    IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

  2. Purification of a 53kD pI 4. 8 cytosolic phosphoprotein from HL60

    SciTech Connect

    Biser, P.S.; Spearman, T.N.; Bruzzone, M.; Durham, J.P.

    1987-05-01

    In order to study the potential role of a 53kD pI 4.8 phosphoprotein in the differentiation of HL60 using monoclonal antibodies, a partial purification has been carried out. Cytosol from cells differentiated with 1 M retinoic acid was applied to a DEAE-cellulose column and eluted with a linear NaCl gradient. Fractions were screened by in vitro phosphorylation of aliquots using /sup 32/P ATP and highly purified protein kinase C, SDS-PAGE, and autoradiography. Fraction which showed autoradiographic bands of the correct molecular weight were further analyzed using 2-D electrophoresis involving isolectric focusing over a pH range of 4-6 followed by SDS-PAGE on a 10% slab gel. Autoradiograms of these gels showed the 53 kD pI 4.8 phosphoprotein to elute with a peak at 0.24 NaCl. This 53 kD pI 4.8 protein was identified as the 53kD pI 4.8 phosphoprotein whose synthesis and phosphorylation is induced by retinoic acid by DEAE chromatography of cytosol from cells labelled in vivo with /sup 32/PO/sub 4//sup -2/ followed by 2-D electrophoresis. Fractions containing the 53 kD pI 4.8 protein were concentrated and applied to a chromatofocusing column which was eluted with a gradient from pH 6 to 4. Analysis of fractions via in vitro phosphorylation and SDS PAGE showed the 53 kD pI 4.8 protein eluting with a peak at pH 4.8 as a silver-stained band well separated from contaminating proteins. Experiments are currently in progress to produce monoclonal antibodies to the 53 kD pI 4.8 protein using the partially purified antigen.

  3. A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy.

    PubMed

    Mihara, Keiko; Nakayama, Tomofumi; Saitoh, Hisato

    2015-09-01

    Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation. All-trans-retinoic-acid-exposed differentiated HL-60 cells, which have properties similar to neutrophils, can also adhere efficiently to coverslips in FBS-free medium. Because the procedure is not complex and special equipment is not required, the simplicity and cost effectiveness of this FBS-free cell adhesion protocol may be beneficial to researchers who are interested in assessing the structure and function of suspension cells using microscopy. Copyright © 2015 John Wiley & Sons, Inc.

  4. Apoptosis and clonogenic survival in three tumour cell lines exposed to gamma rays or chemical genotoxic agents.

    PubMed

    Kumala, Sławomir; Niemiec, Paweł; Wideł, Maria; Hancock, Ronald; Rzeszowska-Wolny, Joanna

    2003-01-01

    We compared the extent to which apoptosis is induced and clonogenicity reduced in three tumour cell lines - the human melanoma Me45 and promyelocytic leukaemia HL-60, and the rat rhabdomyosarcoma R1 - after exposure to the anticancer drugs etoposide and cis-platinum or to gamma radiation; each induces different types of DNA damage. Cells which readily underwent apoptosis did not necessarily show a correlated loss of clonogenicity; for example, Me45 cells showed the highest sensitivity to all three agents in clonogenic assays but much lower levels of apoptotic cells than R1 or HL-60 cells. These results show that the efficiency of the eradication of clonogenic cells by genotoxic agents does not solely depend on the induction of apoptotic processes, and suggest that the induction of apoptosis and suppression of clonogenicity are independent processes.

  5. Targeting SLUG sensitizes leukemia cells to ADR-induced apoptosis.

    PubMed

    Wei, Chang-Rong; Liu, Jun; Yu, Xiao-Jun

    2015-01-01

    Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug's ability to silence expression suppresses the growth of leukemia HL-60 cells and/or sensitizes leukemia HL-60 cells to adriamycin (ADR) through induction of apoptosis. SLUG siRNA was transfected into the HL-60 and HL-60(ADR) cell lines (an adriamycin resistant cell line). The stably SLUG siRNA transfected HL-60 and HL-60(ADR) cells was transiently transfected with PUMA siRNA. The mRNA and protein expression of SLUG and PUMA were determined by Quantitative real-time RT-PCR and Western blot assay. The effects of SLUG siRNA alone or combined with ADR or PUMA siRNA on growth and apoptosis in HL-60 and HL-60(ADR) cells was detected by MTT, ELISA and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The results showed that SLUG was less expressed in the HL-60 cells, and high expressed in the HL-60(ADR) cells. Obvious down-regulation of SLUG mRNA and protein levels and up-regulation of PUMA mRNA and protein levels after SLUG siRNA transfection was showed in the HL-60(ADR) cells. Treatment with ADR induced SLUG mRNA and protein in the HL-60 cells. Significant positive correlation was observed between basal SLUG mRNA and protein and ADR sensitivity. SLUG gene silencing by SLUG siRNA transfection inhibited growth and induced apoptosis, and increased ADR killing of the HL-60 and HL-60(ADR) cell lines. After the SLUG siRNA transfected HL-60 and HL-60(ADR) cells was transiently transfected with PUMA siRNA, did not increase ADR killing of the HL-60 and HL-60(ADR) cell lines. SLUG level positively correlated with sensitivity to ADR. SLUG siRNA could effectively reduce SLUG expression and induce PUMA expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents.

  6. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  7. Sulindac and its metabolites: sulindac sulfide and sulindac sulfone enhance cytotoxic effects of arsenic trioxide on leukemic cell lines.

    PubMed

    Stępnik, Maciej; Ferlińska, Magdalena; Smok-Pieniążek, Anna; Gradecka-Meesters, Dobrosława; Arkusz, Joanna; Stańczyk, Małgorzata

    2011-08-01

    The effects of arsenic trioxide (ATO) in combination with sulindac (SUL), sulindac sulfide (SS) or sulindac sulfone (SF) on human (Jurkat, HL-60, K562 and HPB-ALL) and mouse (EL-4) leukemic cell lines were investigated. The cells showed different sensitivity to sulindacs (2.5-200 μM) with SS being the most cytotoxic (72 h WST-1 reduction test). The cytotoxicity of ATO was enhanced by combination with sulindacs. The combination of ATO (1 μM) with SS or SF at concentrations over 50 μM induced considerable cytotoxicity in all cell lines. Normal human lymphocytes exposed for 48 h to the combinations showed smaller decrease in viability. Measurements of Jurkat, HL-60 and K562 cells exposed to ATO (1 μM) and sulindacs (100 μM or 200 μM for K562 cells) indicated apoptosis as the main cell death mechanism. The mitochondrial membrane potential measurements (JC-1 probe) indicated an active involvement of mitochondria in the process. The results did not indicate involvement of an inhibitory effect of the combinations on NF-κB activity in Jurkat, HL-60 and K562 cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Neutrophil elastase activity in differentiating HL-60 promyelocytes is decreased by culture with ethanol and elastase deficient neutrophils are produced in alcoholics

    SciTech Connect

    Sachs, C.; Christianson, R.; Pratt, P.; Lynn, W.

    1987-05-01

    Serum-free culture of HL-60 in the presence of recombinant Granulocyte-Macrophage Colony Stimulating Factor in four days elicits a five-fold increase in esterolytic neutrophil elastase (NE) like activity measured with methoxy-succinyl-ala-ala-pro-val p-nitroanilide and purified NE standard but does not cause terminal differentiation. Simultaneous exposure to 0.2, 0.4, or 0.6% (vol./vol.) ethanol blocks this increase in NE activity. Exposure to 0.85% ethanol promotes terminal differentiation to elastase-deficient granulocytes which as been described using DMSO. To ascertain if ethanol may have similar effects on granulocytic differentiation in vivo, they compared oxidase and elastase activities of PMN's in male alcoholics on a binge (ethanol > 200 mg/dl.). In 29 patients an average of 872 (+/- 237) (SD) ng./10/sup 6/ PMN's of active NE was found compared to 1571 (+/- 177) in 13 controls. Patients admitted for treatment of alcoholism had similar NE activity in 3-4 days, showed a slight increase in activity within one week and had NE activity comparable to controls within 2-3 weeks. These findings support the previous observation that smoking related emphysema is less prevalent and severe in patients who regularly consume alcohol. They conclude that ethanol may visibly alter responsiveness of promyelocytic precursors to regulatory differentiating factors.

  9. Influence of high-frequency electromagnetic fields on different modes of cell death and gene expression.

    PubMed

    Port, M; Abend, M; Römer, B; Van Beuningen, D

    2003-09-01

    International thresholds for exposure to non-ionizing radiation leading to non-thermal effects were conservatively set by the International Commission on Non-Ionizing Radiation Protection (ICNIRP). The aim of this study was to examine whether biological effects such as different modes of cell death and gene expression modifications related to tumorgenesis are detectable above the threshold defined. Human leukaemia cells (HL-60) grown in vitro were exposed to electromagnetic fields (EMF; t 1/2(r) about 1 ns; field strength about 25 times higher than the ICNIRP reference levels for occupational exposure) leading to non-thermal effects using a high-voltage-improved GTEM cell 5302 (EMCO) connected to a pulse generator NP20 (C = 1 nF, U(Load) = 20kV). HL-60 cells were harvested at 0, 24, 48 and 72 h after radiation exposure. Micronuclei, apoptosis and abnormal cells (e.g. necrosis) were determined using morphological criteria. In parallel, the expression of 1176 genes was measured using Atlas Human 1.2. Array. Based on high data reproducibility calculated from two independent experiments (> 99%), array analysis was performed. No significant change in apoptosis, micronucleation, abnormal cells and differential gene expression was found. Exposure of HL-60 cells to EMFs 25 times higher than the ICNIRP reference levels for occupational exposure failed to induce any changes in apoptosis, micronucleation, abnormal morphologies and gene expression. Further experiments using EMFs above the conservatively defined reference level set by the ICNIRP may be desirable.

  10. SMT-A07, a 3-(Indol-2-yl) indazole derivative, induces apoptosis of leukemia cells in vitro.

    PubMed

    Qian, Shijing; Cao, Ji; Yan, Yan; Sun, Maotang; Zhu, Hong; Hu, Yongzhou; He, Qiaojun; Yang, Bo

    2010-12-01

    N-(2-(1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide (SMT-A07) is a novel 3-(Indol-2-yl) indazole derivative. The anticancer activities in vitro and the cell apoptosis-induction abilities of SMT-A07 on human leukemia HL60 and NB4 cell lines were investigated in this study. The results of MTT assay showed SMT-A07 was a potential and highly efficient antitumor compound with IC(50) values ranging from 0.09 to 1.19 μM in five leukemia cell lines. SMT-A07 treatment for 24 h caused the increment of apoptosis rate from 6.88 to 49.72% in HL60 cells and from 8.72 to 56.28% in NB4 cells by flow cytometry analysis. Agarose gel electrophoresis showed DNA fragmentation that appeared after cells were exposed to SMT-A07. After SMT-A07 incubation, DAPI staining revealed the presence of DNA fragmentation, and perinuclear apoptotic body. SMT-A07 also resulted in a loss of ΔΨm in both HL60 and NB4 cells by JC-1 staining. Moreover, apoptosis-related proteins were examined by western blotting to explore the mechanism of its cytotoxicity. SMT-A07 exposure caused down-regulation and cleavage of procaspase-8, procaspase-3, Bid, PARP and up-regulation of cleaved caspase-8, cleaved caspase-3, PARP (Cleaved Fragment). In addition, the presence of pan-caspase inhibitor BOC-D-FMK prevented cells from caspase-3 activation, PARP cleavage, and subsequent apoptosis. Our study demonstrates that SMT-A07 displays an apparent antitumor activity with extensive anti-leukemia spectrum, and SMT-A07 can induce the apoptosis of HL60 and NB4 cells activation of the caspase cascade, which deserves further development.

  11. Comparative analysis of microRNA and mRNA expression profiles in cells and exosomes under toluene exposure.

    PubMed

    Lim, Jung-Hee; Song, Mi-Kyung; Cho, Yoon; Kim, Woong; Han, Sung Ok; Ryu, Jae-Chun

    2017-02-27

    Recent studies have illustrated the growing importance of exosomes (small extracellular vesicles) and their constituent microRNAs (miRNAs) in the fields of toxicology and pathology. The mechanism of toxicity of toluene, a highly-prevalent and volatile organic compound, is largely unknown. To examine the role of miRNAs in toluene-induced toxicity, we investigated miRNAs and toluene-induced gene expression in HL-60 human promyelocytic leukemia cells and exosomes using microarrays. A total of 54 miRNAs were differentially expressed in HL-60 cell lines exposed to toluene and exosomes from the cells. Four out of the 54 miRNAs (hsa-miR-1290, hsa-miR-718, hsa-miR-3663-3p, and hsa-miR-320c) were subsequently validated by qRT-PCR. Integrated analysis of miRNA and mRNA expression profiles identified 8 miRNA-mRNA correlations. By performing Comparative Toxicogenomics Database analysis, we found that the ei