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Sample records for hl60 cells undergoing

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

    Science.gov (United States)

    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%)(PHL-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 preparation and identification of cytoplast from suspension culture cells.

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

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    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.

  3. Proliferation inhibition, cell cycle arrest and apoptosis induced in HL-60 HL-60HL-60 HL-60 cells by a natural diterpene ester from Daphne mucronata

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    R Yazdanparast

    2011-05-01

    Full Text Available "n  "nBackground and the purpose of the study: Gnidilatimonoein (Gn, a new diterpene ester from Daphne mucronata, possesses strong anti-metastasis and anti-tumor activities. In this study, its apoptosis and differentiation capabilities were evaluated by using the leukemia HL-60 cell line. "nMaterial and methods: Cell prolifaration inhibition was estimated by MTT assay. The occurrence of apoptosis was evaluated by EtBr/AO double staining technique, cell cycle analyses and detection of apoptotic cells by Annexin V-FITC and propodium iodide (PI. Differentiation of the cells was determined by NBT reduction assay and the expression of specific cell surface markers such as CD14 and CD11b, were analyzed by flow cytometry.   "nResults: The drug decreased the growth of the cells dose- and time- dependently and the IC50 was found to be 1.3 µM. Our data suggested that Gn induced both monocytic differentiation  and apoptosis among HL-60 cells. In addition, cell cycle analyses showed an increase in G1 phase population by 24 hrs, which was gradually replaced by Sub-G1 cell population (apoptotic cells by 72 hrs. "nConclusion: Based on these data, the Gn-treated HL-60 cells displayed differentiation-dependent apoptosis. Thus, Gn might be a good candidate for differentiation therapy of leukemia, pending full biological evaluation of the compound among the wide array of leukemia cells. "nevaluation of the compound among the wide array of leukemia cells.

  4. Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells

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    Duan Yingli

    2010-05-01

    Full Text Available Abstract Background Pyk2 is a non-receptor cytoplasmic tyrosine kinase that belongs to the focal adhesion kinase family and has been implicated in neutrophil spreading and respiratory burst activity caused by TNF-α. However, the role of Pyk2 in neutrophil migration is incompletely defined. In this study, we tested the hypothesis that Pyk2 regulates the migration of neutrophil-like differentiated HL-60 cells subsequent to β2-integrin mediated cell adhesion. Methods HL-60 cells were induced to differentiate into neutrophil-like cells (dHL60 by incubation in medium containing 1.25% DMSO for up to 4 days. Pyk2 expression and tyrosine phosphorylation was measured by Western blot analysis. Adhesion of dHL60 cells to plated fibrinogen was measured by residual myeloperoxidase activity. dHL60 cell migration was evaluated using a 96-well chemoTx chamber. Results Western blot analysis demonstrated that hematopoietic Pyk2 was predominantly expressed after HL60 cell differentiation. Pyk2 was tyrosine phosphorylated upon adhesion of dHL60 cells to plated fibrinogen in the presence of fMLP. By contrast, tyrosine phosphorylation of Pyk2 was insignificant in dHL60 cells treated in suspension with fMLP. Antibodies against CD18 blocked both phosphorylation of Pyk2 and adhesion of dHL60 cells to fibrinogen, demonstrating that phosphorylation of Pyk2 was β2-integrin dependent. TAT-Pyk2-CT, a dominant negative fusion protein in which the TAT protein transduction domain was fused to the c-terminal Pyk2, attenuated fMLP-stimulated spreading, migration and phosphorylation of endogenous Pyk2 without blocking adhesion of dHL-60 cells to fibrinogen. Similarly, silencing of Pyk2 expression by siRNA in dHL60 cells also attenuated dHL60 cell migration caused by fMLP. Phospho-Pyk2 was evenly distributed around cell membrane circumferentially in unstimulated dHL-60 cells adherent to plated fibrinogen. In dHL60 cells treated with fMLP to cause cell spreading and polarization

  5. Microarray analysis of responsible genes in increased growth rate in the subline of HL60 (HL60RG) cells.

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    Luan, Yang; Kogi, Mieko; Rajaguru, Palanisamy; Ren, Jin; Yamaguchi, Teruhide; Suzuki, Kazuhiro; Suzuki, Takayoshi

    2012-03-01

    HL60RG, a subline of human promyelocytic leukemia HL60 cells, has a increased growth rate than their parental cells. To gain information of the mechanisms involved in the increased growth rate of HL60RG, we performed a multiplex fluorescence in situ hybridization (M-FISH), standard cytogenetics analysis (G-banding) and genome scan using 10K SNP mapping array on both cell types. Characteristic genomic alterations in HL60RG cells were identified including uniparental disomy (UPD) of chromosome 1, and hemizygous deletion in 10p and 11p. However, no such defects were observed in HL60 cells. Changes in gene expression in HL60RG cells were determined using expression arrays (Affymetrix GeneChip, HU133A). Candidate genes associated with the rapid growth of HL60RG cells were identified. Two tumor necrosis factor receptors, TNFRSF1B (type II tumor necrosis factor-α receptor) and TNFRSF8 (also known as a tumor marker CD30), which are adjacently located on chromosome 1 showed opposing changes in gene expression in HL60RG cells-over-expression of TNFRSF8 and repression of TNFRSF1B. Differences in the DNA methylation status in the transcriptional regulatory regions of both genes between HL60 and HL60RG was detected by a methylation-specific PCR assay. In conclusion, alterations in chromosome and gene expression in HL60RG may be associated with increased growth rate.

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

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    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.

  7. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) HL-60 cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  8. Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

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    Martínez-Zárate, Alma Delia; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Cisneros, Bulmaro; Winder, Steve J; Cerecedo, Doris

    2014-06-01

    Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

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

    Science.gov (United States)

    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.

  10. ARSENIC TRIOXIDE DOWNREGULATES TELOMERASE ACTIVITY IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    何冬梅; 张洹

    2002-01-01

    Objective: To evaluate whether arsenic trioxide (AS2O3) could downregulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity during induction of apoptosis of HL-60 cells. Methods: Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. The expression of hTERT at mRNA and protein levels was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label, respectively. Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: Treatment of 2 μmol/L at As2O3 could induce apoptosis of HL-60 cells. hTERT was decreased at both mRNA and protein levels during apoptosis of HL-60 cells. Telomerase activity of HL-60 cells was significantly inhibited. Conclusion:It is suggested that telomerase activity of HL-60 cells might be specifically inhibited by AS2O3 through the downregulation of hTERT gene expression.

  11. Identification of genes responsive to apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Wei JIN; Le-feng QU; Ping MIN; Shan CHEN; Hong LI; He LU; Yong-tai HOU

    2004-01-01

    AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways.CONCLUSION: TGFβ and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells.TIEG and VDUP1 play important roles in mediating apoptosis.

  12. Tetramethylpyrazine potentiates arsenic trioxide activity against HL-60 cell lines

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    Wu, Yuni; Xu, Youhua; Shen, Yali; Wang, Cuicui; Guo, Gaili; Hu, Tiantian [Key Laboratory of Developmental Diseases in Childhood, Chongqing (China); Key Laboratory of Pediatrics in Chongqing, Chongqing (China); Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing (China)

    2012-02-17

    The objective of this study was to evaluate the effects of tetramethylpyrazine (TMP) in combination with arsenic trioxide (As{sub 2}O{sub 3}) on the proliferation and differentiation of HL-60 cells. The HL-60 cells were treated with 300 µg/mL TMP, 0.5 µM As{sub 2}O{sub 3}, and 300 µg/mL TMP combined with 0.5 µM As{sub 2}O{sub 3}, respectively. The proliferative inhibition rates were determined with MTT. Differentiation was detected by the nitroblue tetrazolium (NBT) reduction test, Wright's staining and the distribution of CD11b and CD14. Flow cytometry was used to analyze cell cycle distribution. RT-PCR and Western blot assays were employed to detect the expressions of c-myc, p27, CDK2, and cyclin E1. Combination treatment had synergistic effects on the proliferative inhibition rates. The rates were increased gradually after the combination treatment, much higher than those treated with the corresponding concentration of As{sub 2}O{sub 3} alone. The cells exhibited characteristics of mature granulocytes and a higher NBT-reducing ability, being a 2.6-fold increase in the rate of NBT-positive ratio of HL-60 cells within the As{sub 2}O{sub 3} treatment versus almost a 13-fold increase in the TMP + As{sub 2}O{sub 3} group. Cells treated with both TMP and As{sub 2}O{sub 3} expressed far more CD11b antigens, almost 2-fold compared with the control group. Small doses of TMP potentiate As{sub 2}O{sub 3}-induced differentiation of HL-60 cells, possibly by regulating the expression and activity of G0/G1 phase-arresting molecules. Combination treatment of TMP with As{sub 2}O{sub 3} has significant synergistic effects on the proliferative inhibition of HL-60 cells.

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

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    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.

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

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    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.

  15. Efflux of potassium ion is an important reason of HL-60 cells apoptosis induced by tachyplesin

    Institute of Scientific and Technical Information of China (English)

    Hai-tao ZHANG; Jun WU; Hai-feng ZHANG; Qi-feng ZHU

    2006-01-01

    Aim: To investigate the role of intercellular potassium in tachyplesin-induced HL-60 cells apoptosis. Methods: The concentration of intercellular potassium, cell volume and mitochondrial membrane potential were examined by flow cytometry. Results: The concentration of intercellular potassium reduced in a time-dependent manner in tachyplesin-treated HL-60 cells. In addition, the loss of mitochondrial membrane potential was tightly coupled with the shrinkage of cell volume. Different caspase inhibitors protected against DNA degradation but did not prevent the loss of HL-60 cell viability induced by tachyplesin. Ba2+, which was a kind of blocker of volume-regulatory K+ channels, increased the viability of tachyplesin-treated HL-60 cells and maintained mitochondrial membrane potential and cell volume. Conclusion: Efflux of K+ was an important reason for apoptosis in tachyplesin-treated HL-60 cells. Efflux of K+ affected the viability of tachyplesin-treated HL-60 cells independent of the process of caspase activation.

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

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    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 HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P HL-60 cells were reduced (P 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.

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

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    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.

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

    Science.gov (United States)

    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.

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

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    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 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 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.

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

    Science.gov (United States)

    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 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.

  1. MG132 Induced Apoptosis Pathway in HL-60 Cells and Impact of Allogeneic Mixed Lymphocyte Reaction

    Institute of Scientific and Technical Information of China (English)

    Yong-ming Zhou; Wei Guo; Hao Zhou; Jin-hua Zhang; Zhi-ping Liu; Mei-xia Yu

    2009-01-01

    Objective: To investigate the proteasome inhibitor MG132-induced apoptosis pathway in HL-60 cells and the role of allogeneic mixed lymphocyte reaction.Methods: Cell apoptosis was analyzed by flow cytometry. The expressions of p21 protein, p27 protein and p53 protein in HL-60 cells treated with MG132 were measured by Western blot. The proliferation of, peripheral blood mononuclear cells (PBMNCs) after treatment with 75 Gy irradiated HL-60 cells treated with MG132 was measured with CCK-8.Results: High-dose MG132 induced apoptosis in HL-60 cells. No significant change was observed in MG132-induced apoptosis after inhibiting caspase-8 and caspase-9 pathway. The expressions of p21 protein and p27 protein increased in MG132-induced apoptosis. HL-60 cells treated with low-dose MG132 improved the proliferation of PBMNCs from healthy volunteers.Conclusion: High-dose MG132 induced apoptosis and directly killed HL-60 cells. MG132 induced apoptosis in a caspase-8- and caspase-9-independent pathway. p21 protein and p27 protein were involved in MG132-induced apoptosis in HL-60 cells. HL-60 cells treated with Low-dose MG132 improved the effect of promoting the proliferation of PBMNCs from healthy volunteers.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 HL-60 cells were up-regulated (P HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.

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

    Science.gov (United States)

    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.

  5. Regulation of Histone Acetylation and Apoptosis by Trichostatin in HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    李新刚; 陈维凯; 谷俊侠; 崔国惠; 陈燕

    2004-01-01

    In order to examine the strong anticancer action and low toxicity of Trichostatin A(TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dosedependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.

  6. Bioreductive activation of mitoxantrone by NADPH cytochrome P450 reductase does not change its apoptotic stimuli properties in regard to sensitive and multidrug resistant leukaemia HL60 cells.

    Science.gov (United States)

    Kostrzewa-Nowak, Dorota; Tarasiuk, Jolanta

    2013-12-05

    The objective of this study was to examine the effect of bioreductive activation of antitumour drug, mitoxantrone (MX), by liver NADPH cytochrome P450 reductase (CPR) on inducing apoptosis of human promyelocytic sensitive leukaemia HL60 cell line and its multidrug resistance (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). It was found that non-activated as well as CPR-activated form of MX used at IC90 were able to influence cell cycle of sensitive HL60 as well as resistant cells and induce apoptosis. Interestingly, it was evidenced that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by both studied forms of MX compared to HL60 and HL60/DOX cells. However, the examined agent did not change the expression of Fas receptors on the surface of HL60 sensitive as well as resistant cells regardless of its form used in the study. Obtained results suggest that CPR-dependent reductive activation of MX does not change its apoptotic stimuli properties in regard to sensitive HL60 and multidrug resistant (HL60/VINC and HL60/DOX) leukaemia cells. Nevertheless, taking into account that side toxic effects observed in course of patient treatment with antitumour drugs are dose-dependent, it seems that the reported increase in antiproliferative activity and ability to induce apoptosis of MX after its reductive activation by exogenous CPR against the MDR cells overexpressing both P-glycoprotein and MRP1 at much more lower concentrations of this drug could be of clinical importance for the treatment of tumours resistant to classical chemotherapy. © 2013 Elsevier B.V. All rights reserved.

  7. EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    王宝燕; 张海涛; 李静

    2002-01-01

    Objective To understand the mechanism of the hype rthermia and harringtonine in purging of leukemia cells In Vitro. telomerase activity of HL-60 cells treated by hyperthermia (42℃ for one hour) and di fferent concentrations of Harringtonine were investigated.Methods Using Telomeric Repeats Amplification Protocol (TRAP) a nd ELISA techniques to analyze the telomerase activity of HL-60 cells. Results Our results showed that harringtonine inhibited the tel omerase activity of HL-60 cells in a dosage related manner. Moreover, the telom erase activity of HL-60 cells was significantly decreased after the hyperthermi a treatment as compared with untreated cells.Conclusion The effect of the hyperthermia and Harringtonine on purging leukemia cells In Vitro may be mediated by down regulation of telome rase activity of tumor cells.

  8. CLONING AND EXPRESSION OF A GENE MEDIATINGγ-RADIATION-INDUCED APOPTOSIS IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To identify the member of the caspase family proteases involved in γ-radiation-induced apoptosis in HL-60 cells and to study the expression of the caspase gene in normal, apoptotic cells and in immortal tu mor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were pre sent in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-60 cells. Caspase-3 mRNA in apoptotic HL-60 cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identified with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly ex pressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-60 cells than in control HL-60 cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-60 cells. The high level of expression of caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radiation, their inherent ability to survive, and apop tosis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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 HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.

  13. Rare Coumarins Induce Apoptosis, G1 Cell Block and Reduce RNA Content in HL60 Cells

    Directory of Open Access Journals (Sweden)

    Widelski Jarosław

    2017-02-01

    Full Text Available The rare coumarins stenocarpin, stenocarpin isobutyrate, oficinalin, oficinalin isobutyrate, 8-methoxypeucedanin and the known xanthotoxin, isoimperatorin, bergapten, peucedanin and 8–methoxyisoimperatorin were isolated from Peucedanum luxurians Tamamsch. (Apiaceae and identified by means of spectral data (1D and 2D NMR. Their immunomodulating activity was evaluated by flow cytometry and their influence on HL60 cells as well as on PHA-stimulated PBLs was tested. All tested coumarins induce apoptosis (maximal in the 48 h culture and decrease cell proliferation in a time- and dose-dependent manner, especially in HL60 cells. They also induce partial G1 block, but only in HL60 cells (at 100 µM concentrations. Dose-dependent reduction of RNA content was also found in G1 cells treated by the coumarins. All of the tested coumarins also possessed immunomodulatory activities. Bergapten and xanthotoxin were found to be the best candidates for further evaluation as anti-cancer drugs.

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

    Science.gov (United States)

    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 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.

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

    Science.gov (United States)

    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.

  16. 14-3-3ξ对HL-60HL-60/VCR细胞增殖的抑制作用%Inhibitory Effect of 14-3-3ξ on the Proliferation of HL-60 Cells and HL-60/VCR Cells

    Institute of Scientific and Technical Information of China (English)

    梁蓉; 陈协群; 王哲; 熊华; 白庆咸; 高广勋; 董宝侠; 朱华锋

    2013-01-01

    This study was aimed to investigate the expression and role of 14-3-3ξ 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,14-3-3ξ,and anti-apoptosic protein BCL-2,MCL-1 proteins.Immunofluorescence assay was used to detect the subcellular location of 14-3-3ξ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy.Transduction with siRNA was used to silence 14-3-3ξ 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 14-3-3(o),the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells,especially 14-3-3ξ.The higher expression of 14-3-3ξ,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 14-3-3ξ was located in the cytoplasma and nuclei of AML cell lines,especially over-expressed in HL-60/VCR cells.Furthermore,suppression of 14-3-3ξ 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 14-3-3ξ 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 14-3-3ξ may provide novel,effective strategies for refractory and relapsed AML.%本研究旨在进一步探讨14-3-3ξ在急性髓系白血病(acute myeloid leukemia,AML)敏感细胞HL-60和耐药细胞HL-60/VCR中的表达和作用.用半定量RT-PCR和Western blot分

  17. ANTI-PROLIFERATION EFFECT OF ORIDONIN ON HL-60 CELLS AND ITS MECHANISM

    Institute of Scientific and Technical Information of China (English)

    Jia-jun Liu; Xin-yao Wu; Hui-ling Lu; Xiang-lin Pan; Jun Peng; Ren-wei Huang

    2004-01-01

    Objective To investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism.Methods HL-60 cells in vitro in culture medium were given different concentrations oforidonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM),morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was detected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred.Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly.The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especially after cells were treated 48-60 hours by oridonin.Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells in vitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.

  18. EFFECTS OF CURCUMIN ON PROLIFERATION AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA CELLS HL-60

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To investigate the curcumin killing leukemia cells in vitro,. Methods: The myeloid leukemic cell line HL-60 was studied by using cell culture, flow cytometrydetermining DNA content and TUNEL method measuring apoptotic cell percentage. Results: The data showed that curcumin selectively inhibited proliferation of acute myeloid leukemia (AML) HL-60 cell lines in a dose- and time-dependent manner. The growth inhibition rate was gradually increased and reached the peak at concentration of 25 m mol/L curcumin at 24h. The sub-G1 peak appeared after 12h treatment and was increased to 34.4% at 24h. The TUNEL method further certified that apoptotic cells reached 41% at the same phase. Conclusion: curcumin possesses obvious potent of anti-leukemia cell proliferation, which is contributed to the induction of HL-60 cells apoptosis. The concentration and action time of curcumin in vitro provide some reference for clinical use.

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

    Science.gov (United States)

    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

  20. OPTIMIZATIONS FOR 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY IN PURGING LEUKEMIA CELL HL60

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.

  1. Growth inhibition and differentiation of promyelocytic cells (HL-60) induced by BC-4, an active principle from Boswellia carterii Birdw.

    Science.gov (United States)

    Jing, Y; Xia, L; Han, R

    1992-03-01

    The induction of cell differentiation and growth inhibition of HL-60 cells by Bc-4, an active principle from Boswellia carterii, was studied in vitro and in vivo. The proliferation of HL-60 cells was found to be inhibited by Bc-4 at a concentration of 5-10 micrograms/ml. Under the action of Bc-4, the acid phosphatase and NBT reduction activities in HL-60 cells were increased, and phagocytosis of cells was also induced. All these activities were concentration dependent. The HL-60 cells were induced by Bc-4 to differentiate into more mature cells morphologically. The in vivo growth of HL-60 cells in mouse subrenal capsules (SRC) and in diffusion chambers inoculated into mice was inhibited by Bc-4 at a dose of 50 mg/kg. The morphology of HL-60 cells treated by Bc-4 in diffusion chambers exhibited characteristics of mature granulocytic cells.

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

    Science.gov (United States)

    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

  3. Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells.

    Science.gov (United States)

    Lee, Younghyun; Kim, Yang Jee; Choi, Young Joo; Lee, Joong Won; Lee, Sunyeong; Chung, Hai Won

    2012-07-01

    Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely used chemotherapeutic drugs, but its effectiveness is limited by tumor cell resistance and the severe side effects it causes. One strategy for overcoming this problem is the concomitant use of natural dietary compounds as therapeutic agents. Benzyl isothiocyanate (BITC) is a promising chemopreventive agent found in cruciferous vegetables and papaya fruits. The aim of this study was to investigate the effects of BITC on cisplatin-induced cytotoxicity in human promyelocytic leukemia cells and normal human lymphocytes. The combined treatment of HL-60 cells with BITC followed by cisplatin (BITC/cisplatin) caused a significant decrease in cell viability. BITC also increased apoptotic cell death compared to cisplatin treatment alone. In normal human lymphocytes, BITC did not enhance the cytotoxic effects of cisplatin. Cellular exposure to BITC/cisplatin increased reactive oxygen species (ROS) generation but decreased the total glutathione (GSH) level in HL-60 cells. Pretreatment of HL-60 cells with N-acetylcysteine or glutathione monoethyl ester effectively decreased BITC/cisplatin-induced cell death. The addition of the extracellular signal-regulated kinase (ERK) inhibitor PD98059 abolished BITC/cisplatin-induced apoptosis. Taken together, our results suggest that BITC enhances cisplatin-induced cytotoxicity through the generation of ROS, depletion of GSH, and ERK signaling in HL-60 cells.

  4. Pinoresinol inhibits proliferation and induces differentiation on human HL60 leukemia cells.

    Science.gov (United States)

    Sepporta, Maria Vittoria; Mazza, Teresa; Morozzi, Guido; Fabiani, Roberto

    2013-01-01

    Pinoresinol (PIN), one of the simplest lignans, is the precursor of other dietary lignans that are present in whole-grain cereals, legumes, fruits, and other vegetables. Several experimental and epidemiological evidences suggest that lignans may prevent human cancer in different organs. In this study we investigated the chemopreventive properties of PIN on cell lines derived from different sites either expressing or not the functional tumor suppressor protein p53. It was found that PIN inhibited the proliferation of p53 wild type colon and prostate tumor cells (HCT116 and LNCaP) while in breast cells the inhibition of growth was observed only in p53 mutant cells (MDA-MB-231). A potent antiproliferative activity of PIN was also observed on p53 null cells HL60 (IC50% 8 μM), their multidrug resistant variant HL60R (IC50% 32 μM) and K562. On HL60 cells, PIN caused a block of cell cycle in the G0/G1 phase, induced a weak proapoptotic effect but it was a good trigger of differentiation (NBT reduction and CD11b expression). PIN caused an upregulation of the CDK inhibitor p21(WAF1/Cip1) both at mRNA and protein levels so suggesting that this could be a mechanism by which PIN reduced proliferation and induced differentiation on HL60 cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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-regulation of the PI3K/Akt/mTOR signal pathway. The combination of baicalin and VP16

  7. Effects of red orpiment on cell morphology and expression of PML mRNA and protein in NB4 and HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2003-01-01

    Objective To investigate the effects of red orpiment on cell morphology, expression of promyelocytic leukemia (PML) mRNA and its protein localization in NB4 and HL-60 cell lines.Methods Cell morphology was assayed by Wright's staining and fluorescence staining, while PML mRNA expression was determined by RT-PCR. PML protein localization by evaluated by immunofluorescence staining. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with red orpiment. The fusion protein was no longer observed in NB4 cells, PML protein was relocated, and then degraded. In HL-60 cells, PML protein underwent a similar progress. The expression of promyelocytic leukemia (PML) mRNA was not changed in the treated cells.Conclusion Red orpiment inhibits the proliferation of leukemia cells by inducing them to undergo apoptosis.

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

    Science.gov (United States)

    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 HL-60 cells obviously decreased after transfection (P HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响%Influence of TIEG1 on Apoptosis of HL-60 Cells and Expression of Bcl-2/Bax

    Institute of Scientific and Technical Information of China (English)

    姚鲲; 杨莹; 胡荣; 苗苗; 廖爱军; 杨威; 刘卓刚

    2013-01-01

    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 increasied,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 assosciated with HL-60 cell apoptosis induced by TIEG1.%本研究旨在观察TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响.用不同浓度的TIEG1处理HL-60细胞,采用MTT法检测细胞生长抑制率.用12.03 ng/ml TIEG1处理HL-60细胞,以流式细胞术检测细胞凋亡,以RT-PCR方法检测Bcl-2及Bax的表达变化.结果表明,TIEG1对HL-60细胞具有增殖抑制作用,其抑制效应呈时间及剂量依赖性.TIEG1 12.03 ng/ml增殖抑制作用较为明显.在细胞凋亡过程中,Bax表达呈升高趋势,而Bcl-2呈下降趋势(P<0.05).结论:TIEG1可以抑制HL-60细胞增殖,进而诱导其凋亡,且呈时间、剂量依赖性.在TIEG1诱导HL-60细胞凋亡的过程中,Bcl-2/Bax与TIEG1诱导HL-60细胞凋亡相关.

  14. THE EFFECTS OF LOW CONCENTRATIONS OF ZINC ON ETOPOSIDE INDUCED HL-60 CELLS APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    盛晓阳; 沈立松; 徐翀; 吴湘如; 陈宏新; 洪照毅

    2001-01-01

    Objective Observation of the effects of lower concentrations of zinc (20~400μmol/L), which is nearby the physiological concentration, on etoposide induced HL-60 cells apoptosis. Methods Using the flow cytometry , DNA extraction, electrophoresis , and fiuoresence microscopic observation. Results We demonstrated that the low concentrations of zinc also affect cells apoptosis. If zinc was added at early time, even 200μmol/L could inhibit VP16 induced HL-60 cell apoptosis completely in 4h. But at this concentration, zinc also seems to have cell toxicity, not prolong the time of protection effect. Conclusion Zinc plays multiple roles in cellular functions and in protecting cells from exogenous deleterious factors. Zinc plays a complex, dose- and time-dependent role in apoptosis.

  15. Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification.

    Science.gov (United States)

    Gervasoni, J E; Taub, R N; Yu, M T; Warburton, D; Sabbath, M; Gilleran, S; Coppock, D L; D'Alessandri, J; Krishna, S; Rosado, M

    1992-10-01

    Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp). The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986. This change did not affect drug sensitivity. The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype. The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere. Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome. Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp. Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

  16. Apoptosis induction of Persicae Semen extract in human promyelocytic leukemia (HL-60) cells.

    Science.gov (United States)

    Kwon, Hee-Young; Hong, Seon-Pyo; Hahn, Dong-Hoon; Kim, Jeong Hee

    2003-02-01

    The major ingredient of Persicae Semen is a cynogenic compound, amygdalin (D-mandelonitrile-beta-gentiobioside). Controversial results on the anticancer activity of amygdalin were reported due to its conversion to its inactive isomer, neoamygdalin. In order to inhibit the epimerization of amygdalin, we used newly developed simple acid boiling method in preparation of Persicae Semen extract. HPLC analysis revealed most of amygdalin in Persicae Semen extract was active D-form. Persicae Semen extract was used to analyze its effect on cell proliferation and induction of apoptosis in human promyelocytic leukemia (HL-60) cells. Persicae Semen extract was cytotoxic to HL-60 cells with IC50 of 6.4 mg/mL in the presence of 250 nM of beta-glucosidase. The antiproliferative effects of Persicae Semen extract appear to be attributable to its induction of apoptotic cell death, as Persicae Semen extract induced nuclear morphology changes and internucleosomal DNA fragmentation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  20. Structure-activity relationship of lysophosphatidylcholines in HL-60 human leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Eun-heeLEE; Mi-ranYUN; Wei-hongWANG; JeeHJUNG; Dong-soonIM

    2004-01-01

    AIM: To explore the structure-activity relationship of lysophosphatidylcholine (LPC) and lysolipid molecules from a marine sponge and ladybirds. METHODS: We tested three synthetic LPCs and four natural lysolipids on Ca2+ mobilization in HL-60 human leukemia cells. RESULTS: We observed lysolipid-mediated Ca2+ mobilization. The activity was the same in both ester-and ether-linked lysolipids, and introduction of a double bond or methoxy group on the alkyl chain did not significantly modulate the activity. However, replacement of trimethylammonium moiety in the choline structure with ammonium moiety reduced the activity. Furthermore, change of the alkyl chain length influenced the Ca2+ response. CONCLUSION: LPC-induced Ca2+ mobilization might be dependent on the length of alkyl chain and the presence of choline moiety in HL-60 leukemia cells.

  1. Structure-activity relationship of lysophosphatidylcholines in HL-60 human leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Eun-hee LEE; Mi-ran YUN; Wei-hong WANG; Jee H JUNG; Dong-soon IM

    2004-01-01

    AIM: To explore the structure-activity relationship of lysophosphatidylcholine (LPC) and lysolipid molecules from a marine sponge and ladybirds. METHODS: We tested three synthetic LPCs and four natural lysolipids on Ca2+mobilization in HL-60 human leukemia cells. RESULTS: We observed lysolipid-mediated Ca2+ mobilization. The activity was the same in both ester- and ether-linked lysolipids, and introduction of a double bond or methoxy group on the alkyl chain did not significantly modulate the activity. However, replacement of trimethylammonium moiety in the choline structure with ammonium moiety reduced the activity. Furthermore, change of the alkyl chain length influenced the Ca2+ response. CONCLUSION: LPC-induced Ca2+ mobilization might be dependent on the length of alkyl chain and the presence of choline moiety in HL-60 leukemia cells.

  2. GENE EXPRESSION PROFILING OF HUMAN PROMYELOCYTIC LEUKEMIA HL-60 CELL TREATED BY AJOENE

    Institute of Scientific and Technical Information of China (English)

    方志俊; 黄文秀; 黄明辉; 梁润松; 崔景荣; 王夔; 杨梦苏

    2002-01-01

    Objective: Ajoene, a major compound extracted from crashed garlic, has been shown to have antitumor, antimycotic, antimicrobial, antimutagenic functions in vivo or in vitro and treated as a potential antitumor drug. However, the molecular mechanisms underlying the tumor cytotoxicity of ajoene and even garlic substances are poorly defined. In the present study, we aimed to generate gene expression profiles of HL-60 cell treated by ajoene. Methods: A cDNA microarray presenting 2400 of genes amplified from human leukocyte cDNA library was constructed and the gene expression profiles of HL-60 cell induced by ajoene were generated. Results: After data analysis, 28 differentially expressed genes were identified and sequenced. These genes include 21 known genes and 7 ESTs. Most of the known genes are related to cell apoptosis, such as secretory granule (PRG1), beta-2 microglobulin (B2M), 16S ribosomal RNA gene and ribosomal protein S12. Several genes are related to cell differentiation, including the genes similar to H3 histone and ribosomal protein L31. Northern blot analysis was used to verify and quantify the expression of selected genes. Conclusion: Ajoene can induce HL-60 cell apoptosis significantly and may play a role in differentiation. cDNA microarray technology can be a valuable tool to gain insight into molecular events of pharmacological mechanism of herbal medicine.

  3. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line%8-氯腺苷对未分化HL-60细胞增殖的影响(图片更正)

    Institute of Scientific and Technical Information of China (English)

    崔景荣; 惠峪; 相幼青; 张礼和

    2004-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line.Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope.The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay.The cycle of HL-60 cells was analyzed by flow cytometry.Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order: KB (0.05 μmol·L-1) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56 μmol·L-1) < MCF-7 (0.65 μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89 μmol·L-1) < BGC-823 (1.149 μmol·L-1) < PG (2.50 μmol·L-1).The scanning and transmission electron microscopy showed that the microvilli of HL-60 cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA.Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 cells in a time and concentration-dependent manner.Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase.Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  4. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

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    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  5. Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspas-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

  6. Dose- and Time-Dependent Response of Human Leukemia (HL-60 Cells to Arsenic Trioxide Treatment

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2006-06-01

    Full Text Available The treatment of acute promyelocytic leukemia (APL has been based on the administration of all-trans retinoic acid plus anthracycline chemotherapy, which is very effective as first line therapy; however 25 to 30% of patients will relapse with their disease becoming refractory to conventional therapy. Recently, studies have shown arsenic trioxide to be effective in the treatment of acute promyelocytic leukemia. In this study, we used the human leukemia (HL-60 cell line as a model to evaluate the cytoxicity of arsenic trioxide based on the MTT assay. Data obtained from this assay indicated that arsenic trioxide significantly reduced the viability of HL-60 cells, showing LD50 values of 14.26 + 0.5μg/mL, 12.54 + 0.3μg/mL, and 6.4 + 0.6μg/mL upon 6, 12, and 24 hours of exposure, respectively; indicating a dose- and time-dependent response relationship. Findings from the present study indicate that arsenic trioxide is highly cytotoxic to human leukemia (HL-60 cells, supporting its use as an effective therapeutic agent in the management of acute promyelocytic leukemia.

  7. EFFECT OF ARSENIC TRIOXIDE OR ATRA ON PRIMARY APL CELL OR HL-60 CELLS AND THEIR VALUE ANALYSIS IN HYPERLEUKOCYTOSIS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To detect the effect of arsenic trioxide or ATRA on APL cells or HL-60 cells and to investigate the mechanism of the hyperleukocytosis and detect the cross resistance between ATRA and arsenic trioxide. Methods: The number of promyelocytes or more matured granulocytes were counted by regular method, MTT test was used to measure the proliferation of HL-60 cells or APL cells, flow cytometry analysis to measure the apoptosis, NBT method to detect the differentiation of HL-60 cells or APL cells. Results: The proliferation of primary APL cells or HL-60 cells could be inhibited in vitro by either arsenic trioxide or ATRA, which could induce obvious apoptosis or obvious differentiation of primary APL cells or HL-60 cells. Inhibition of proliferation or apoptosis of ATRA resistant HL-60 cells were achieved by exposure toarsenic trioxide in vitro. On the other hand, the results of in vivo treatment showed that arsenic trioxide also induce of hyperleukocytosis. Conclusion: The results indicated that the hyperleukocytosis induced by ATRA is not contributed to the mechanism of more differentiation than apoptosis, there was not cross resistance between ATRA and arsenic trioxide.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  11. Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances

    DEFF Research Database (Denmark)

    Timm, Michael; Hansen, Erik W; Moesby, Lise;

    2006-01-01

    species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The events of cell death and the expression of nuclear matrix protein(NMP)have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide.By means of TUNEL assay,the nuclei displayed a characteristic morphology change,and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h.Nucleosomal DNA fragmentation was observed after treatment for 4 h.The morphological change of HL-60 cells,thus,occurred earlier than the appearance of DNA ladder.Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis.Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells.Western blotting was then performed on three nuclear matrix acssociated proteins,PML,HSC70 and NuMA.The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment,while NuMA,a nuclear mitotic apparatus protein,was down regulated.These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  17. [Combination induction of cell differentiation of HL-60 cells by daidzein (S86019) and BC-4 or Ara-C].

    Science.gov (United States)

    Jing, Y K; Han, R

    1993-01-01

    Experiments demonstrated that the cell differentiation of HL-60 cells induced by low concentrations of daidzein (S86019), BC-4 (active principle of Boswellia carterii Birdw) and Ara-C was not impressive. However, when they were used in combination 80% of HL-60 cells exhibited NBT reduction and 82% of the cells showed phagocytosis after four days exposure to daidzein and BC-4. When HL-60 cells were exposed to daidzein and Ara-c, 70% of the cells exhibited NBT reduction and phagocytosis. Flow cytometry indicated that the majority of the cells were blocked at G1 phase under the induction of combination of daidzein with BC-4 or Ara-C.

  18. Molecular events induced in the HL 60 cell line following treatment with gamma-interferon.

    Science.gov (United States)

    Dubreuil, P; Courcoul, M; Mannoni, P

    1988-01-01

    Gamma-interferon (gamma IFN), like the phorbol ester TPA, is able to induce the differentiation of the HL 60 human promyelocytic cell line in the monocytic pathway. Morphological and serological data show a differential expression of cell surface markers upon treatment with these two inducers. It was reported that TPA treatment alters the expression of some protooncogenes, like c-myc and c-fos, involved in the induction of cell proliferation, and c-fos and c-fms (CSF.1 receptor), linked to monocytic differentiation. We have analyzed the variations in the levels of expression of these oncogenes upon gamma IFN treatment of HL 60 cells. c-myc expression was unchanged during the whole induction period. The early increase reported in c-fos expression was not observed; however, c-fos levels increased upon 24 hr of gamma IFN treatment. These results suggest either that TPA and gamma IFN act at different steps, or, alternatively, that different pathways exist in the monocytic differentiation.

  19. Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT on human leukemia HL-60 cells

    Directory of Open Access Journals (Sweden)

    Jiang Pingping

    2010-04-01

    Full Text Available Abstract Background Polysaccharopeptide (PSP from Coriolus versicolor (Yunzhi is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT. Methods We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI flow cytometry analysis to measure the relative movement (RM of the BrdUrd positively labeled cells and DNA synthesis time (Ts on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. Results PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. Conclusion The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells.

  20. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells.

    Science.gov (United States)

    Smith, James; Bunaciu, Rodica P; Reiterer, Gudrun; Coder, David; George, Thaddeus; Asaly, Michael; Yen, Andrew

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  1. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Coder, David; George, Thaddeus [Amnis Corporation, Seattle, Washington (United States); Asaly, Michael [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Yen, Andrew, E-mail: ay13@cornell.edu [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States)

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  2. Antileukemic Potential of Momordica charantia Seed Extracts on Human Myeloid Leukemic HL60 Cells

    Directory of Open Access Journals (Sweden)

    Ramani Soundararajan

    2012-01-01

    Full Text Available Momordica charantia (bitter gourd has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity of M. charantia extracts has been demonstrated by numerous in vitro and in vivo studies. In the present study, we investigated the differentiation inducing potential of fractionated M. charantia seed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation of c-myc transcript, indicating the involvement of c-myc pathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts of M. charantia seeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Barbora Šalovská

    2014-07-01

    Full Text Available 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-(methylsulfonylphenyl-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.

  5. The Critical Role of Redox Homeostasis in Shikonin-Induced HL-60 Cell Differentiation via Unique Modulation of the Nrf2/ARE Pathway

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2012-01-01

    Full Text Available Among various cancer cell lines, the leukemia cell line HL-60 was most sensitive to Shikonin, with evidence showing both the prooxidative activities and proapoptotic effects of micromolar concentrations of Shikonin. However, the mechanism involved in the cytotoxicity of Shikonin in the submicromolar range has not been fully characterized. Using biochemical and free radical biological experiments in vitro, we identified the prodifferentiated profiles of Shikonin and evaluated the redox homeostasis during HL-60 differentiation. The data showed a strong dose-response relationship between Shikonin exposure and the characteristics of HL-60 differentiation in terms of morphology changes, nitroblue tetrazolium (NBT reductive activity, and the expression level of surface antigens CD11b/CD14. During drug exposure, intercellular redox homeostasis changes towards oxidation are necessary to support Shikonin-induced differentiation, which was proven by additional enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P<0.05 was recorded with regard to the unique expression levels of the Nrf2/ARE downstream target genes in HL-60 cells undergoing late differentiation, which were restored with further antioxidants employed with the Shikonin treatment. Our research demonstrated that Shikonin is a differentiation-inducing agent, and its mechanisms involve the Nrf2/ARE pathway to modulate the intercellular redox homeostasis, thus facilitating differentiation.

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

    Science.gov (United States)

    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.

  7. NADPH oxidase-dependent production of reactive oxygen species induces endoplasmatic reticulum stress in neutrophil-like HL60 cells.

    Directory of Open Access Journals (Sweden)

    Wilson Mitsuo Tatagiba Kuwabara

    Full Text Available 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.

  8. NADPH oxidase-dependent production of reactive oxygen species induces endoplasmatic reticulum stress in neutrophil-like HL60 cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. NPM1基因沉默的HL-60及其耐药细胞株的建立%Establishment of Nucleophosmin Gene Silenced HL-60 and Its Resistant Cell Line

    Institute of Scientific and Technical Information of China (English)

    林敏辉; 胡建达

    2011-01-01

    本研究旨在构建针对HL-60细胞及其耐药细胞株(HL-60/ADR)的NPM1-RNAi的细胞模型,为研究NPM1基因在白血病耐药过程中的作用奠定实验基础.通过针对NPM1基因的shRNA与线性化的pGCSIL-GFP 载体进行连接转化,对获得的重组子(pGCSIL-GFP-NPM1-shRNA)进行PCR和测序鉴定.采用慢病毒载体系统将pHelper 1.0载体、pHelper 2.0载体与pGCSIL-GFP-NPM1-shRNA共转染293T细胞,包装成NPM1 -RNAi-LV,将慢病毒载体感染HL-60HL-60/ADR细胞.采用实时定量RT-PCR和Western blot法分别从mRNA和蛋白水平验证建立的细胞株的转染效率.结果表明,成功构建了重组真核表达载体pGCSIL-GFP-NPM1-shRNA,并通过慢病毒载体系统包装成NPM-1-RNAi-LV;将NPM1-RNAi-LV转染至HL-60HL-60/ADR细胞后,从mRNA水平上看,对细胞的NPM1 mRNA表达有显著抑制,达到90%以上(p<0.05);从蛋白水平上看,对细胞的NPM蛋白表达具有显著的抑制效果,表明转染后的HL-60HL-60/ ADR细胞的NPM1基因特异性沉默.NPM1基因沉默后,HL-60/ADR对阿霉素的耐药性有一定程度的下降.结论:成功构建了NPM1-RNAi的HL-60HL-60/ADR细胞模型,为进一步研究NPM1基因在白血病耐药过程中的作用建立了良好的实验基础.%This study was aimed to construct model cell line of NPMl-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E. Coli DH5a. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2. 0 and pGCSIL-GFP-NPMl-shRNA were cotransformed into 293T cells by lentivius vector system. NPMl-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic

  11. Proapoptotic activity of heterocyclic compounds containing succinimide moiety in the promyelocytic leukemia cell line HL-60.

    Science.gov (United States)

    Kuran, Bozena; Krawiecka, Mariola; Kossakowski, Jerzy; Koronkiewicz, Mirosława; Chilmonczyk, Zdzisław

    2013-01-01

    In the present paper, we describe proapoptotic activity of several heterocyclic compounds 9, 12, 18, 19 and 20 possessing succinimide (as well as succinimide related) moieties. The compounds properties were examined with the aid of flow cytometry on the promyelocytic leukemia cell line HL-60. The highest proapoptotic activity exhibited compound 12 (4-{4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl}-1,7-diethyl-8,9-diphenyl-4-azatricyklo[5.2.1.0(2,6)]-dec-8-ene-3,5,10-trione). The synthesis of compounds 1-17 is also described. The structures of obtained compounds were characterized by 1H NMR, 13C NMR, ESI MS and/or elemental analyses.

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

    Science.gov (United States)

    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.

  13. Dynamic effects of autophagy on arsenic trioxide-induced death of human leukemia cell line HL60 cells

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Zhong-qin LIANG; Bo GAO; Yan-li JIA; Zheng-hong QIN

    2008-01-01

    Aim: To evaluate the contribution of an autophagic mechanism to the As2O3-induced death of human acute myeloid leukaemia cell line HL60 cells. Methods: The growth inhibition of HL60 cells induced by As2O3 was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazohum bromide colorimetric assay. The ac-tivation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As2O3-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Results: After treatment with As2O3, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutraliz-ing agent NH4C11 h before As2O3 potentiated the As2O3-induced death of HL60 cells. In contrast, 3-MA attenuated As2O3-induced death when administered 30 min after As2O3. 3-MA and NH4Cl also inhibited As2O3-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As2O3, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As2O3 was suggested by the collapse of mitochondrial membrane potential, re-lease of cytochrome c from mitochondria, and the activation of caspase-3. Pre-treatment with 3-MA prior to As2O3 amplified these apoptotic signals, while post-treatment with 3-MA 30 min after As2O3 attenuated the apoptotic pathways. Conclusion: Autophagy plays complex roles in the As2O3-induced death of HL60 cells; it inhibits As2O3-induced apoptosis in the initiation stage, but amplifies the AS2O3-mediated apoptotic program if it is persistently activated.

  14. Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

    DEFF Research Database (Denmark)

    Stervbo, Ulrik; Vang, Ole; Bonnesen, Christine

    2006-01-01

    , an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration......- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    1987-10-01

    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.

  17. Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns.

    Science.gov (United States)

    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/cm(2)), 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

  18. SENSITIVITY OF LEUKEMIC CELL LINE HL-60 TO COMBINATION OF NEFERINE AND ARSENIC

    Institute of Scientific and Technical Information of China (English)

    LIU Ge-xiu; ZHANG Yuan; HE Dong-mei

    2006-01-01

    Objective: To determine whether neferine (Nef) enhances the sensitivity of human myeloid leukemia HL-60 cells to arsenic trioxide (ATO). Methods: Apoptosis was detected by DNA electrophoresis. Giemsa staining was used to observe the apoptotic cells under microscope. The apoptotic rates of cells were analyzed using flow cytometry. The inhibitory rates of cell growth were assayed by MTT, and the expression of P-gp was determined by flow cytometry. Results: Low doses of ATO (1.0 (mol/L) only partially inhibitory percentage of cell growth at 72 h was (9.92(3.03) % (P>0.05, vs control). The combination of 1.0 (mol/L ATO with 2.0 (mol/L Nef inhibited (45.27(4.93) % of cell growth, and induced apoptosis in leukemic cells more significantly than wither ATO or Nef on their own (P<0.01). Moreover, ATO-induced expression of P-gp in leukemic cells was inhibited significantly by Nef. Conclusion: These results indicate that Nef significantly increases sensitivity of leukemic cells to ATO, which might be associated with inhibitory expression of P-gp gene. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.

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

    Science.gov (United States)

    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.

  20. Ethanolic extract of propolis induces apoptosis of HL-60 cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Shi; Yana Li; Co-first author Naie Li; Min Yu; Dong Wang; Lijun Kong

    2016-01-01

    Objective The aim of the study was to investigate whether ethanolic extract of propolis inhibits the growth and induces apoptosis of HL-60 cel s. Methods HL-60 cel s were treated for 24, 48, 72 h with various concentrations ethanolic extracts of prop-olis (0, 50, 100, and 200 μg/mL). The proliferation of HL-60 cels was determined using the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, Hochest 33258 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to test the apoptosis of HL-60 cel s. We observed the expression levels of Bax and Bcl-2 in HL-60 cel s by immunohistochemistry. Results MTT assay showed that various concentrations of ethanolic extract of propolis had significant inhibitory efect on HL-60 cel proliferation (P Conclusion Ethanolic extract of propolis inhibits leukemia cel proliferation and induces apoptosis in vitro. Its mechanism may be related to the regulation of Bax and Bcl-2 expression and up-regulation of Bcl-2/Bax ratio.

  1. Effect of fractionation and rate of radiation dose on human leukemic cells, HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Rhee, J.G.; Song, C.W.; Kim, T.H.; Levitt, S.H.

    1985-03-01

    The capacity of HL-60 cells, human acute promyelocytic leukemic cells established in culture, to repair sublethal radiation damage was estimated from the response of the cells to fractionated irradiation or to a single irradiation at difference dose rates. After exposure of cells to a single dose of X rays at a dose rate of 78 rad/min, the survival curve was characterized by n = 2.5, D/sub q/ = 80 rad, and D/sub 0/ = 83.2 rad. Split-dose studies demonstrated that the cells were able to repair a substantial portion of sublethal radiation damage in 2 hr. The response of the cells to irradiation at different dose rates decreased with a decrease in the dose rates, which could be attributed to repair of sublethal radiation damage. The possibility that some of the malignant hemopoietic cells, if not all, may possess a substantial capacity to repair sublethal radiation damage should not be underestimated in planning total-body irradiation followed by bone marrow transplantation.

  2. Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

    Institute of Scientific and Technical Information of China (English)

    李登举; 张瑶珍; 胡向荣; 曹文静; 黄伟

    2004-01-01

    Summary: The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell' nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells.

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

    Science.gov (United States)

    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.

  4. Proliferation inhibition, cell cycle arrest and apoptosis induced in HL-60 cells by a natural diterpene ester from Daphne mucronata

    Science.gov (United States)

    Nouri, K.; Yazdanparast, R.

    2011-01-01

    Background and the purpose of the study Gnidilatimonoein (Gn), a new diterpene ester from Daphne mucronata, possesses strong anti-metastasis and anti-tumor activities. In this study, its apoptosis and differentiation capabilities were evaluated by using the leukemia HL-60 cell line. Material and methods Cell prolifaration inhibition was estimated by MTT assay. The occurrence of apoptosis was evaluated by EtBr/AO double staining technique, cell cycle analyses and detection of apoptotic cells by Annexin V-FITC and propodium iodide (PI). Differentiation of the cells was determined by NBT reduction assay and the expression of specific cell surface markers such as CD14 and CD11b, were analyzed by flow cytometry. Results The drug decreased the growth of the cells dose- and time-dependently and the IC50 was found to be 1.3 µM. Our data suggested that Gn induced both monocytic differentiation and apoptosis among HL-60 cells. In addition, cell cycle analyses showed an increase in G1 phase population by 24 hrs, which was gradually replaced by Sub-G1 cell population (apoptotic cells) by 72 hrs. Conclusion Based on these data, the Gn-treated HL-60 cells displayed differentiation-dependent apoptosis. Thus, Gn might be a good candidate for differentiation therapy of leukemia, pending full biological evaluation of the compound among the wide array of leukemia cells. PMID:22615651

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  7. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  8. Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

    Science.gov (United States)

    Smolen, J E; Hessler, R J; Nauseef, W M; Goedken, M; Joe, Y

    2001-08-01

    Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

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

    Science.gov (United States)

    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.

  10. Loss of TRF2 by radiation-induced apoptosis in HL60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakagami, Yoshihiro; Ito, Megumi; Hara, Takamitsu; Inoue, Tomio [Yokohama City Univ. (Japan). School of Medicine

    2002-06-01

    A telomere consists of the short tandem DNA repeats of (T2AG3)n localized to the distal ends of chromosomes. Telomere repeat binding factor 2 (TRF2) has been implicated in the protection of chromosome ends. Recently, it has been reported that the loss of TRF2 induces apoptosis by various stimuli or genetic technique, however, the effects of radiation are not known. Therefore, this study investigated the interaction between TRF2 and radiation. Western blot analysis, immunohistochemistry, and a DNA fragmentation assay for the detection of apoptosis were performed. The interaction between elastase and TRF2 was also investigated in vitro. Western blot analyses and immunohistochemistry showed that {gamma}-rays induce the temporary accumulation and subsequent loss of TRF2 protein in the nuclei of irradiated HL60 cells. Following DNA fragmentation, the loss of TRF2 could be detected. TRF2 was broken down by elastase, which was translocated into the nucleus before the loss of TRF2. The results of the study showed that irradiation first induces activation of TRF2, consequently protecting the end of the chromosome. Subsequently, translocation of elastase into the nucleus results in the breakdown of TRF2 after DNA fragmentation has occurred. (author)

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

    Science.gov (United States)

    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.

  12. Force-dependent bond dissociation govern rolling of HL-60 cells through E-selectin.

    Science.gov (United States)

    Li, Quhuan; Fang, Ying; Ding, Xiaoru; Wu, Jianhua

    2012-08-15

    E-selectin-mediated rolling on vascular surface of circulating leukocyte on vascular surface is a key initial event during inflammatory response and lymphocyte homing. This event depends not only on the specific interactions of adhesive molecules but also on the hemodynamics of blood flow. Little is still understood about whether wall shear stress or shear rate regulates the rolling. With flow chamber techniques, we here measured the effects of transport, shear stress and cell deformation on rolling of both unfixed and fixed HL-60 cells on E-selectin either in the absence or in the presence of 3% Ficoll in medium at various wall shear stresses from 0.05 to 0.7 dyn/cm(2). The results demonstrated a triphasic force-dependent rolling, that is, as increasing of force, the rolling would be accelerated firstly, then followed a decelerating phase occurred at the initial shear threshold of about 0.1 dyn/cm(2), and lastly returned to an accelerating process starting at the optimal shear threshold of 0.35 dyn/cm(2) approximately. The catch bond regime was completely reflected to rolling behaviors, such as tether lifetime, cell stop time and rolling velocity, meaning that the dominant factor to govern rolling is force. The initial shear threshold might be the minimum level of wall shear stress to sustain a stationary rolling, and the optimal shear threshold would make rolling to the most stable and regular. These findings strongly elucidate the catch bond mechanism for flow-enhanced rolling through E-selectin since longer bond lifetimes led to slower and stabler rolling.

  13. Apoptosis-inducing potential of Myrothamnus flabellifolius, an edible medicinal plant, on human myeloid leukemia HL-60 cells

    Directory of Open Access Journals (Sweden)

    J. Dhillon

    2014-02-01

    Full Text Available Summary. Conventional therapies for treating acute myeloid leukemia involve chemotherapy and radiation. This approach causes damage to both normal and cancerous cells resulting in several side effects. There is a dire need to discover novel drugs that selectively targets only the cancer cells with minimal effects on normal cells. Our research is an effort to identify a novel plant based drug which is edible and selectively targets only the leukemic cells with negligible effects on the normal cells. In this study, extracts from Myrothamnus flabellifolius, a South African resurrection plant was used against human leukemic cells (HL-60. M. flabellifolius is known for its anti-viral, anti-microbial and anti-inflammatory properties. Extracts from this plant also contain derivatives of galloyl and quinic acid. In literature, galloyl and quinic acid have been demonstrated to show anti-cancerous effects. Here, we investigated the anti-cancerous effects of the methanolic and petroleum ether extract of this plant on human leukemic cells (HL-60 compared to non-leukemic lymphocytes (TK6. The methanolic extract depicted reduced HL-60 cell viability while the petroleum ether extract did not. The loss in HL-60 viability in response to the methanolic extract was accompanied by the induction of caspase-dependent apoptosis by way of caspase-7 and Poly (ADP-ribose polymerase cleavage. This study establishes an IC50 of 62.5 µg/ml of dry Myrothamnus extract on HL-60 leukemic cells.Industrial Relevance. The outcome of our study depicts the potential of M. flabellifolius as a cancer drug due to its selective biological activity against cancer cells. The anti-cancer effects of this plant extract did not manifest toxic side effects as it did not harm the normal lymphocytic cells. The edible nature of M. flabellifolius marks it as having a potential role in cancer treatment as a complementary medicine to the existing treatment options.Keywords. Myrothamnus

  14. 肉桂醛对HL-60细胞的生长抑制及诱导凋亡作用研究%Proliferation Inhibitory and Apoptosis-inducing Effects of Cinnamaldehyde on HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    魏练平; 罗美; 蒋瑾; 蒋立科

    2011-01-01

    Objective: To determine the proliferation inhibitory and apoptosis-inducing effects of cinnamaldehyde on HL-60 cells.Methods: The proliferation inhibitory effect was evaluated using trypan-blue staining and MTT assay,and the apoptosis-inducing effect using flow cytometry,scanning electron microscope(SEM) and transmission electron microscope(TEM).Results: Cinnamaldehyde had an oblivious inhibitory effect on the proliferation of HL-60 cells with dependence upon its concentration and treatment time and the LC50,median lethal concentration was 34.87 μg/mL.The rate of apoptosis in HL-60 cells was 32.6 % after being treated with cinnamaldehyde at the dose of 20 μg/mL.for 16 h.Typical apoptotic morphological changes in the cells was observed under fluorescence microscopy,SEM and TEM.Conclusion: cinnamaldehyde cna notably inhibit the proliferation and induce the apoptosis of HL-60 cells.%目的:探讨肉桂醛对HL-60细胞的增殖及凋亡的影响。方法:采用台盼蓝细胞计数和四甲基偶氮唑蓝(MTT)法研究肉桂醛对HL-60细胞增殖的抑制作用;采用流失细胞仪(FCM)、荧光显微镜(FM)、扫描电镜(SEM)以及透射电镜(TEM)探讨肉桂醛对HL-60细胞的诱导凋亡作用。结果:台盼蓝细胞计数和MTT法结果表明:肉桂醛对HL-60细胞的增殖有明显抑制作用,抑制作用与肉桂醛质量浓度及作用时间呈依赖关系,其半数致死质量浓度为34.87μg/mL;流式细胞仪检测结果表明:肉桂醛(终质量浓度20μg/mL)作用16h后,HL-60细胞早期凋亡比例为32.6%;该组细胞在荧光显微镜、扫描电镜以及透射电镜观察下均呈现早期凋亡特征。结论:肉桂醛能抑制HL-60细胞增殖并促进其凋亡。

  15. Induction of apoptosis by furanodiene in HL60 leukemia cells through activation of TNFR1 signaling pathway

    Institute of Scientific and Technical Information of China (English)

    MA En-long; WANG Xiao-long; LI Yan-chun; TAI Wen-jiao; LI Te; GUO Tao

    2008-01-01

    Objective Curcuma wenyujin, named Ezhu in Chinese, a traditional Chinese medicine, which has been shown to possess antiearcinogenie activity and used for the treatment of tumor in China,for example, hepatic cancer and leukemia. β-elemene, a component of Curcuma wenyujin, was largely reported as a main active component for anti-tumor effect of Curcuma wenyujin. However, furanodiene, another main component of Curcuma wenyujin, was seldom investigated for its anti-tumor activities. In the present study, we aim to investigate the effect of furanodiene on human leukemia HL60 cells and to study the accurate molecular mechanisms of its action. Methods Trypan blue exclusion experiment was used for cell growth inhibition assay. The apoptotic characterizations were assessed by flow cytometry analysis, AO/EB staining assay and agarose gel eleetrophoresis assay. The expression of apoptosis-related proteins and the release of cytochrome c from mitochondrial were detected by western blotting, and the mRNA levels of TNF-α and TNFR1 were probed by RT-PCR. Formation of TNFR1 complex was analyzed by using immunoprecipitation of TNFR1 with TRRF1/2 and RIP. Results Furanodiene (10-100μM) treatment inhibited the growth of HL60 cells in a concentration-dependent manner. The effect of furanodiene on HL60 cells was associated with the induction of apoptosis, which was characterized by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8 and caspase-9. In the Bcl-2 family proteins, Bid protein (a substrate of caspase-8) was activated by furanodiene, but Bcl-2, Bax and Bcl-xL proteins were not inuenced by furanodiene stimulation. Furanodiene elicited cytochrome c release from mitochondria into the cytosol. Moreover, furanodiene treatment caused the upregulation of TNFR1, the formation of TNFR1 complex and an obvious production of TNF-a in HL60 cells. The soluble TNFR1 receptor effectively inhibited furanodiene-induced apoptosis. Conclusions Furanodiene

  16. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    Science.gov (United States)

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  17. Using cell fate attractors to uncover transcriptional regulation of HL60 neutrophil differentiation

    Directory of Open Access Journals (Sweden)

    Kauffman Stuart A

    2009-02-01

    Full Text Available Abstract Background The process of cellular differentiation is governed by complex dynamical biomolecular networks consisting of a multitude of genes and their products acting in concert to determine a particular cell fate. Thus, a systems level view is necessary for understanding how a cell coordinates this process and for developing effective therapeutic strategies to treat diseases, such as cancer, in which differentiation plays a significant role. Theoretical considerations and recent experimental evidence support the view that cell fates are high dimensional attractor states of the underlying molecular networks. The temporal behavior of the network states progressing toward different cell fate attractors has the potential to elucidate the underlying molecular mechanisms governing differentiation. Results Using the HL60 multipotent promyelocytic leukemia cell line, we performed experiments that ultimately led to two different cell fate attractors by two treatments of varying dosage and duration of the differentiation agent all-trans-retinoic acid (ATRA. The dosage and duration combinations of the two treatments were chosen by means of flow cytometric measurements of CD11b, a well-known early differentiation marker, such that they generated two intermediate populations that were poised at the apparently same stage of differentiation. However, the population of one treatment proceeded toward the terminally differentiated neutrophil attractor while that of the other treatment reverted back toward the undifferentiated promyelocytic attractor. We monitored the gene expression changes in the two populations after their respective treatments over a period of five days and identified a set of genes that diverged in their expression, a subset of which promotes neutrophil differentiation while the other represses cell cycle progression. By employing promoter based transcription factor binding site analysis, we found enrichment in the set of divergent

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

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Tomonobu [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Okumura, Fumihiko [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Iguchi, Akihiro; Ariga, Tadashi [Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Hatakeyama, Shigetsugu, E-mail: hatas@med.hokudai.ac.jp [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan)

    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.

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

    Directory of Open Access Journals (Sweden)

    Zahra Tayarani-Najaran

    2017-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  1. EFFECTS OF QUERCETIN ON CELL MORPHOLOGY AND EXPRESSION OF PML mRNA AND PROTEIN OF NB4 AND HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2001-01-01

    Objective To investigate the effects of quercetin on cell morphology, expression of promyelocytic leukemia ( PML ) mRNA and PML protein localization of NB4, HL-60 cells. Methods Cells morphology assayed by Wright's stain, fluorescence stain, and PML mRNA expression by RT-PCR , PML protein localization by immuno-fluorescence. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with quercetin . Immuno-fluorescence analysis showed after treatment with quercetin , the fusion protein disappeared in NB4 cells, PML protein relocated, then degraded, and that also seen in HL-60 cells. The expression of PML mRNA is not changed in quercetin-treated cells. Conclusion PML play the role of apoptosis inducer in leukemia cells at the translational level, quercetin can inhibit the proliferation of leukemia cells, and induce NB4, HL-60

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2015-09-01

    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.

  4. The effect of cytoplasmic regions of LIF receptor on activating MAPK p42/44 in HL-60 cell

    Institute of Scientific and Technical Information of China (English)

    LIU Hou-qi; SHAN Ji-kuan; TANG Shu-ping; LIU Shan-rong; JI Kai-hong

    2001-01-01

    Objective: To study the relation between the cytoplasmic region of each subunit of leukemia inhibitory factor (LIF) receptor and mitogen-activated protein kinase (MAPK) in human leukemia line HL-60 for studying the mechanism of leukemia cell proliferation disorder. Methods: We prepared the chimerical receptor with exchanged cytoplasmic domain (190/130,130/190), expressed it on the membrane of HL-60, and then bound LIF with the wild type receptor competitively. The cytoplasmic region homodimerization (190cyt- 190cyt, 130cyt- 130cyt) initiated intracellular signal transduction, cell proliferation and differentiation. MAPK phosphorylation level was observed by means of immunoblotting and immunobiochemistry. Results: Higher level ofMAPK p42/p44 phosphorylation (Thr202Tyr204) and faster cell proliferation were determined in group 190/130 compared with the wild type receptor. Those in group 130/190 were lower than the others. Conclusion: The cytoplasmic domain ofgpl30 involves in the induction of MAPK activation and the cell proliferation of HL-60.

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

    Science.gov (United States)

    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 HL-60 treated with MG132 was up-regulated time-dependently (P HL-60 cells treated with MG132 and reached its peak when the concentration

  6. Diallyl disulfide suppresses growth of HL-60 cell through increasing his-tone acetylation and p21WAF1 expression in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    Jie ZHAO; Wei-guo HUANG; Jie HE; Hui TAN; Qian-jin LIAO; Qi SU

    2006-01-01

    Aim: To examine the differentiation induction and growth inhibition of HL-60 cells by diallyl disulfide (DADS), and its relationship with the alterations of histone acetylation and p21WAF1 expression in vitro and in vivo. Methods: Differentiation was studied by nitroblue tetrazolium (NET) reduction of HL-60 cell in vitro. HL-60 cells 5xl06 were injected into the right side of the peritoneal cavity of severe combined immunodeficiency (SCID) mice. When the peritoneal neoplasms were detected, the SCID mice were randomly divided into 3 groups and received an ip injection of vehicle alone (NS), DADS or sodium butyrate (SB). The growth inhibition of peritoneal neoplasms induced by DADS was observed by a growth curve. The cycle distribution of HL-60 cells in SCID mice was monitored by flow cytometry. The expression of acetylated histone H3, H4 and p21WAF1 were measured by Western blot. Results: After treatment with DADS for 0-72 h, the NET reduction ability of HL-60 cells increased in a time-dependent manner, compared with no treatment of HL-60 cells. In the HL-60 cells treated with DADS for 24 h, the expression of acetylated histone H3, H4, and p21WAF1 increased obviously. After treatment with DADS, tumor growth was markedly suppressed. HL-60 cells from mice treated with DADS were blocked in the G1 phase, from 25.4% to 63.4%. The tumors from the mice treated with DADS showed an increase of acetylated histone H3, H4, and p21WAF1. Conclusion: DADS could induce differentiation and inhibit the growth of HL-60 cells through increasing the expression of acetylated histone H3, H4, and p21WAF1 in vitro and in vivo.

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

    Science.gov (United States)

    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.

  8. Monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells induced by the glutamine antagonist acivicin.

    Science.gov (United States)

    Nichols, K E; Chitneni, S R; Moore, J O; Weinberg, J B

    1989-10-01

    Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N-formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability

  9. Diesel exhaust particle-induced cell death of human leukemic promyelocytic cells HL-60 and their variant cells HL-NR6.

    Science.gov (United States)

    Matsuo, M; Uenishi, R; Shimada, T; Yamanaka, S; Yabuki, M; Utsumi, K; Sagai, M

    2001-04-01

    The cytotoxicity of diesel exhaust particles (DEPs) toward human leukemic promyelocytic cells HL-60 was examined. DEPs were toxic and cytotoxicity increased in a dose-dependent manner. All cells died with 750 microg/ml DEPs in culture media. Apoptosis occurred in HL-60 cells exposed to DEPs. The cytotoxicity of DEP extracts with organic solvents was much lower than those of DEPs and organic solvent-washed residual DEPs. HL-NR6 cells, an HL-60 variant cell line, having higher superoxide dismutase and catalase activities than HL-60 cells, were more resistant to DEP cytotoxicity. When preincubated with the fluorescent probe diacetoxymethyl 6-carboxy-2',7'-dichlorodihydrofluorescinate diacetate and then exposed to DEPs, HL-60 cells emitted green fluorescence under blue illumination, indicating that reactive oxygen species were generated within the cells. The DEP cytotoxicity correlated inversely with the cellular concentration of reduced glutathione (GSH), which had been attenuated with L-buthionine-(R,S)-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, and was lowered with ethyl reduced glutathionate, a GSH carrier across biomembranes. Further, DEPs themselves decreased the cellular concentration of GSH in a dose-dependent manner. The alpha-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol decreased DEP cytotoxicity, while alpha-tocopherol had no effect. In addition, quinacrine, an endocytosis inhibitor, decreased DEP cytotoxicity. These results show that DEPs are cytotoxic and suggest that the cytotoxicity results from generation of reactive oxygen species by DEPs which have been incorporated into cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 24- and 26-homo-1,25-dihydroxyvitamin D/sub 3/: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro inducing differentiation of human leukemia cells HL-60 in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ostrem, V.K.; Tanaka, Y.; Prahl, J.; DeLuca, H.F.; Ikekawa, N.

    1987-05-01

    1,25-Dihydroxyvitamin D/sub 3/, the hormonal form of vitamin D/sub 3/, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. The authors have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ and their ..delta../sup 22/ analogues are 10-fold more potent than 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxy-vitamin D/sub 3/ in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxy-vitamin D/sub 3/ intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ have the same effectiveness as 1,25-dihydroxyvitamin D/sub 3/ in displacing the tritiated hormone from its receptor in rat intestine of HL-60 cells. These analogues of 1,25-dihydroxyvitamin D/sub 3/ may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation.

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

    Directory of Open Access Journals (Sweden)

    Hairong Song

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. Mangiferin increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in human HL60 myeloid leukemia cells.

    Science.gov (United States)

    Zhao, Jie; Zhang, Benping; Li, Shanshan; Zeng, Linglan; Chen, Yan; Fang, Jun

    2014-05-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway is a key target for cancer chemoprevention. Recent studies have that Nrf2 activation may be the result of an increase in Nrf2 protein stability. Mangiferin (MA), a compound monomer extracted from the mango plant, has antioxidant and cytoprotective activities. Our previous study demonstrated that MA increased Nrf2 expression and activated Nrf2 signaling in hematopoietic cells. Thus, in the present study, we aimed to investigate the mechanisms by which MA increases Nrf2 expression in human HL60 myeloid leukemia cells in vitro. Our western blot analysis results revealed that MA markedly increased Nrf2 expression in dose- and time-dependent manner. However treatment with MA did not affect the Nrf2 mRNA level. The results of cycloheximide (CHX)-chase analysis demonstrated that the Nrf2 protein half-life was prolonged to 58 min when the HL60 cells were pre-incubated with 50 µM MA for 4 h, whereas its half-life was only 20 min in the non-MA treated control cells. Further experiments revealed that MA mainly enhanced non-ubiquitinated Nrf2 protein levels when increasing Nrf2 protein stability; these effects differed from those induced by the proteasome inhibitor, MG132. Subsequent immunoprecipitation experiments confirmed that MA inhibited Nrf2 ubiquitination in HL60 cells. These results provide evidence that MA increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in hematopoietic cells. This may be one of the mechanisms through which MA activates the Nrf2-mediated antioxidant response and exerts cytoprotective effects.

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

    Science.gov (United States)

    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.

  17. Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

    DEFF Research Database (Denmark)

    Boje, Alex; Moesby, Lise; Timm, Michael;

    2012-01-01

    that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest...... concentration of testosterone (120 µM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further...

  18. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells

    National Research Council Canada - National Science Library

    Lee, Wei-Jiunn; Chen, Yun-Ru; Tseng, Tsui-Hwa

    2011-01-01

    .... 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...

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

    Science.gov (United States)

    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.

  20. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    Science.gov (United States)

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

    2012-08-30

    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

  1. Bryostatin, an activator of the calcium phospholipid-dependent protein kinase, blocks phorbol ester-induced differentiation of human promyelocytic leukemia cells HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Kraft, A.S.; Smith, J.B.; Berkow, R.L.

    1986-03-01

    Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the induction of phorbol ester biologic effects. The authors' research indicates that bryostatin, a macrocyclic lactone that binds to the phorbol ester receptor in human polymorphonuclear leukocytes, also binds to this receptor in the human promyelocytic leukemia cell line, HL-60. Bryostatin activates partially purified C kinase from HL-60 cells in vitro, and when applied to HL-60 cells in vivo, it decreases measurable cytoplasmic C kinase activity. Unlike the phorbol esters, bryostatin is unable to induce a macrophage-like differentiation of HL-60 cells; however, bryostatin, in a dose-dependent fashion, blocks phorbol ester-induced differentiation of HL-60 cells and, if applied within 48 hr of phorbol esters, halts further differentiation. These results suggest that activation of the C kinase by some agents is not sufficient for induction of HL-60 cell differentiation and imply that some of the biologic effects of phorbol esters may occur through a more complex mechanism than previously thought.

  2. The Effect of Telomerase Antisense RNA on Telomerase Activity and Cell Proliferation of HL-60%端粒酶反义RNA 对HL-60细胞端粒酶活性及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    刘利; 孙秉中; 梁英民; 张传山; 张惠中; 阎严; 张方信

    2001-01-01

    Objective To study the effect of telomerase antisense RNA ontelomerase activity and cell proliferation of HL-60 cells.Methods By using lipofectin mediated DNA transfection,telomerase antisense RNA was successfully introduced into HL-60 cells.By means of TRAP、dynamics of cell growth、flucytometry the changes of HL-60 cells in aspect of telomerase activity,proliferation were detected.Results Telomerase antisense RNA can significantly inhibit the telomerase activity and the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells.Conclusion It seems to be a new methods to treat leukemia by using telomerase antisense to inhibit telomerase activity and the proliferation of HL-60 cells.telomerase is a new latent target to treat leukemia.%目的 探讨端粒酶反义RNA对HL-60细胞端粒酶活性及细胞增殖的影响。方法 用脂质体介导法将pBBS212-hTR(反义端粒酶RNA真核表达载体)导入人髓性白血病细胞系HL-60中,采用TRAP法测定端粒酶活性,细胞生长动力学检测,FCM分析等方法观察其对HL-60细胞的端粒酶活性及细胞增殖影响。结果 端粒酶反义RNA能显著抑制HL-60细胞的端粒酶活性,抑制HL-60细胞的增殖并诱导其凋亡。结论 以端粒酶反义RNA抑制端粒酶活性是治疗白血病的潜在途径。

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Effect of myeloperoxidase inhibition on gene expression profiles in HL-60 cells exposed to 1,2,4,-benzenetriol.

    Science.gov (United States)

    Miyahara, Emiko; Nishikawa, Takuro; Takeuchi, Toru; Yasuda, Kaori; Okamoto, Yasuhiro; Kawano, Yoshifumi; Horiuchi, Masahisa

    2014-03-20

    While it is known that benzene induces myeloid leukemia in humans, the mechanism has yet to be clarified. Previously, we suggested that myeloperoxidase (MPO) was the key enzyme because it promotes generation of powerful oxidant hypochlorous acid (HOCl) which, reacting with DNA, causes leukemogenesis. In this study, using a whole-human-genome oligonucleotide microarray to clarify the relationships between myelotoxicity of benzene and MPO, we analyzed the genome-wide expression profiles of HL-60 human promyelocytic cell lines exposed to 1,2,4-benzenetriol (BT) with or without MPO inhibition. The microarray analysis revealed that short (1 h) and longer (4 h) exposure to BT changed the expression in HL-60 cells of 1,213 or 1,214 genes associated with transcription, RNA metabolic processes, immune response, apoptosis, cell death, and biosynthetic processes (|Z-score|> 2.0), and that these changes were dramatically lessened by MPO-specific inhibition. The presence of functionally important genes and, specifically, genes related to apoptosis, carcinogenesis, regulation of transcription, immune responses, oxidative stress, and cell-cycle regulation were further validated by real-time RT-PCR. Gene expression profiles along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis suggest that BT-induced DNA halogenation by MPO is a primary reaction in the leukemogenesis associated with benzene.

  6. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P. [Harvard Medical School, Boston, MA (United States)

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  7. A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 cells.

    Science.gov (United States)

    Khan, Imran; Guru, Santosh K; Rath, Santosh K; Chinthakindi, Praveen K; Singh, Buddh; Koul, Surrinder; Bhushan, Shashi; Sangwan, Payare L

    2016-01-27

    In an attempt to arrive at more potent cytotoxic agent than the bioactive natural product betulinic acid, influence of small structural modifications of its 1, 2, 3 triazole derivatives tethered at C-28 and both C3, C-28 using click chemistry approach has been studied. The chemically characterized triazoles have been screened for in vitro cytotoxicity against four human cancer cell lines HL-60, MiaPaCa-2, PC-3 and A549 which has allowed to identify triazole derivative 28{1N (4-fluoro phenyl)-1H-1, 2, 3-triazol-4-yl} methyloxy betulinic ester having better potency profile than the parent compound with IC50 values in the range of 5-7 μM. It caused disruption of mitochondrial membrane potential, rendered Bcl-2 cleavage, Bax translocation and decrease Bcl-2/Bax ratio. These events are accompanied by activation of caspases -9, -3, which cleave the PARP-1. It also induces caspase-8, which is involved in extrinsic apoptotic pathway. Therefore, it induces apoptosis through both intrinsic and extrinsic pathways in human leukemia HL-60 cells.

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

    Science.gov (United States)

    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

  9. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

    Full Text Available Abstract Background Compound K [20-O-β-(D-glucopyranosyl-20(S-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. Methods We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Results Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1 the activation of caspases-3, -8, and -9; (2 the loss of mitochondrial membrane potential; (3 the release of cytochrome c and Smac/DIABLO to the cytosol; (4 the translocation of Bid and Bax to mitochondria; and (5 the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. Conclusions The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through

  10. Monitoring magnesium efflux cyclic AMP-induced in HL60 cells by using a new hydroxyquinoline fluorescent chemosensor

    Directory of Open Access Journals (Sweden)

    Chiara Marraccini

    2014-01-01

    Full Text Available Cellular homeostasis of magnesium is still unclear. Several studies documented the occurrence of fluxes of magnesium across the plasmamembrane within minutes from the application of metabolic or hormonal stimuli. These fluxes, however, result in limited variation of free Mg2+ intracellular concentration and large changes in total Mg content. It has been reported that a stimulation with cyclic AMP caused a movement of total magnesium within 10 min after treatment in cardiomyocytes. In this study we tested this hypothesis in HL60 leukemic cells, not excitable but highly proliferating cell model. We evaluated Mg flux by DCHQ5, the phenyl-derivative of hydroxyquinoline fluorescent probe family. We observed a drastic decrease of intracellular total magnesium in the first 3 min. We also verified that at least 10% of the total intracellular amount of magnesium moved in the supernatant of stimulated cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  14. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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...

  16. Affection of Boswellia Carterii Birdw on Telomerase Activityexpression in HL60 Cells%乳香提取物对HL60细胞端粒酶活性影响的研究

    Institute of Scientific and Technical Information of China (English)

    齐振华; 谭柏林; 彭旭阳; 张国平

    2000-01-01

    为研究乳香提取物对HL60细胞端粒酶活性影响,应用端粒酶重复序列扩增技术及聚丙烯胺凝胶电泳对HL60细胞端粒酶活性的表达进行检测,并应用乳香提取物及全反式维甲酸下调HL60细胞端粒酶活性.结果乳香提取物能降低HL60细胞端粒酶活性.其降低HL60细胞端粒酶活性作用与全反式维甲酸相似.

  17. Effects of Quercetin and Resveratrol combined Reversing Drug Resistance of Leukemia Cell Line HL-60-ADM%槲皮素联合白藜芦醇逆转白血病细胞株HL-60-ADM耐药性的影响

    Institute of Scientific and Technical Information of China (English)

    张复华; 尹松梅; 牛国敏; 杨国雷; 凌奕文; 李蕊; 徐嘉愉

    2014-01-01

    Objective The recent study showed that querctin and resveratrol can reverse multidug resistance in leukemia. The aim of this study was to investigate the reversal multidug resistance effect by querctin and resveratrol. Methods CCK-8 assay was used to compare the toxic effect of querstin or resveratrol alone with that in combination with adriamycin on HL-60-A cells. Results Compared to HL-60 cell line, the IC50 value of adriamycin for HL-60-A increased signiifcantly, it showed that HL-60-A was multidug resistance cell line;querctin or resveratrol could inhibite cell proliferation of HL-60-A in a dose-dependent pattern, while the toxic effect of querctin combined with resveratrol was greater than that of the cells treated with querstin or resveratrol alone;the low-dose of querctin and resveratrol could enhance the sensitivity of adriamycin on HL-60-A, and querstin combined with resveratrol could further enhance the toxic effect of adriamycin. Conclusion Querctin and resveratrol both illustrated signiifcant reversal effect on the leukemia cell line, while the reversal effect of querctin was greater than resveratrol, the synergistic reversal effect of querstin combined with resveratrol was determined.%目的:研究槲皮素(Quercetin,Que)及白藜芦醇(Resveratrol,Res)单用及联用对HL-60-A细胞株耐药性的影响。方法不同浓度阿霉素(adriamycin,ADM)孵育HL-60HL-60-A细胞48 h后CCK-8法测定细胞存活率;Que、Res单用或联用,及加用ADM 48 h测定HL-60-A细胞存活率。结果 ADM作用48 h后,HL-60-A较HL-60细胞IC50明显增高增加提示为耐药细胞株;Que及Res在12.5~150μmol/L时呈剂量依赖性杀伤HL-60-A细胞,Que毒性作用更强,二者可以进一步增强杀伤效应;Que及Res低剂量时可增强ADM对HL-60-A敏感性,二者联用进一步增强ADM的杀伤效应。结论 Que及Res均可逆转白血病细胞耐药性,Que作用较强,二者联合可更有效逆转肿瘤耐药。

  18. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confo-cal laser scanning microscopy

    Institute of Scientific and Technical Information of China (English)

    张春阳; 李艳平; 马辉; 李素文; 薛绍白; 陈瓞延

    2001-01-01

    Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

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

    Science.gov (United States)

    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.

  1. Inhibition of NF-κb by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-XL expression

    Institute of Scientific and Technical Information of China (English)

    曹文静; 张瑶珍; 张东华; 李登举; 唐锦治

    2004-01-01

    Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB) activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism.Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. The cytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner.Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the anti-leukemia effects of TNF-α or even of other cytotoxic agents.

  2. 6-C-methyl flavonoids isolated from Pinus densata inhibit the proliferation and promote the apoptosis of the HL-60 human promyelocytic leukaemia cell line.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. Effect of frankincense extract on VEGF and its receptor-1 expression in acute promyelocytic leukemia cell lines HL-60 cells%乳香提取物对HL-60细胞株VEGF分泌及其Flt-1受体表达的影响

    Institute of Scientific and Technical Information of China (English)

    张勇; 齐振华; 李乐赛; 符晓华; 彭小宁; 张娜; 于才红

    2011-01-01

    目的:探索不同浓度乳香提取物作用于HL-60细胞后,白血病细胞株HL-60细胞VEGF mRNA表达及VEGF蛋白分泌及受体Flt-1的变化.方法:MTT法检测乳香提取物对细胞增殖的抑制作用,选择适当的药物浓度及作用时间:RT-PCR法检测乳香提取物处理前后HL-60细胞中VEGF mRNA表达水平;Western Blot法检测乳香提取物处理前后HL-60细胞VEGF分泌和受体Flt-1蛋白表达水平.结果:乳香提取物在15.0 mg/L时在体外能下调HL-60细胞VEGF mRNA的表达和VEGF蛋白的分泌并呈时间与剂量依赖性(P<0.05).结论:乳香提取物在体外可以抑制HL-60细胞分泌VEGF,同时抑制HL-60细胞内VEGF及其受体Flt-1 mRNA及蛋白表达下降,具有潜在抑制急性早幼粒细胞白血病血管新生的作用,是一种可供选择的抗白血病药物.%Objective To assess the effects of Frankincense extract on VEGF and Fit-1 expression in acute promyelocytic leukemia Cell Lines HL-60 cells. Methods HL-60 cells were treated with Frankincense extract, then cell proliferation were examined by the method of MTT. The level of VEGF-mRNA expression was distinguished by semi -quantitative RT -PCR technique. Western Blot was applied to examine the protein expression of VEGF and Fit-1. Results Within the concentration of (10.0~15.0) mg/L, Frankincense extract strongly inhibited proliferation of HL-60 cells. When the drug is 15.0 mg/L, α-boswellic acid can inhibit the mRNA expression of VEGF and the protein expression of VEGF and Fit-1. Conclusion Within a certain drug concentration, Frankincense extract can inhibit HL-60 cells proliferation. Its role of inhibiting angiogenesis maybe relate with down regulating the expression of VEGF and Fit-1.

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

    Energy Technology Data Exchange (ETDEWEB)

    Perri, Mariarita; Yap, Jeremy L.; Yu, Jianshi [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States); Cione, Erika [Department of Pharmacy, Health and Nutritional Sciences, Ed. Polifunzionale, University of Calabria, 87036 Rende, CS (Italy); Fletcher, Steven [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States); Kane, Maureen A., E-mail: mkane@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States)

    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.

  6. CHANGES OF POLYAMINE METABOLISM IN HL-60 CELLS DURING THE INDUCTION OF DIFFERENTIATION BY RETINOIC ACID AND DIMETHYLSULFOXIDE

    Institute of Scientific and Technical Information of China (English)

    缪金明; 潘瑞彭; 欧阳仁荣

    1992-01-01

    The polyamines putrescine, spermidine and spermine have been implicated in the regulation of cell proliferation and differentiation. In this study, the changes of intracellular polyamine contents and activity of ornithine decarboxylase, a rate-limiting enzyme in the polyamine synthetic pathway, were studied. The results showed that both retinoic acid (RA) and dimethylsulfoxide (DMSO) could elevate intracellular putrescine level by more than 2-fold over control value, then it declined gradually. In RA-treated cells, transient increase in spermidine and spermine levels was noted. In contrast, the spermidine and spermine levels in DMSO-treated cells declined to about 50% of the level of control cells at 96 h. The measurement of ornithine decarboxylase activity demonstrated that the increase of intracellular putrescine in RA and DMSO treated cells was due to the polyamine synthesis by inducing ornithine decarboxylase which reached 2 to 4-fold higher over basic level at 2 h, and above 6-fold at 16 h. These results suggest that the polyamine metabolism may be involved in RA and DMSO-induced granulocytic differentiation of HL-60 promyelocytic leukemia cells.

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

    Science.gov (United States)

    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.

  8. Effect of Eucheuma gelatinae polysaccharide on apoptosis of human leukemia HL-60 cells%琼枝麒麟菜多糖对白血病细胞株HL-60凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    吴显劲; 欧超伟; 孟庆勇

    2007-01-01

    目的 观察琼枝麒麟菜多糖(EGP)对白血病细胞株HL-60凋亡的影响.方法 分别采用100、200、400 mg/L的EGP处理HL-60细胞.采用荧光显微镜观察HL-60细胞形态、MTT法测定细胞株的增殖抑制情况,流式细胞仪测定细胞凋亡率,逆转录-聚合酶链反应(RT-PCR)检测凋亡相关基因Bcl-2、Fas表达情况.结果 200、400 mg/kg的EGP作用后HL-60细胞出现典型的凋亡形态学变化,HL-60细胞抑制率增加;凋亡抑制基因Bcl-2表达下调,促凋亡基因Fas表达上调.结论 琼枝麒麟菜多糖能诱导HL-60细胞发生典型凋亡.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. The enhancement of sensitivity of HL-60 cells to arsenic trioxide by Bcl-2 siRNA%以Bcl-2为靶标siRNA提高HL-60细胞对三氧化二砷敏感性的探讨

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study whether the small-interference RNA (siRNA) targeting against Bcl-2 gene can enhance sensitivity of HL-60 cell to arsenic trioxide. Methods: SiRNA was transferred into the HL-60 cells. At 6 h after transfection, the cells were cultured with arsenic trioxide. The cell growth of the HL-60 cells was detected using MTT at 24, 48 and 72 h, respectively.The levels of the Bcl-2 protein and reactive oxygen species (ROS), as well as of the membrane potential of the mitochondrion were determined by flow cytometry. Results: The Bcl-2 siRNA significantly increased the inhibitory action of arsenic trioxide on growth of HL-60 cells. The combination of siRNA with arsenic trioxide resulted in decrease of the Bcl-2 protein level and increase of the ROS level, as well as significant descending of the membrane potential of mitochondrion of HL-60 (P < 0.05).Conclusion: The siRNAtargeting Bcl-2 can increase the sensitivity of the HL-60 leukemia cells to arsenic trioxide by inhibiting the expression of Bcl-2 protein.

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

    Science.gov (United States)

    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.

  15. Effects of anti-CXCR4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

    Institute of Scientific and Technical Information of China (English)

    WEI Li; KONG Pei-yan; SHI Zhan-zhong; ZENG Dong-feng; CHEN Xing-hua; CHANG Cheng; PENG Xian-gui; ZHANG Yi; LIU Hong

    2007-01-01

    Objective: To investigate the expression of CXCR4 on HL-60 cell line and the proliferation,apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5, an anti-CXCR4 monoclonal antibody, in eradicating the minimal residual disease. Methods: The activity of SDF-1 was inhibited by 10 μg/ml 12G5. After treatment with 12G5, the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohistochemical technique. Results: (1) There was middling degree expression of CXCR4 on HL-60 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR4 decreased gradually. (2)After treatment for 24 h, the adhesion rates in the experiment group and the control were (39.4±7.9)%and (51.4±5.9)%, respectively. (3)After treatment for 24 h, the percentage of HL-60 cells in G0/G1 phase were (55.21±4.9)%, and that in S phase and G2/M phase were (30.40±4.1)% and (14.39±5.2)%, respectively, with the corresponding proportions being (44.67±2.2)%, (45.30±3.7)%, and (10.03±2.6)% in the control. (4) The percentage of apoptotic HL-60 cells was (8.95±1.7)% in the experiment group, compared to (3.97±2.4)% in the control. (5)The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. (6)The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion: 12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G0/G1 phase and promote apoptosis. It may be helpful by inhibiting the bioactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.

  16. The Photocatalytic Inactivation Effect of Fe-Doped TiO2 Nanocomposites on Leukemic HL60 Cells-Based Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Kangqiang Huang

    2012-01-01

    Full Text Available The Fe-doped TiO2 nanocomposites synthesized by a deposition-precipitation method were characterized by X-ray diffraction (XRD, transmission electron microscope (TEM, X-ray photoelectron spectroscopy (XPS, and UV-vis adsorption spectra and then were taken as a new “photosensitizer” for photodynamic therapy (PDT. The photocatalytic inactivation of Fe-doped TiO2 on Leukemic HL60 cells was investigated using PDT reaction chamber based on LED light source, and the viability of HL60 cells was examined by Cell Counting Kit-8 (CCK-8 assay. The experimental results showed that the growth of leukemic HL60 cells was significantly inhibited by adding TiO2 nanoparticles, and the inactivation efficiency could be effectively enhanced by the surface modification of TiO2 nanoparticles with Fe doping. Furthermore, the optimized conditions were achieved at 5 wt% Fe/TiO2 at a final concentration of 200 μg/mL, in which up to 82.5% PDT efficiency for the HL60 cells can be obtained under the irradiation of 403 nm light (the power density is 5 mW/cm2 within 60 minutes.

  17. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Interaction between {alpha}5{beta}1 integrin and secreted fibronectin is involved in macrophage differentiation of human HL-60 myeloid leukemia cells.

    Energy Technology Data Exchange (ETDEWEB)

    Laouar, A.; Collart, F. R.; Chubb, C. B. H.; Xie, B.; Huberman, E.; Center for Mechanistic Biology and Biotechnology; anl-cmb

    1999-01-01

    We examined the role of fibronectin (FN) and FN-binding integrins in macrophage differentiation. Increased FN and {alpha}5{beta}1 integrin gene expression was observed in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or macrophage-CSF-treated blood monocytes before the manifestation of macrophage markers. After treatment of HL-60 cells and monocytes, newly synthesized FN was released and deposited on the dishes. An HL-60 cell variant, HL-525, which is deficient in the protein kinase C{beta} (PKC-{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Transfecting HL-525 cells with a PKC-{beta} expression plasmid restored PMA-induced FN gene expression and macrophage differentiation. Untreated HL-525 cells (which have a high level of the {alpha}5{beta}1 integrin) incubated on FN differentiated into macrophages. The percentage of cells having a macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525 cells, or by either PMA or macrophage-CSF in monocytes was reduced in the presence of mAbs to FN and {alpha}5{beta}1 integrin. The integrin-signaling nonreceptor tyrosine kinase, p72{sup Syk}, was activated in PMA-treated HL-60 and FN-treated HL-525 cells. We suggest that macrophage differentiation involves the activation of PKC-{beta} and expression of extracellular matrix proteins such as FN and the corresponding integrins, {alpha}5{beta}1 integrin in particular. The stimulated cells, through the integrins, attach to substrates by binding to the deposited FN. This attachment, in turn, may through integrin signaling activate nonreceptor tyrosine kinases, including p72{sup Syk}, and later lead to expression of other genes involved in evoking the macrophage phenotype.

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

    Science.gov (United States)

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

    2017-03-02

    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.

  1. microRNA-181b targets MLK2 in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    microRNAs(miRNAs) play critical roles in many different cellular processes,including metabolism,apoptosis,differentiation and development.We showed miR-181b to be highly expressed in acute myeloid leukemia(AML).Furthermore,miR-181b contributed to proliferation of AML cells by targeting MLK2.Our results demonstrated that miR-181b plays an important role in the biology of AML and may be useful in developing therapies targeting miRNAs.

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

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavo-nol 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.

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

    Science.gov (United States)

    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 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.

  5. Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

    Science.gov (United States)

    Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

    2015-05-12

    This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

  6. Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage.

    Science.gov (United States)

    Levay, Konstantin; Slepak, Vladlen Z

    2010-04-15

    Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

  7. BETULINIC ACID WAS MORE CYTOTOXIC TOWARDS THE HUMAN BREAST CANCER CELL LINE MDA-MB-231 THAN THE HUMAN PROMYELOCYTIC LEUKAEMIA CELL LINE HL-60

    Directory of Open Access Journals (Sweden)

    LATIFAH SAIFUL YAZAN

    2009-01-01

    Full Text Available Betulinic acid (BA is a pentacyclic triterpene found in several botanical sources that has been shown to cause apoptosis in a number of cell lines. This study was undertaken to determine the in vitro cytotoxic properties of BA towards the human mammary carcinoma cell line MDA-MB-231 and the human promyelocytic leukaemia cell line HL-60 and the mode of the induced cell death. The cytotoxicity and mode of cell death of BA were determined using the MTT assay and DNAfragmentation analysis, respectively. In our study, the compound was found to be cytotoxic to MDA-MB-231 and HL-60 cells with IC50 values of 58 μg/mL and 134 μg/mL, respectively. Cells treated with high concentrations of BA exhibited features characteristic of apoptosis such as blebbing, shrinking and a number of small cytoplasm body masses when viewed under an inverted light microscope after 24 h. The incidence of apoptosis in MDA-MB-231 was further confirmed bythe DNA fragmentation analysis, with the formation of DNA fragments of oligonucleosomal size (180-200 base pairs, giving a ladder-like pattern on agarose gel electrophoresis. BA was more cytotoxic towards MDA-MB-231 than HL-60 cells, and induced apoptosis in MDA-MB-231 cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

    2016-12-13

    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.

  10. 银杏外种皮多糖对HL-60细胞的体外实验研究%Experimental Study of Ginkgo biloba Exocarp Polysaccharides on HL-60 Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    许爱华; 陈华圣; 孙步蟾

    2004-01-01

    目的:研究银杏外种皮多糖对体外HL-60细胞的作用.方法:运用MTT方法和流式细胞技术,检测HL-60细胞体外增殖、凋亡以及相关基因的表达.结果:银杏外种皮多糖(40~160 m/L,48h)可抑制HL-60细胞增殖及增殖促进基因c-myc的表达,可诱导HL-60细胞凋亡并下调凋亡抑制基因bcl-2的表达.结论:银杏外种皮多糖对HL-60细胞增殖及凋亡的影响涉及增殖促进基因c-myc和凋亡抑制基因bcl-2.

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

    Science.gov (United States)

    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.

  12. BCL-xL/MCL-1 inhibition and RARγ antagonism work cooperatively in human HL60 leukemia cells.

    Science.gov (United States)

    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 As2O3 has increased survival further, patients that experience relapse and are refractory to atRA and/or As2O3 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-xL) 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-xL/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. 2 (±)-7, 8, 3′, 4′, 5′-Pentamethoxyflavan induces G2/M phase arrest and apoptosis in human leukemia HL60 cells

    Institute of Scientific and Technical Information of China (English)

    TAI Wen-jiao; LI Yan-chun; LI Te; ZHANG Wei-ge; MA En-long

    2008-01-01

    Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus, which are widely distributed in the plant kingdom. A number of flavan compounds exhibit antitumor activities. In our previous report, a straightforward synthetic procedure for 2 (±)-7, 8, 3′, 4′, 5′-pentamethoxyflavan (PMF) was developed. To be more important, PMF showed growth inhibitory effect on various human tumor cell lines, especially against HL60 ceils. In the present study, we aim to investigate the molecular mechanisms of action of PMF in HL60 cells. This is the first report of the molecular mechanisms on anti-tumor effect of flavan compounds. Methods Trypan blue exclusion experiment was used for cell growth inhibition assay. Cell apoptosis, cell cycle distribution and the mitochondrial membrane potential (MMP) were assessed by flowcytometric analysis after AO/EB, PI and Rh123 flurescence staining, respectively. Cell cycle- and apoptosis-related proteins were detected using western blotting analysis. Results PMF (1-30 μM) inhibited the growth of HL60 cells in a time- and concentration-dependent manner. Antiproliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest, which was mediated by regulating the expression of p21, Cdc25C and cyclin A proteins and inhibiting the phosphorylation of Cdc2 at Thr161. The prolonged PMF treatment also induced apoptosis of HL60 cells, which was characterized by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase, easpase-3, caspase-8 and caspase-9, changes of Bcl-2 and Bax expression and a decrease in the mitochondrial membrane potential (MMP). Furthermore, caspase-3 inhibitor, not caspase-8 inhibitor and caspase-9 inhibitor, completely blocked PMF-caused apoptosis. Conclusions PMF inhibited the growth of HL60 cells via induction of G2/M arrest and apoptosis. Blockade of cell cycle was associated with the downregulation of Cdc2 complex activity. Both death receptor and mitochondrial

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

    Directory of Open Access Journals (Sweden)

    Judit Molnár

    2016-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

    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 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.

  19. 肉苁蓉总苷在HL-60细胞中的抗氧化活性研究%Antioxidant property of general cistanosides in human HL-60 cell line

    Institute of Scientific and Technical Information of China (English)

    木合布力·阿布力孜; 毛新民; 热娜·卡斯木; 马淑燕; Leininger-Muller B; Gérard SIEST

    2008-01-01

    目的 观察肉苁蓉总苷(general cistanosides,GCs)在HL-60细胞中的抗氧化生物活性.方法 GCs从新疆盐生肉苁蓉中提取得到;利用HL-60细胞中的氧化系统,对GCs的抗氧化活性进行测定研究.HL-60细胞中自由基诱发程度由探针物DCFH-DA的氧化产物DCF的浓度来检测.银杏叶提取物EGB 761用作阳性对照物.结果 GCs以0.25、0.50 g·L-1和0.75 g·L-1浓度,对自由基的抑制率分别为56%、62%和67%.其抗氧化活性与EGB 761类似.结论 GCs在HL-60细胞氧化系统中具有很强的抗自由基氧化活性.

  20. 18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

    Science.gov (United States)

    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.

  1. 槲皮素联合硼替佐米或雷利度胺对抑制HL-60细胞增殖的影响%Effects of quercetin in combination with bortezomib or lenalidomide on inhibition of proliferation of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    肖洁; 牛国敏; 尹松梅; 谢双峰; 李益清; 聂大年; 马丽萍; 王秀菊; 吴裕丹

    2014-01-01

    目的:前期研究证实槲皮素可有效抑制白血病细胞株HL-60细胞增殖,本研究旨在进一步筛选与槲皮素有协同抗白血病作用的药物。方法:不同浓度硼替佐米与HL-60细胞孵育48 h,槲皮素联合硼替佐米孵育HL-60细胞48 h。不同浓度雷利度胺与HL-60细胞孵育48 h,槲皮素联合雷利度胺孵育HL-60细胞48 h。CCK-8法检测细胞存活率。结果:硼替佐米可显著抑制HL-60细胞的增殖。槲皮素联合硼替佐米的IC50值是49.24μmol/L,比单用槲皮素下降13.44μmol/L;等辐射分析法显示两药有协同作用。低浓度雷利度胺(5、10、20、40、80μmol/L)的细胞存活率与对照组比较无统计学差异,高浓度雷利度胺(160、320μmol/L)细胞存活率显著降低;雷利度胺联合槲皮素的IC50值较单用槲皮素无明显下降;等辐射分析法显示两药无协同作用。结论:硼替佐米可显著抑制HL-60细胞的增殖,槲皮素与硼替佐米有协同抑制HL-60细胞增殖的作用。雷利度胺抑制HL-60细胞增殖作用较弱,与槲皮素在抑制HL-60细胞增殖方面无协同作用。%Objective Our preliminary study demonstrates that quercetin can inhibit the proliferation of HL-60 cells. This sudy aimed to find some drugs which could have synergistic effects with quercetin on apoptosis of HL-60 cells. Methods HL-60 cells were cultured with bortezomib at different concentrations (1, 2, 4, 8, 16, and 32μmol/L) alone or combined with quercetin at different concentrations for 48 h. HL-60 cells were cultured with lenalidomide at different concentrations (5, 10, 20, 40, 80, 160, and 320 μmol/L) alone or in combination with quercetin at different concentrations for 48 h. The CCK-8 assay was used to determine the effects on proliferation of HL-60 cells. Results Bortezomib significantly inhibited the proliferation of HL-60 cells (P 0.05). The results of cell viability of HL-60 cells treated by lenalidomide

  2. Influence of Radix Isatidis on the Endotoxin-induced Release of TNF-α and IL-8 from HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The effects of active antiendotoxin chemical fraction isolated from Radix Isatidis (fraction D) on TNF-α and IL-8 secretion in HL-60 cells induced by lipopolysaccharide (LPS) were studied. The appropriate densities of cell suspension and fraction D solution were determined by MTT colorimetric method. Fraction D and LPS were added to HL-60 cell suspension with three different methods respectively. The contents of TNF-α and IL-8 in the cultured supernatant induced by LPS were detected by using ELISA method. The results showed that the absorbance (A) was directly proportional to the number of cells and the linearity was good in the range from 0.25 × 105 to 2 ×105 cell/mL cell suspension. The fraction D significantly inhibited the oversecretion of TNF-α and IL-8 in HL-60 cells induced by LPS at the concentration of 7. 812 mg/mL which had no cytotoxicity. It was indicated that the antiendotoxin mechanism of the active fraction from Radix Isatidis was contributed to the inhibition of the oversecretion of cytokines induced by LPS.

  3. In vitro anticancer property of a novel thalidomide analogue through inhibition of NF-κB activation in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Min LI; Wan SUN; Ya-ping YANG; Bo XU; Wen-yuan YI; Yan-xia MA; Zhong-jun LI; Jing-rong CUI

    2009-01-01

    Aim: To investigate the anticancer property and possible mechanism of action of a novel sugar-substituted thalidomide derivative (STA-35) on HL-60 cells in vitro. Methods: TNF-α-induced NF-κB activation was determined using a reporter gene assay. The MTT assay was used to mea-sure cytotoxicity of the compound. The appearance of apoptotic Sub-G1 cells was detected by flow cytometry analysis. PARP cleavage and protein expression of NF-κB p65 and its inhibitor IκB were viewed by Western blotting. Results: STA-35 (1-20 μmol/L) suppressed TNF-α-induced NF-κB activation in transfected cells (HEK293/pNiFty-SEAP) in a dose- (1-20 μmol/L) and time-dependent (0-48 h) manner. It was also shown that STA-35 exerted a dose-dependent inhibitory effect on HL-60 cell proliferation with an IC50 value of 9.05 μmol/L. In addition, STA-35 induced apoptosis in HL-60 cells, as indicated by the appearance of a Sub-G1 peak in the cell cycle distribution, as well as poly ADP-ribose polymerase (PARP) cleavage. Subsequently, both NF-κB p65 and its inhibitor IκB gradually accumulated in cytoplasmic extracts in a dose- and time-dependent manner, indicating the blockage of NF-κB translocation induced by TNF-α from the cytoplasm to the nucleus. Conclusion: A novel sugar-substituted thalidomide derivative, STA-35, is potent toward HL-60 cells in vitro and induces apoptosis by the suppression of NF-κB activation.

  4. Juglone, from Juglans mandshruica Maxim, inhibits growth and induces apoptosis in human leukemia cell HL-60 through a reactive oxygen species-dependent mechanism.

    Science.gov (United States)

    Xu, Hua Li; Yu, Xiao Feng; Qu, Shao Chun; Qu, Xiang Ru; Jiang, Yan Fang; Sui, Da Yuan

    2012-03-01

    Juglone, a major chemical constituent of Juglans mandshruica Maxim, is a promising anticancer agent that has shown a strong activity against cancer cells in vitro. Our previous study showed that juglone inhibited the proliferation of HL-60 cells with an IC50 value ∼8 μM. To further explore the proapoptotic mechanism of juglone, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by juglone in HL-60 cells. The generation of ROS was about 2 to 8-fold as compared to control cell after treatment with juglone (2, 4 and 8 μM) for 24 h. The glutathione (GSH) depletion was consistent with ROS generation after treatment with juglone. Reversal of apoptosis in antioxidants (NAC and catalase) pretreated cells indicated the involvement of ROS in juglone-induced apoptosis. The cleavage of PARP and procaspase-3 and -9, loss of mitochondrial membrane potential (△Ψm), and release of cytochrome c (Cyt c) and Smac induced by juglone were significantly blocked by NAC. NAC also prevented the inhibition the phosphorylation of Akt and mTOR proteins by juglone. Collectively, these results indicated that ROS played a significant role in the apoptosis induced by juglone in human leukemia cell HL-60.

  5. Viral fusogenic membrane glycoprotein induces HL-60 cell death via inhibiting NF-κB activity%病毒融合基因包膜糖蛋白通过抑制NF-κB活性诱导HL-60细胞死亡

    Institute of Scientific and Technical Information of China (English)

    谭丽; 谭获; 李小龙

    2012-01-01

    目的 探讨猿猴白血病病毒融合基因包膜糖蛋白(GALV-FMG)诱导白血病HL-60细胞的死亡效应与NF-κB的关系.方法 将HL-60细胞分为pcDNA3.1(+)-GALV-FMG质粒转染(A)组、pcDNA3.1(+)空载体对照(B)组和空白对照(C)组.脂质体转染后,应用乳酸脱氢酶释放实验检测GALV-FMG引起HL-60细胞的死亡效应,免疫荧光和凝胶电迁移检验分析GALV-FMG在诱导HL-60细胞死亡中NF-κB的活性.结果 与B、C组相比,A组细胞中NF-κB的活性受到了明显的抑制,细胞死亡的发生显著增加(P<0.05).结论 GALV-FMG的表达可诱导HL-60细胞发生死亡;其机制可能与NF-κB的活性抑制有关.%Objective To investigate the relationship between HL-60 cell death induced by gibbon ape leukemia virus fusion gene envelope glycoprotein (GALV-FMG) and NF-kB activity. Methods HL-60 cells were divided into three groups of A[transfected with pcDNA3. l( + )-GALV-FMG],B[transfected with empty pcDNA3.1( + ) vector] and C(blank controls). After lipofectamine transfection, HL-60 cell death induced by GALV-FMG was detected by lactate dehydrogenase release test, and NF-kB activity was analyzed by immunofluorescence and electrophoretic mobility shift assay. Results Compared with groups of B and C, NF-kB activity was significantly inhibited and cell death was remarkably increased in group C(P<0. 05). Conclusion GALV-FMG expression can induce HL-60 cell death, which may be related with inhibition of NF-kB activity.

  6. A new 2-aminosteroid induces cellular differentiation and upregulates the expression of MafB and Egr-1 genes respectively in HL-60 and K562 leukemia cells

    Institute of Scientific and Technical Information of China (English)

    HE Qun; LI Qiong; YUAN Lin-bo; HE Jun

    2005-01-01

    Background In previous work, we suggested that some 2-aminosteroids inhibited proliferation and induced differentiation of both human and murine leukemia cells. Here, we reported the actions of another new 2-aminosteroid designated as H89712 on human leukemia cells. Methods Cell colony counting and MTT assay were used to determine proliferation. Cell morphology, histochemical staining, UV detection and cytometry were used to determine differentiation. RT-PCR was used to detect gene expression. Standard statistical method was used to analyze data.Results H89712 inhibited proliferation of HL-60 leukemia cells and the inhibition percentage in MTT assay was 18% at the dose of 10-8 mol/L and 65% at the dose of 10-5 mol/L, respectively. The inhibition for HL-60 in colony assay was 23% at the dose of 10-8 mol/L and 96% at the dose of 10-5 mol/L, respectively. H89712 also induced HL-60 cells toward macrophage-like differentiation. It was verified by flow cytometry that the percentage of positive CD14 expression in differentiated HL-60 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 20 times higher than that of the control at the dose of 10-5 mol/L respectively, and this action involved upregulation of MafB gene in HL-60 leukemia cells. On the other hand, H89712 inhibited proliferation of K562 leukemia cells and the inhibition of K562 leukemia cells in MTT assay was shown by 34% at the dose of 10-8 mol/L and 88% at the dose of 10-5 mol/L respectively. The inhibition of K562 leukemia cells in colony assay was 53% at the dose of 10-8 mol/L and 100% at the dose of 10-5 mol/L respectively. H89712 also induced K562 cells toward erythroid-like differentiation and it was verified by flow cytometry that the percentage of positive CD71 expression in differentiated K562 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 16 times higher than that of the control at the dose of 10-5 mol/L respectively. This action

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

    Science.gov (United States)

    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.

  8. Analysis of Spontaneous,gamma ray-and ethylnitrosourea-induced hprt mutants in HL-60 cells With multiplex PCR

    Institute of Scientific and Technical Information of China (English)

    Sheng-Xue Liu; Jia Cao; Hui An; Hua-Min Shun; Lu-Jun Yang; Yong Liu

    2003-01-01

    AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-rays.METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200μg/ml, P<0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P<0.05; in the group of 60Co γ-ray with 1-4 Gy, P<0.05) significantly. In the 13spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %,P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gammaray-induced mutants, 88.1% in ENU-induced mutants).CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-ray.Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

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

    Science.gov (United States)

    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

  10. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. Role of alpha class glutathione transferases (GSTs) in chemoprevention: GSTA1 and A4 overexpressing human leukemia (HL60) cells resist sulforaphane and curcumin induced toxicity.

    Science.gov (United States)

    Sharma, Rajendra; Ellis, Bryan; Sharma, Abha

    2011-04-01

    Alpha-class glutathione transferases (α-GSTs) have been shown to protect cells from the harmful effects of reactive oxygen species (ROS) induced lipid peroxidation (LPO) during oxidative stress caused by various physico-chemical agents. While GSTA1-1/A2-2 isozymes exhibit high activity towards lipid and fatty acid hydroperoxides through their selenium independent glutathione peroxidase (GPx) activity, the GSTA4-4 isozyme efficiently metabolizes the LPO product 4-hydroxynonenal (4-HNE) by conjugating it with glutathione (GSH). Because of the fact that ROS generated by the chemopreventive agents, sulforaphane (SFN) and curcumin (Cur), are implicated in the mechanisms of cancer cell killing, the present studies were designed to investigate the contribution of ROS induced LPO in the cytotoxic effects of these agents and the role of α-class GSTs in modulating their toxicity. Human erythroleukemic (HL60) cells were stably transfected with the cDNA encoding the hGSTA1-1 and mGsta4-4 isozymes. After analysing the expression and activities of the respective GST isozymes, the effects of SFN and Cur on the extent of LPO, cytotoxicity and apoptosis were compared in empty vector (VT), hGSTA1-1 and mGsta4-4 expressing HL60 cells. These studies demonstrate that when compared with SFN, Cur was relatively more cytotoxic to HL60 cells. The ectopic expression of hGSTA1-1 and mGsta4-4 isozymes provided resistance to SFN and Cur induced cytotoxicity and apoptosis through a significant suppression of LPO in these cells. Overall, the results suggest that the expression of α-class GSTs in cancer cells can modulate the therapeutic efficacy of chemopreventive agents.

  13. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

    Science.gov (United States)

    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

  14. 瑞香狼毒诱导HL-60细胞凋亡和调节SGC-7901细胞bcl-2蛋白表达%Stellera chamaejasme induced apoptosis of HL-60 cells and regulated expression of bcl-2 protein in SGC-7901 cells

    Institute of Scientific and Technical Information of China (English)

    贾正平; 王彦广; 樊俊杰; 谢景文; 徐丽婷; 刘盛

    2001-01-01

    Object To explore the antitumor mechanism of Stellera chamaejasme Linn.(SC).Methods SC containing-serum(SCCS)was derived from mice pretreated with different doses of SC.Cultured human leukemia HL-60 and human gastric adenocarcinoma SGC-7901 cells were used.Inhibition of proliferation was measured using MTT assay.Morphological assessment of apoptosis was performed with fluorescence microscope.DNA fragmentation was assessed by agarose gel electrophoresis and flow cytometry.Expression of bcl-2 protein was measured with immunohistochemistry.Results Exposure of exponentially growing HL-60 cells to mice serum containing 10% SC(pretreated with SC3,6, and 12 g/kg)for 48h resulted in growth inhibition in a dose-dependent manner.Typical morphological changes of apoptosis and DNA fragmentation in HL-60 cells were induced."Apobodies'in the apoptotic cells were observed,'ladder"pattern of agarose gel electrophoresis of DNA from 11.7% to 57.4%.Treatment with SC containing serum decreased the percentage of SGC-7901 cell of bcl-2 protein positive expression from 78.3% to 32.9%.Conclusion SC could induce apoptosis of HL-60 cells and decrease the expression of bcl-2 protein of gastric adenocarcinoma SGC-7901 cells.%目的探索瑞香狼素(SC)抗肿瘤机制.方法以HL-60和SGC-7901为靶细胞,用MTT比色法测定细胞增殖抑制,荧光显微镜观察凋亡细胞的形态学改变,DNA电泳和流式细胞仪检测DNA断裂,免疫组化检测bcl-2蛋白表达.结果含SC药物血清处理细胞48 h后,HL-60细胞增殖呈剂量依赖性抑制,并表现出典型的凋亡细胞形态学改变及DNA断裂:即染色体聚集、核固缩、断裂及阶梯状DNA电泳条带,G1期前细胞从11.7%增至57.4%;而SGC-7901细胞bcl-2蛋白表达率从78.3%下降到32.9%.结论 SC可诱导肿瘤细胞凋亡,降低bcl-2蛋白表达.

  15. 4-Acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isariajaponica Yasuda induces apoptosis of human leukemia cells (HL-60).

    Science.gov (United States)

    Oh, G S; Hong, K H; Oh, H; Pae, H O; Kim, I K; Kim, N Y; Kwon, T O; Shin, M K; Chung, H T

    2001-07-01

    The fruiting bodies of Isaria fungi have been traditionally used in Korea to treat cancer. An apoptosis-inducing compound, 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol, was isolated from the methanol extract of fruiting bodies of Isaria japonica Yasuda by bioassay-guided fractionation. The apoptosis of the human leukemia cells (HL-60) by the compound was accessed by propidium iodide-staining flow cytometric analysis, and apoptosis-inducing activity at IC50 concentration (10 nmol/l) was further confirmed by a nuclear morphological change, a ladder pattern of internucleosomal DNA fragmentation, and an activation of caspase-3.

  16. Opposite effects of two trichothecene mycotoxins, deoxynivalenol and nivalenol, on the levels of macrophage inflammatory protein (MIP)-1α and MIP-1β in HL60 cells.

    Science.gov (United States)

    Nagashima, Hitoshi; Nakagawa, Hiroyuki; Kushiro, Masayo

    2012-11-01

    To elucidate the mechanisms underlying the toxicities of the trichothecene mycotoxins deoxynivalenol and nivalenol, their effects on the secretion of anti-hematopoietic chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β in human promyelocytic leukemia cell line HL60 were investigated. Exposure to deoxynivalenol for 24h significantly induced the secretion of chemokines. The induction of these chemokines may account for the leukopenia after exposure to trichothecene mycotoxins. Treatment with nivalenol decreased the secretion of these chemokines. Our finding that deoxynivalenol induces the secretion of these chemokines, whereas nivalenol has the opposite effect, clearly indicates that the toxicity mechanisms of deoxynivalenol and nivalenol differ.

  17. Pellitorine, a Potential Anti-Cancer Lead Compound against HL60 and MCT-7 Cell Lines and Microbial Transformation of Piperine from Piper Nigrum

    Directory of Open Access Journals (Sweden)

    Gwendoline Cheng Lian Ee

    2010-04-01

    Full Text Available Pellitorine (1, which was isolated from the roots of Piper nigrum, showed strong cytotoxic activities against HL60 and MCT-7 cell lines. Microbial transformation of piperine (2 gave a new compound 5-[3,4-(methylenedioxyphenyl]-pent-2-ene piperidine (3. Two other alkaloids were also found from Piper nigrum. They are (E-1-[3’,4’-(methylenedioxycinnamoyl]piperidine (4 and 2,4-tetradecadienoic acid isobutyl amide (5. These compounds were isolated using chromatographic methods and their structures were elucidated using MS, IR and NMR techniques.

  18. MNP-端粒酶反义寡核苷酸复合物诱导HL-60细胞凋亡%The apoptosis of HL - 60 cells induced by magnetic nano - particulate with telomerase antisense oligonucleotides complex

    Institute of Scientific and Technical Information of China (English)

    程呈; 忻茜; 陈祥峰; 李兴玉

    2008-01-01

    反义寡核苷酸具有抑制细胞基因表达作用,对于病毒感染及癌症的治疗具有潜在疗效,然而由于其在生物介质中的不稳定性和低穿透率,常需要修饰于一定的载体上,纳米粒子便是其良好的载体,修饰后可提高反义寡核苷酸的稳定性.研究应用FACS,MTT比色法,电子显微镜,原子力显微镜,细胞集落法研究MNP-端粒酶反义寡核苷酸复合物体外诱导HL-60细胞(人急性早幼粒细胞白血病细胞系)细胞凋亡情况.研究结果表明:通过MTT比色法检测可知不同的药物浓度对细胞的生长有明显的抑制影响;实验组细胞在诱导后于流式细胞术检测中产生了亚二倍体峰(凋亡峰);通过集落形成及电镜观察都有明显的凋亡现象产生,因此认为MNP-端粒酶反义寡核苷酸复合物具有诱导HL-60细胞凋亡的作用.

  19. 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.

    Science.gov (United States)

    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.

  20. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  1. Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

    Science.gov (United States)

    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.

  2. Pyrrocidine A, a metabolite of endophytic fungi, has a potent apoptosis-inducing activity against HL60 cells through caspase activation via the Michael addition.

    Science.gov (United States)

    Uesugi, Shota; Fujisawa, Nozomi; Yoshida, Jun; Watanabe, Mitsuru; Dan, Shingo; Yamori, Takao; Shiono, Yoshihito; Kimura, Ken-ichi

    2016-03-01

    Pyrrocidine A is a known antimicrobial compound produced by endophytic fungi and has a unique 13-membered macrocyclic alkaloid structure with an α,β-unsaturated carbonyl group. We have previously reported that pyrrocidine A shows potent cytotoxicity against human acute promyelocytic leukemia HL60 cells, and the activity is 70-fold higher than that of pyrrocidine B which is the analog lacking the α,β-unsaturated carbonyl group. Pyrrocidine A induced nuclear condensation, DNA fragmentation and caspase activation in HL60 cells. Since the DNA fragmentation was suppressed by pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk), caspase-mediated apoptosis contributes to pyrrocidine A-induced cytotoxicity. JFCR39 human cancer cells panel indicated that the mechanism of action of pyrrocidine A is different from other clinical anticancer drugs, and this compound broadly inhibited the growth of various cancer cell lines. The apoptosis induction by pyrrocidine A was suppressed by both N-acetyl-l-cysteine (NAC) and glutathione, both of which are thiol-containing antioxidants. Furthermore, pyrrocidine A directly bound to N-acetyl-l-cysteine methyl ester (NACM) through the Michael-type addition at the α,β-unsaturated carbonyl group and was detected by HPLC and liquid chromatography-ESI-tandem MS (LC-ESI-MS/MS) analyses. This indicates that this moiety is crucial for the potent apoptosis-inducing activity of pyrrocidine A.

  3. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells.

    Science.gov (United States)

    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.

  4. 冬凌草甲素联合丙戊酸钠对HL-60细胞的生长抑制和诱导凋亡作用研究%Growth inhibition and apoptosis induced by oridonin combined with valproic acid in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    邹慧娟; 姜国胜

    2011-01-01

    Objective To investigate the apoptosis-inducing effect of oridonin combined with valproic acid( VPA)on leukemic cell line HL-60,and study the feasibility of oridonin combined with VPA to be used in clinical practice. Methods Oridonin of 6-12 μmol/L combined with VPA of 0. 5-1 mmol/L were added in exponential growth HL-60 cells respectively. Cell count assays were used to measure the growth inhibitory effect of oridonin combined with VPA or alone. Flow cytometry was used to evaluate apoptosis with Annexin V and propidium iodide (PI) double staining. Results Combined use of oridonin and VPA could synergistically inactivate HL-60 cells,and inhibit the cell proliferation and induce apoptosis in a dose-and time-dependent manner (P < 0.05 ). Conclusion Oridonin has a synergistic effect combined with VPA. Oridonin has a promising prospect in clinical use of leukemia.%目的 研究中药单体冬凌草甲素(ORI)联合组蛋白去乙酰化酶抑制剂丙戊酸钠(VPA)诱导急性早幼粒白血病细胞株HL-60凋亡,探讨其应用于临床的可行性.方法 在对数生长期的HL-60细胞中分别加入6~12 la,mol/L的ORI和0.5~1 mmol/L的VPA,采用细胞计数法测定ORI和VPA单独和联合应用时对细胞的生长抑制,并用Annexin V/PI双标法流式细胞术检测细胞凋亡.结果 ORI联合VPA可协同降低HL-60细胞活力,抑制细胞增殖,诱导细胞凋亡,比单独用药凋亡作用更为显著(P<0.05).结论 ORI和VPA具有协同作用,能高效杀灭白血病细胞.ORI和VPA是一种有望应用于白血病临床治疗的新型生物制剂.

  5. 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.

    Science.gov (United States)

    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.

  6. 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.

    Science.gov (United States)

    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 (pHL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway.

  7. 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.

    Science.gov (United States)

    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.

  8. Enhancement Effect of human acute myeloid leukemia HL-60 cell line supernatant on Angiogenesis of Human Umbilical Vein Endothelial Cell%人急性白血病细胞株HL-60细胞培养上清液对人脐静脉内皮细胞血管形成能力的影响

    Institute of Scientific and Technical Information of China (English)

    潘小兵; 陈建萍

    2011-01-01

    目的 研究人急性白血病细胞株(HL-60)上清液对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVECs)血管形成能力的影响.方法 以单独培养HUVECs为对照组,以HUVECs加HL-60细胞培养上清为实验组.采用MTT比色法分别在24h、48h、72h检测两组中HUVECs的吸光值;采用RT-PCR技术检测两组中HUVECs周期蛋白E(cyclinE )mRNA及血管内皮生长因子(VEGF)mRNA的表达情况.结果 实验组与对照组相比HUVECs增殖速度加快,72小时后差异有显著性意义(p〈0.05).72小时后,实验组中HUVECs CyclinE mRNA及VEGFmRNA表达明显增加,与对照组相比差异具有显著性意义(p〈0.01).结论 HL-60细胞培养上清液能够增强HUVECs的血管形成能力.

  9. Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells.

    Directory of Open Access Journals (Sweden)

    Islam Husain

    Full Text Available 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.

  10. Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1 receptors and is an incomplete secretagogue.

    Science.gov (United States)

    Seifert, R; Höer, A; Offermanns, S; Buschauer, A; Schunack, W

    1992-08-01

    Human neutrophils and dibutyryl-cAMP (Bt2cAMP)-differentiated HL-60 cells possess receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), which mediate activation of phospholipase C, with subsequent increase in cytosolic Ca2+ concentration ([Ca2+]i) and activation of specific cell functions. In many cell types, histamine, via H1 receptors, activates phospholipase C, but it is unknown whether neutrophilic cells possess functional H1 receptors. We compared the effects of histamine with those of fMet-Leu-Phe on activation of these cells. In Bt2cAMP-differentiated HL-60 cells, substances increased [Ca2+]i in the effectiveness order fMet-Leu-Phe greater than histamine greater than betahistine. Pertussis toxin diminished fMet-Leu-Phe-induced rises in [Ca2+]i to a greater extent than those induced by histamine. H1 but not H2 antagonists inhibited histamine- and betahistine-induced rises in [Ca2+]i. fMet-Leu-Phe and histamine activated phospholipase C and increased [Ca2+]i through release of Ca2+ from intracellular stores and sustained influx of Ca2+ from the extracellular space. The substances also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). The stimulatory effects of histamine on [Ca2+]i were more sensitive to inhibition by 4 beta-phorbol 12-myristate 13-acetate than were those of fMet-Leu-Phe. Unlike fMet-Leu-Phe, histamine did not activate superoxide anion formation, release of beta-glucuronidase, and tyrosine phosphorylation. In neutrophils, histamine and betahistine did not induce rises in [Ca2+]i. Our data show that (i) in Bt2cAMP-differentiated HL-60 cells, histamine increases [Ca2+]i via H1 receptors coupled to pertussis toxin-sensitive and possibly, pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (ii) histamine activates nonselective cation channels, and (iii

  11. 黄芩苷对高致瘤HL-60细胞裸鼠移植瘤模型的体内抑瘤作用和机制探讨%Effects of Baicalin on HL-60 Cell Xenografts in Nude Mice and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    郑静; 胡建达; 黄毅; 陈英玉; 李静; 陈步远

    2012-01-01

    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% NaHCO3), 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 PDK/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

  12. Effects of co-treatment with puerariae radix flavones and arsenic trioxide on proliferation and apoptosis of Kasumi-1 cells and HL-60 cells%葛根总黄酮联合三氧化二砷对Kasumi-1和HL-60细胞增殖和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    唐宇宏; 邵化敏; 朱红青; 姜鹏君; 季建敏; 沈群

    2011-01-01

    目的 探讨葛根总黄酮(PRF)联合三氧化二砷(ATO)对M2型急性髓系白血病细胞株Kasumi-1和HL-60细胞增殖和凋亡的影响.方法 以100 μg/mL PRF和1μmoL/L ATO联合处理Kasumi-1和HL-60细胞48 h(RRF+ ATO组),MTT法检测细胞增殖抑制率,Annexin V-FITC/PI双染法流式细胞术检测细胞早期凋亡率,Real-Time PCR检测凋亡抑制基因Bcl-2、Survivin mRNA表达,Western blotting检测细胞凋亡相关蛋白酶Caspase-3、-8、-9的表达.设立单用100μg/mL PRF的PRF处理组和空白对照组.结果 RRF+ ATO组Kasum-1细胞增殖抑制率、早期凋亡率以及Caspase-3、-9表达均显著高于PRF处理组(P<0.05),细胞中Bcl-2 mRNA表达的下调趋势明显.RRF+ ATO组HL-60细胞各项指标检测结果 与PRF处理组比较差异均无统计学意义(P>0.05).结论 PRF联合ATO能有效抑制Kasumi-1细胞增殖并诱导早期凋亡,而对HL-60细胞则无明显影响.%Objective To investigate the effects of co-treatment with puerariae radix flavones (PRF) and arsenic trioxide ( ATO) on proliferation and apoptosis of Kasumi-1 cells and HL-60 cells of acute myeloid leukemia Ml. Methods Kasumi-1 cells and HL-60 cells were treated with 100 jig/mL PRF and 1 funol/L ATO for 48 h (RRF + ATO group). MTT assay was employed to measure the inhibition rate of cell proliferation, Annexin V-FITC/PI double staining was used to determine the early apoptosis rate by flow cytometry, the expression of Bcl-2 and Survivin mRNA was detected by Real-Time PCR, and the expression of Caspase-3, Caspase-8 and Caspase-9 protein was detected by Western blotting. Cells only treated with 100 (ig/mL PRF were served as PRF group, and blank control group was also established. Results The inhibition rate of cell proliferation, early apoptosis rate and expression of Caspase-3 and Caspase-9 protein of Kasumi-1 cells in RRF + ATO group were significantly higher than those in PRF group (P 0.05). Conclusion Co-treatment with PRF and ATO can effectively

  13. Rab27a is essential for the formation of neutrophil extracellular traps (NETs in neutrophil-like differentiated HL60 cells.

    Directory of Open Access Journals (Sweden)

    Tatsumi Kawakami

    Full Text Available Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs. NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an

  14. Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells

    DEFF Research Database (Denmark)

    Winzen, R; Wallach, D; Engelmann, H;

    1992-01-01

    Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol d...

  15. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    Science.gov (United States)

    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.

  16. Berberine and a Berberis lycium extract inactivate Cdc25A and induce {alpha}-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Musa [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Giessrigl, Benedikt; Vonach, Caroline; Madlener, Sibylle [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Prinz, Sonja [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Herbaceck, Irene; Hoelzl, Christine [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Bauer, Sabine; Viola, Katharina [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Mikulits, Wolfgang [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Quereshi, Rizwana Aleem [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Knasmueller, Siegfried; Grusch, Michael [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Kopp, Brigitte [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Krupitza, Georg, E-mail: georg.krupitza@meduniwien.ac.at [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2010-01-05

    Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H{sub 2}O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 {mu}g BuOH extract (that was gained from 1 mg dried root) contained 2.0 {mu}g berberine and 0.3 {mu}g/ml palmatine. 1.2 {mu}g/ml berberine inhibited cell proliferation significantly, while 0.5 {mu}g/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of {alpha}-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.

  17. Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells.

    Science.gov (United States)

    Kanayasu-Toyoda, Toshie; Yamaguchi, Teruhide; Oshizawa, Tadashi; Kogi, Mieko; Uchida, Eriko; Hayakawa, Takao

    2002-09-01

    Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.

  18. EGCG抑制HL-60细胞增殖作用的机制研究%Study of EGCG Inhibited Proliferation of HL-150 Cells

    Institute of Scientific and Technical Information of China (English)

    欧阳琳; 姜浩

    2011-01-01

    目的 探讨EGCG抑制白血病HL-60细胞增殖的作用机制.方法 采用软琼脂集落形成实验和绘制细胞生长曲线,检测EGCG对HL-60细胞增殖的影响;FCM及DNA凝胶电泳法检测ECCG处理HL-60细胞后的凋亡情况;实时荧光定量RT-PCR和Wester blotting检测AKT1的表达.结果 细胞生长曲线和软琼脂集落形成实验结果均显示EGCG呈浓度依赖性抑制HL-60细胞的增殖(P<0.05);FCM及DNA凝胶电泳法结果显示EGCG呈浓度依赖性诱导HL-60细胞的凋亡(P<0.05).实时荧光定量RT-PCR和Wester blotting结果显示EGCG浓度升高,HL-60细胞中AKT1的表达降低.结论 EGCG可能通过下调AKT1的表达,促进细胞凋亡,发挥其对HL-60细胞增殖的抑制作用.

  19. Desmosdumotin-C A环衍生物TEP诱导HL-60细胞凋亡作用机制研究%The Effects and Mechanisms of an A ring Derivative of Desmosdumotin-C (TEP) induced Apoptosis in HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    郭宏举; 向卓; 常李荣; 史宁; 梁海; 郑凯音; 吴久鸿

    2016-01-01

    This study focused on the effects of TEP,an A ring derivative of desmosdumotin-C,on the human acute leukemia HL-60 cell apoptosis,and the underlying mechanisms.TEP-induced apoptosis and its effect on Fas,FasL,Bax,Bcl-2 expression were measured by flow cytometry,and the morphological changes of the apoptosis cells were further observed by transmission electron microscop.The results showed that 24 h after 40 μg/mL TEP treatment,the typical apoptotic morphological changes were induced in HL-60 cell.Meantime,40 μg/mL TEP significantly increased Fas,FasL,and Bax expression (P < 0.05),and decreased Bcl-2 expression (P < 0.05).All the results suggested that TEP effectively induced apoptosis in HL-60 cells,and the mechanism may be involved in upregulating Fas,FasL,Bax expression,and reducing Bcl-2 expression.%本研究主要探讨毛叶假鹰爪素C(Desmosdumotin C)A环衍生物TEP诱导人急性白血病HL-60细胞凋亡作用及机制.流式细胞技术检测TEP诱导细胞凋亡及其对凋亡细胞中Fas、FasL、Bax、Bcl-2表达率的影响,透射电镜观察凋亡细胞形态学改变.结果显示40μg/mL TEP作用细胞24h后,细胞可呈现典型的凋亡形态学变化;40 μg/mL TEP可明显提高凋亡细胞中Fas、FasL、Bax的表达(P<0.05),并可明显降低抗凋亡细胞Bcl-2的表达(P<0.05).以上实验结果表明毛叶假鹰爪素C(Desmosdumotin C,Des C)A环衍生物TEP可有效地诱导HL-60细胞凋亡,其作用机制可能与上调Fas、FasL、Bax表达以及下调Bcl-2表达有关.

  20. Safrole induces G0/G1 phase arrest via inhibition of cyclin E and provokes apoptosis through endoplasmic reticulum stress and mitochondrion-dependent pathways in human leukemia HL-60 cells.

    Science.gov (United States)

    Yu, Chun-Shu; Huang, An-Cheng; Yang, Jai-Sing; Yu, Chien-Chih; Lin, Chin-Chung; Chung, Hsiung-Kwang; Huang, Yi-Ping; Chueh, Fu-Shin; Chung, Jing-Gung

    2012-05-01

    Safrole, a component of Piper betle inflorescence, is a carcinogen which has been demonstrated to induce apoptosis on human oral cancer HSC-3 cells in vitro and to inhibit HSC-3 cells in xenograft tumor cells in vivo. In our previous study, safrole promoted phagocytosis by macrophages and natural killer cell cytotoxicity in normal BALB/c mice. The cytotoxic effects of safrole on HL-60 cells were investigated by using flow cytometric analysis, comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting and confocal laser microscopy. The obtained results indicate that safrole induced a cytotoxic response through reducing the percentage of viable cells and induction of apoptosis in HL-60 cells in a dose-dependent manner. DAPI staining and comet assay also showed that safrole induced apoptosis (chromatin condensation) and DNA damage in HL-60 cells. The flow cytometric assay showed that safrole increased the production of reactive oxygen species (ROS) and Ca(2+) and reduced the mitochondrial membrane potential in HL-60 cells. Safrole enhanced the levels of the pro-apoptotic protein BAX, inhibited those of the anti-apoptotic protein BCL-2 and promoted the levels of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in HL-60 cells. Furthermore, safrole promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and of activating transcription factor 6α (ATF-6α). Based on these findings, we suggest that safrole-induced apoptosis in HL-60 cells is mediated through the ER stress and intrinsic signaling pathways.

  1. 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.

    Science.gov (United States)

    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.

  2. 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.

    Science.gov (United States)

    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.

  3. Programmed Cell Death Induced by (−)-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

    Science.gov (United States)

    Fuwa, Haruhiko; Sato, Mizuho; Sasaki, Makoto

    2014-01-01

    (+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion. PMID:25419998

  4. Programmed Cell Death Induced by (−-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

    Directory of Open Access Journals (Sweden)

    Haruhiko Fuwa

    2014-11-01

    Full Text Available (+-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−-8,9-dehydroneopeltolide (8,9-DNP, a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe-fluoromethylketone (zVAD. Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose polymerase (PARP. These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF, although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.

  5. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    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.

  6. Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

    Science.gov (United States)

    Pae, H O; Oh, G S; Choi, B M; Seo, E A; Oh, H; Shin, M K; Kim, T H; Kwon, T O; Chung, H T

    2003-02-01

    Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.

  7. Stimulation of proliferation of HL60 cells by low concentrations of 12-O-tetradecanoylphorbol-13-acetate and its relationship to the mitogenic effects of insulin

    Energy Technology Data Exchange (ETDEWEB)

    Trayner, I.D.; Clemens, M.J. (St. George' s Hospital Medical School, London (United Kingdom))

    1992-03-01

    The effects of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived in insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine by three- to fourfold, as compared to a seven fold stimulation by insulin. The proliferative response to low TPA concentrations provides a useful model for dissecting the signaling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.

  8. 27. Molecular spectra of acrylamide-induced mutation at hprt locus in human promyelocytic leukemia HL-60 and NB4. cell lines

    Institute of Scientific and Technical Information of China (English)

    刘胜学; 曹佳; 杨梦苏; 方志俊; 安辉

    2001-01-01

    The genotoxicity of acrylamide was investigated by methods of single cell clone culturing, two-way screening count, multiplex PCR amplification and electrophoresis technique. Acrylamide only showed clear mutagenesis until dose raised 700 mg*L-1 in HL-60 cells. The most frequent spontaneous mutation were point mutation (≥90.0%) and acrylamide-induced mutation mainly included partial deletion and point mutation (respectively 40.0%~66.7%/33.3%~60.0%). Total gene deletion wasn't discovered in both of cells. There were deletion mutation in all exons of hprt gene(except 7/8 exon), and toward the 3' end of the hprt gene. The most frequent acrylamide-induced mutation were point mutation and single exon deletion (93.3%/86.1%). There were not clear difference in both of cells. The results suggest that the spectra of spontaneous and acrylamide-induced mutants were different. and the smaller changes in genetic structure have something to do with mechanism.

  9. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Growth inhibition and apoptosis induction of Scutellaria luteo-coerulea Bornm. & Sint. on leukemia cancer cell lines K562 and HL-60

    Directory of Open Access Journals (Sweden)

    Mahsa Motaez

    2015-10-01

    Full Text Available Objective: Scutellaria (Lamiaceae has been implicated for medicinal purposes both in modern and traditional medicine. Some species of the genus Scutellaria has extensively been studied for anticancer activity. Scutellaria luteo-coerulea (S. luteo-coerulea is one of the Iranian species of the genus Scutellaria. Materials and Methods: In the present study, cytotoxic and apoptogenic properties of CH2Cl2, EtOAc, n-BuOH, and H2O fractions of S. luteo-coerulea were investigated on K562. Moreover, HL-60. DNA fragmentation in apoptotic cells were determined by propidium iodide (PI staining (sub-G1 peak. Results: Scutellaria luteo-coerulea inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. luteo-coerulea, the CH2Cl2 fraction was found to be the most cytotoxic one among others. Sub-G1 peak in flow cytometry histogram of treated cells suggested the induction of apoptosis in S. luteo-coerulea. Conclusion: Scutellaria luteo-coerulea could be a novel candidate for further analytical elucidation in respect to fine major components responsible for the cytotoxic effect of the plant also clinical evaluations.

  11. Inhibitory effect of compound 6F isolated from Pteris semipinnata L. on the activity of protein kinase C in HL-60 cells%半边旗提取物6F抑制HL-60细胞蛋白激酶C活性

    Institute of Scientific and Technical Information of China (English)

    何承伟; 梁念慈; 莫丽儿; 李金华; 张晓

    2003-01-01

    目的探讨半边旗提取物6F的细胞毒作用及诱导DNA片段化与蛋白激酶C(PKC)信号转导途径的关系,检测6F对PKC活性的影响.方法受试对象为HL-60细胞,超速离心法获得的胞液(可溶部分)及颗粒(不可溶部分,包括细胞膜系统及胞核)部分用作PKC活性测定.经0.4 g@L-1磷脂酰丝氨酸,0.04g@L-1甘油二油酸酯激动剂作用酶粗提物后,用液体闪烁计数仪计数[γ-32P]ATP参入外源底物的量以测定PKC活性.MTT法测定HL-60细胞的活力,二苯胺法测定6F诱导DNA片段化程度.结果在所测试的浓度范围内(0.5~312 μmol@L-1),化合物6F显著抑制胞液及颗粒部分PKC活性,最大抑制率达88.6%,呈浓度依赖关系(胞液部分r=0.781,P<0.05,颗粒部分r=0.931,P<0.01).6F诱导HL-60细胞DNA片段化及对细胞的毒性作用可被具有致癌作用的PKC激活剂肉豆蔻酸酯(PMA,浓度为65 nmol@L-1)拮抗,抑制率分别是30%和44%(P<0.01).PMA单独用使HL-60细胞线粒体将MTT还原为甲臜的能力增强14%(P<0.01),即增强细胞活力.结论化合物6F是PKC的抑制剂.6F对HL-60细胞DNA片段化的诱导作用及其细胞毒作用至少可部分归因于其对PKC活性的抑制作用.%AIM To investigate whether compotmd 6F isolated from Pteris semipinnata L inhibits the activity of protein kinase C (PKC) and whether the DNA fragment induction and cytotoxicity of 6F on HL-60 cells relate to PKC signaling pathway. METHODS HL-60 cells were used as in vitro model and its cytosol (soluble sample)and particle( insoluble sample including membrane system and nuclei) fractions obtained by ultracentrifugation were used as samples for PKC assay. PKC activity was measured by incorporation of [ γ-32p]ATP into exogenous substrate after stimulated by phosphatidylserine and diolein.Diphenylamine assay and MTT staining methods were applied for DNA fragmentation detection and cytotoxicity assay, respectively. RESUL TS PKC activities in cytosol and particle

  12. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    Directory of Open Access Journals (Sweden)

    Holly A Jensen

    Full Text Available 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

  13. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    Science.gov (United States)

    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

  14. Toxicological effects of three types of silver nanoparticles and their salt precursors acting on human U-937 and HL-60 cells.

    Science.gov (United States)

    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.

  15. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    Science.gov (United States)

    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

  16. Cytotoxic and DNA-damaging effects of methyl tert-butyl ether and its metabolites on HL-60 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tang, G.H. [Xian Medical Univ. (China); Shen, Y.; Shen, H.M. [National Univ. of Singapore (Singapore)] [and others

    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.

  17. Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor-selective retinoic acid analogs.

    Science.gov (United States)

    Brooks, S C; Kazmer, S; Levin, A A; Yen, A

    1996-01-01

    The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

  18. Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-10-24

    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 11,579 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.

  19. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  20. A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

    Science.gov (United States)

    Orbán, Erika; Manea, Marilena; Marquadt, Andreas; Bánóczi, Zoltán; Csík, Gabriella; Fellinger, Erzsébet; Bosze, Szilvia; Hudecz, Ferenc

    2011-10-19

    Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

  1. 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}

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Cell differentiation and down regulation of nm23 gene expression in HL-60 cells induced by 1,25 dihydroxyvitamin D3%1,25-二羟基维生素D3诱导HL-60细胞分化并下调nm23基因的表达

    Institute of Scientific and Technical Information of China (English)

    朱宁希; 吕庆华; 周莹; 虞荣喜

    2001-01-01

    目的观察1,25-二羟基维生素D3[1,25(OH)2D3]对白血病细胞株HL-60的诱导分化作用并探讨其机制.方法通过MTT试验、细胞形态观察、表面标志分析和硝基四氮唑蓝(NBT)还原反应, 观察1,25(OH)2D3作用后HL-60细胞增殖的改变和分化情况;通过流式细胞仪和RT-PCR检测细胞周期和nm23基因表达的变化.结果 HL-60细胞经1,25(OH)2D3作用后增殖受抑制、向成熟单核细胞系分化、细胞被阻滞在G1期、nm23基因的表达下调.结论 1,25(OH)2D3对HL-60细胞株具有诱导分化作用,其作用机制可能与下调nm23基因的表达有关.

  3. 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.

    Science.gov (United States)

    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.

  4. An antisense oligodeoxynucleotide targeted against the type II sub. beta. regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

    The type II{sub {beta}} regulatory subunit of cAMP-dependent protein kinase (RII{sub {beta}}) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII{sub {beta}} antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII{sub {beta}} antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII{sub {beta}} protein. Exposure to RII{sub {beta}} sense, RI{sub {alpha}} and RII{sub {alpha}} antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII{sub {beta}} regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

  5. 全反式维甲酸与紫杉醇、阿霉素联合诱导HL-60细胞凋亡的研究%Studies on the apoptosis using ATRA in combination with taxol and ADR in HL-60 cell lines

    Institute of Scientific and Technical Information of China (English)

    尤学芬; 丁润生; 谢玉娟; 姚登福; 陆德炎

    2007-01-01

    目的:探讨全反式维甲酸(ATRA)分别与紫杉醇(Taxol)、阿霉素(ADR)单独或联合孵育HL-60细胞株能否增强凋亡诱导作用,并进一步研究bcl-2基因及家族bax、bcl-x基因在此过程中的作用.方法:①用台盼兰拒染法观察细胞生长活力;②在光镜和电镜下观察细胞形态变化;③用流式细胞仪(FCM)检测药物孵育前后细胞凋亡率(AP)的变化;④用RT-PCR法检测药物孵育前后bcl-2基因及其家族bax、bcl-x基因的表达水平.结果:①ATRA能增强Taxol和ADR对HL-60细胞的生长抑制作用;②ATRA能增强HL-60细胞对Taxol和ADR诱导的细胞凋亡;③在ATRA与Taxol、ADR联合诱导的HL-60细胞凋亡中,bcl-2、bcl-xl基因表达下降,bax、bcl-xs基因表达增加.结论:ATRA能增加Taxol和ADR对HL-60细胞的生长抑制作用及凋亡诱导作用,bcl-2基因及家族bax、bcl-x基因参与了ATRA与Taxol、ADR联合诱导细胞凋亡的调控.

  6. Effect of an Intermediate-Frequency Magnetic Field of 23 kHz at 2 mT on Chemotaxis and Phagocytosis in Neutrophil-Like Differentiated Human HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Shin Koyama

    2014-09-01

    Full Text Available Public concerns about potential health risks of intermediate-frequency (IF electromagnetic fields are increasing, especially as the use of induction-heating cooktops has spread extensively in Japan and Europe. In order to investigate the properties of IF electromagnetic fields, we examined the effect of exposure to a 23-kHz IF magnetic field of 2 mT for 2, 3, or 4 h on neutrophil chemotaxis and phagocytosis using differentiated human HL-60 cells. Compared with sham exposure, exposure to the IF magnetic field had no effect on neutrophil chemotaxis or phagocytosis. Previous studies demonstrated that exposure to a 23-kHz IF magnetic field of 2 mT (about 74-times the maximum value recommended by the International Commission for Nonionizing Radiation Protection guidelines may affect the first-line immune responses in humans. To our knowledge, this is the first study to evaluate the effects of IF magnetic fields on cellular immune responses. We found that exposure to an IF magnetic field of 2 mT has minimal if any effect on either the chemotaxis or phagocytic activity of neutrophil-like human HL-60 cells.

  7. 鼠尾草酸联合1,25-二羟基维生素D3诱导HL-60细胞分化作用的研究%The differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3 combined with carnosic acid

    Institute of Scientific and Technical Information of China (English)

    安慧萍; 任立红; 陈娟娟

    2007-01-01

    目的 研究鼠尾草酸与1,25-二羟基维生素D3[1,25(OH)2D3]联合应用对HL-60细胞分化的影响.方法 通过四唑氮蓝(MTT)法观察细胞生长,光学显微镜观察细胞形态,应用流式细胞仪测定细胞周期及成熟单核细胞分化标记CD14的表达,观察联合应用鼠尾草酸与低浓度1,25(OH)2D3对HL-60细胞的影响.结果 低浓度1,25(OH)2D3(1nmol/L)抑制HL-60细胞增殖、诱导细胞分化的作用与对照组比较无统计学意义(P>0.05).同浓度1,25(OH)2D3与10μmol/L鼠尾草酸联合处理HL-60细胞72h后,细胞形态具有成熟单核细胞特征、细胞增殖明显受抑制、细胞阻滞于G0/G1期、CD14的表达率为57.624%;其作用效应与对照组比较有显著性差异(P<0.01),与单独应用高浓度1,25(OH)2D3(100nmol/L)组比较作用相似,无统计学意义(P>0.05).结论 鼠尾草酸可增强1,25(OH)2D3对HL-60细胞的诱导分化、抑制增殖作用.

  8. Enhancement of differentiation induction of HL-60 cells by 1,25-dihydroxyvitaminD3 in combination with carnosic acid%鼠尾草酸增加1,25(OH)2D3诱导HL-60细胞分化及其机制研究

    Institute of Scientific and Technical Information of China (English)

    任立红; 陈娟娟; 安慧萍

    2008-01-01

    目的 已证实1,25(OH)2D3可以诱导HL-60细胞向成熟单核细胞分化,但其不足之处是高钙血症和形成耐药株.该实验应用鼠尾草酸(CA)联合1,25(OH)2D3研究对HL-60细胞分化的影响及引起细胞内活性氧(ROS)水平和细胞内钙离子浓度的改变.方法 应用鼠尾草酸,1,25(OH)2D3单独和联合应用处理HL-60细胞,共分为5组:空白对照组;低浓度1,25(OH)2D3(1 nmol/L)组、鼠尾草酸组;联合处理组(10 μmol/L鼠尾草酸和1 nmol/L 1,25(OH)2D3);高浓度1,25(OH)2D3(100 nmol/L)组.每24 h监测1次,通过四唑氮蓝(MTT)法观察细胞生长,共72 h;收集处理后72 h的HL-60细胞,通过光学显微镜观察细胞形态,用流式细胞仪(FCM)检测不同处理组细胞周期及单核细胞分化标记CD14的表达,ROS和细胞内钙离子浓度.结果 72 h后,联合处理组HL-60细胞与空白对照组相比,细胞增殖明显受抑制(Ab=0.560±0.020;P0.05),但与高浓度1,25(OH)2D3组相比细胞内钙离子水平明显下降(P<0.01).结论 鼠尾草酸可增强1,25(OH)2D3对HL-60细胞的诱导分化、增强抑制HL-60细胞增殖作用,细胞阻滞于G0/G1期,细胞内活性氧水平降低,这可能与细胞的诱导分化机制有关,且这一联合作用不增高细胞内钙离子水平.

  9. Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin%Bcl-2反义核酸增强HL-60、K562细胞对柔红霉素敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    雷小勇; 张洹; 何冬梅

    2003-01-01

    目的:研究bcl-2反义寡核苷酸作用后,白血病细胞株HL60、K562细胞对柔红霉素(DNR)敏感性的影响.方法:MTT法测HL60、K562细胞中DNR的半数抑制率(IC50);免疫荧光标记观测细胞Bcl-2蛋白水平;用Hoechest33258和碘化丙锭双染色法及流式细胞仪检测细胞凋亡.结果:靶向bcl-2 mRNA蛋白编码区反义寡核苷酸(AS-ODN1)和靶向翻译起始区的反义寡核苷酸(AS-ODN2)分别与DNR联合作用于HL60细胞后DNR的IC50值分别为0.124±0.011、0.149±0.012,分别与不加寡核苷酸组DNR的IC50值(0.173±0.021)或无义寡核苷酸联用DNR的IC50值(0.180±0.023)相比有显著差异,P<0.05.AS-ODN1和AS-ODN2分别与DNR联合作用于K562细胞后DNR的IC50值分别为0.078±0.007、0.079±0.008,分别与不加寡核苷酸组DNR的IC50值(0.106±0.011)或无义寡核苷酸联用DNR的IC50值(0.107±0.012)相比有显著差异,P<0.05.AS-ODN1和AS-ODN2分别与DNR同时作用于HL60或K562细胞后抑制Bcl-2蛋白表达及诱导细胞凋亡率分别与单用DNR或无义寡核苷酸联用DNR相比有显著差异,P<0.05.与AS-ODN2比较,AS-ODN1提高HL60细胞对DNR的敏感性作用要强些(P<0.05).结论:靶向bcl-2 mRNA蛋白编码区反义寡核苷酸和靶向翻译起始区的反义寡核苷酸能增强HL60和K562细胞对DNR的敏感性.

  10. 扶正祛邪含药血清对白血病HL60/VCR细胞Bcl-2表达水平的影响%Effects and reversal mechanism of Fuzheng Quxie Prescription serum to the apoptosis gene (Bcl-2) of the human leukemia HL60/VCR cells

    Institute of Scientific and Technical Information of China (English)

    李秀军; 严鲁萍; 姚宇红

    2013-01-01

    目的:观察扶正祛邪中药复方含药血清对长春新碱诱导的急性早幼粒白血病耐药细胞株H L60/VCR0细胞耐药基因Bcl-2表达水平的影响.方法:采用流式细胞术检测法,选择HL60/VCR细胞为靶细胞,观察不同浓度扶正祛邪含药血清对其耐药基因Bcl-2表达的影响.结果:扶正祛邪含药血清对HL60/VCR细胞内凋亡抑制基因Bcl-2的表达有明显抑制作用,其中含药血清高、中、低剂量组荧光强度依次为401.67±0.86、453.69±0.40、516.66±0.40.结论:扶正祛邪复方中药抗白血病多药耐药的作用可能与下调Bcl-2的表达有关.

  11. 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

    Directory of Open Access Journals (Sweden)

    Alessia Arcaro

    2015-01-01

    Full Text Available 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.

  12. 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.

    Science.gov (United States)

    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.

  13. (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

    Energy Technology Data Exchange (ETDEWEB)

    Magalhães, Hemerson I.F. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Paraíba (Brazil); Wilke, Diego V. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Bezerra, Daniel P., E-mail: danielpbezerra@gmail.com [Centro de Pesquisa Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia (Brazil); Cavalcanti, Bruno C. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson [Centro de Ciências Exatas e Tecnológicas (Laboratório LP4), Universidade Federal do Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul (Brazil); Moraes, Manoel O.; Diniz-Filho, Jairo [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Pessoa, Claudia, E-mail: c_pessoa@yahoo.com [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil)

    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.

  14. Exposure to p,p′-DDE Induces Morphological Changes and Activation of the PKCα-p38-C/EBPβ Pathway in Human Promyelocytic HL-60 Cells

    Science.gov (United States)

    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

  15. Exposure to p,p′-DDE Induces Morphological Changes and Activation of the PKCα-p38-C/EBPβ Pathway in Human Promyelocytic HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Nallely A. Torres-Avilés

    2016-01-01

    Full Text Available 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.

  16. 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.

    Science.gov (United States)

    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.

  17. Transcriptional targeting of sphingosine-1- phosphate receptor S1P2 by epigallocatechin- 3-gallate prevents sphingosine-1-phosphate- mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

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    Chokor R

    2014-05-01

    Full Text Available Rima Chokor, Sylvie Lamy, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de recherche BIOMED, Département de Chimie, Université du Québec à Montréal, Montreal, QC, CanadaBackground: Macrophage chemotaxis followed by blood–brain barrier transendothelial migration is believed to be associated with inflammation in the central nervous system. Antineuroinflammatory strategies have identified the dietary-derived epigallocatechin-3-gallate (EGCG as an efficient agent to prevent neuroinflammation-associated neurodegenerative diseases by targeting proinflammatory mediator signaling.Methods: Given that high levels of sphingosine kinase and its product, sphingosine-1-phosphate (S1P, are present in brain tumors, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR and immunoblotting to test whether EGCG may impact on S1P receptor gene expression and prevent S1P response in undifferentiated and in terminally differentiated macrophages.Results: Promyelomonocytic human leukemia (HL-60 cells were differentiated into macrophages, and S1P triggered phosphorylation in extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and P38 mitogen-activated protein kinase (MAPK intracellular signaling, as shown by Western blot analysis. Pretreatment of cells with EGCG prior to differentiation inhibited the response to S1P in all three pathways, while EGCG abrogated P38 MAPK phosphorylation when present only during differentiation. Terminally-differentiated macrophages were, however, insensitive to EGCG treatment. Using qRT-PCR, gene expression of the S1P receptors S1P1, S1P2, and S1P5 was predominantly induced in terminally-differentiated macrophages, while the S1P2 was decreased by EGCG treatment.Conclusion: Our data suggest that diet-derived EGCG achieves efficient effects as a preventive agent, targeting signaling pathways prior to cell terminal differentiation. Such properties could impact on cell chemotaxis

  18. 铁剥夺和铁超负荷对白血病细胞株HL-60凋亡的影响机制%The influence of iron deprivation and rich iron on the apoptosis of human Leukemia-60 cells

    Institute of Scientific and Technical Information of China (English)

    王叨; 李四保; 刘玉峰

    2012-01-01

    目的 探讨铁剥夺和铰超负荷对白血病细胞株HL-60细胞凋亡的影响及其机制,为临床采用铁剥夺策略治疗或辅助治疗白血病提供理论依据.方法 在HL-60细胞培养基中分别加入不同浓度的去铁胺(DFO)或三氯化铁(FeCl3),造成细胞内铁剥夺或铁超负荷状态,采取噻唑蓝(MTT)法、DNA原位末端标记染色法(TUNEL)、免疫组化法检测铁剥夺和铁超负荷状态下HL-60细胞活力、凋亡率、细胞色素C(Cyt C)阳性细胞率.结果 DFO组细胞活力呈明显下降趋势,凋亡率呈显著上升趋势;FeCl3组细胞活力和凋亡率与对照组相比呈下降趋势;DFO组细胞胞浆内Cyt C阳性细胞率与对照组相比明显升高;而FeCl3组细胞浆内CytC阳性细胞率与对照组相比无明显差异.结论 铁剥夺可促进线粒体释放Cyt C,诱导HL-60细胞凋亡;铁超负荷对线粒体释放Cyt C无直接影响作用.%Objective To explore the influence and mechanism of iron deprivation and rich iron on the apoptotic process of human Leukemia-60 ( HL-60) cells, and provide the theoretical basis for the clinical therapy. Methods HL-60 cells were cultivated with different concentration of DFO and FeCl3 to establish intracellular iron-deprivated and rich-iron states. Cell viability, apoptotic rate and the positive rate of Cyt C were detected by MTT, TUNEL and immunohistochemistry. Results The cell viability in DFO-treated group decreased, while the apoptotic rate increased dramatically. In contrast, an opposite result was obtained in FeCL,-treated group. The positive rate of Cyt C in DFO-treated group was elevated, but no difference was found in FeCl3-treated group. Conclusions Iron deprivation induces apoptosis of HL-60 cells by releasing Cyt C from mitochondria, while rich iron has no direct impact on it. The mechanism of HL-60 cell proliferation might be related with the apoptosis-related genes in the upstream of the mitochondrial pathway.

  19. The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1 as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

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    Martin Jakab

    2014-10-01

    Full Text Available Background/Aims: The ATP12A gene codes for a non-gastric H+/K+ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H+/K+ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H+/K+ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi after acute intracellular acid load (NH4Cl prepulsing. Mean cell volumes (MCV and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle, to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining and differentiation (CD86 expression. Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H+/K+ ATPase inhibitor SCH28080 (100 µM diminishes K+-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently

  20. Antitumor Effect of BCG on Growth of Transplanted Human Myeloid Leukemia HL-60 Cells in Nude Mice%卡介苗抑制裸鼠白血病移植瘤生长及抗肿瘤的实验研究

    Institute of Scientific and Technical Information of China (English)

    王媛媛; 王玲珍; 孙立荣

    2011-01-01

    This study was purposed to explore the anti-leukemia effect of bacillus calmette-guerin vaccine (BCG) on the human myeloid leukemia cell xenograft models.An animal model was established by inoculating the human myeloid leukemia HL-60 cells into the BALB/c (8 - 10 weeks of age) nude mice.The mice were randomly divided into two groups: control group and experimental group.Nude mice in control group were injected with physiological saline, while those of experimental group were given BCG and inactivated BCG respectively.The tumor growth was assayed by using caliper.The survival time of nude mice was determined.Necrotic extent and morphological changes of tumor were observed and examined by HE staining and immunohistochemical method.The results indicated that on 5th -7th days after tumor inoculated, 2 -3 mm tumor mass could be observed.The tumor volume increased over the time.HE staining of tumor tissues showed that there were different degrees of tumor necrosis in BCG group and inactivated BCG group.Immunohistochemistry results demonstrated that CD20 positive cells were obviously observed in the necrotic area of BCG group, compared with the control group and inactivated BCG group.It is concluded that human myeloid leukemia HL-60 cells have been successfully transplanted in nude mice, and the systemic metastasis occurs along with the prolongation of time.BCG inoculation can delay the tumor growth and prolong the survival time of nude mice with leukemia, suggesting that BCG has an antitumor effect.%本研究通过建立人白血病突变株细胞异种移植模型探讨卡介苗(bacillus calmette- guerin vaccine,BCG)的抗白血病作用.对8-10周龄的BALB/c裸鼠皮下接种1×107/ml人急性髓系白0细胞,于4-6天可形成皮下浸润的白血病裸鼠模型,随后将其分为2组:对照组和实验组.对照组于肿瘤内接种生理盐水,实验组又分为T1组(BCG组)、T2组(灭活的BCG组).观察各组裸鼠带瘤生存情况,以及通过对肿瘤组织

  1. Synergistic Effect of Decitabine and Valproic Acid on Induction of Differentiation in Leukemic HL-60 Cells%地西他滨联合丙戊酸钠对白血病细胞株的促分化效应研究

    Institute of Scientific and Technical Information of China (English)

    刘波; 邬露丹

    2012-01-01

    OBJECTIVE To investigate the synergistic effect of decitabine(DCA) and valproic acid(VPA) in differentiation induction in leuketnic HL-60 cells. METHODS The drug groups were set as follows: control group; DCA group A(1.0 umol·L‐1); DCA group B(4.0 μmol·L‐1); VPA group(2.0 mmol·L‐1); combination group A(DCA 1.0 μmolL‐1+VPA 2.0 mmol·L‐1); combination group B(DCA 4.0 umol·L‐1+VPA 2.0 mmol·L‐1). The cells were treated for 48 h. The CD117, CD34 mean fluorescence intensity(MFl) and CDI4, CDllb expression were detected by flow cytometry. RESULTS The CD117, CD34 MFI of combination group A and B were significantly lower than their corresponding concentration single drug group(P<0.01). Except CD14 of combination group B, the CD14, CDllb expressions of combination group A and B were significantly lower than their corresponding concentration single drug group(.P<0.01). CONCLUSION There is an enhanced differentiation activity of combinations of DCA and VPA in HL-60 cells.%目的 探讨地西他滨(decitabine,DCA)和丙戊酸钠(valproic acid,VPA)联用对白血病细胞株HL-60的促分化影响.方法 设立分组如下:对照组,DCAA组(1.0 μmol·L-1),DCA B组(4.0 μmol·L-1),VPA组(2.0mmol·L-1),联合用药A组(DCA1.0 μmol·L-1+VPA 2.0 mmol·L-1),联合用药B组(DCA4.0 μmol·L-1+VPA 2.0 mmol·L-1),作用48h.流式细胞术检测CD34、CD117 MF1以及CD11b、CD14表达率、结果 联合用药A、B组的CD1 17和CD34MFI显著低于其各自的单药组(P<0.01);联合用药A组的CD11b和CD14表达率和联合用药B组CDllb表达率显著低于其各自的单药组(P<0.01).结论 VPA与DCA联用对HL-60细胞其促分化作用.

  2. 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.

    Science.gov (United States)

    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.

  3. Characterization of a receptor for interleukin-5 on human eosinophils and the myeloid leukemia line HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Ingley, E.; Young, I.G. (Medical Molecular Biology Group, John Curtin School of Medical Research, Australian National University, Canberra (Australia))

    1991-07-15

    Interleukin-5 (IL-5) promotes the growth and differentiation of human eosinophils and may regulate the selective eosinophilia and eosinophil activation seen in certain diseases. Radiolabeled recombinant human IL-5 (hIL-5) was used to characterize the IL-5 receptor present on normal human eosinophils and on the myeloid leukemia line HL-60, which can be induced to differentiate into eosinophilic cells. Binding studies with eosinophils and HL-60 cells grown under alkaline conditions demonstrated similar high-affinity binding sites for hIL-5 on both cell types with kd values of approximately 400 pmol/L. The binding observed was specific in that it was not inhibited by hIL-3, human granulocyte-macrophage colony-stimulating factor, or hIL-2. Binding studies with a number of other human cell lines, including a B-lymphoma line, and with lymphocyte and neutrophil preparations were also performed, but IL-5 receptors were not detectable on these cells. The number of hIL-5 receptors on HL-60 cells could be correlated with its propensity to differentiate towards an eosinophilic cell type. Expression of hIL-5 receptors on HL-60 cells was upregulated by butyric acid under alkaline conditions, downregulated by hIL-3, virtually eliminated by dimethyl sulfoxide and hIL-5, while hIL-2 had no detectable effect. One major 125I-hIL-5-crosslinked complex of 75 to 85 Kd in Mr was detected on HL-60 cells using crosslinking agents giving a molecular mass of 55 to 60 Kd for the hIL-5 receptor itself. Studies using cellular autoradiography showed that IL-5 receptors were evenly distributed on eosinophils but that receptor distribution on HL-60 cells was noticeably heterogeneous. Eosinophils were the only cells in slides prepared from peripheral blood that had detectable levels of IL-5 receptors in agreement with the specific action of IL-5 on the human eosinophil lineage.

  4. Differentiation of HL-60 promyelocytes to granulocytes induced via the activation of protein kinase-C by benzene

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, C.; O' Connor, A.; Kalf, G. (Rutgers-the State Univ., Piscataway, NJ (United States) Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-03-15

    Benzene is a hematotoxin which affects the development of bone marrow progenitor cells and a leukemogen which causes acute myelogenous leukemia. The authors studied the effect of benzene on the differentiation of progenitors of the myeloid lineage, using HL-60 promyelocytic leukemia cells which can be induced to differentiate to granulocytes via the activation of protein kinase-C (PKC) by DMSO and retinoic acid. Exposure of HL-60 cells to 5 mM benzene for 5 min. results in the activation of PKC as measured by an increases in the phosphorylation of cellular proteins in a whole cell assay including proteins pp17 and pp27 reported by Feuerstein and Cooper to be involved in HL-60 cell differentiation. The increase in protein phosphorylation observed with benzene was equally as great as that observed with 100 ng/mL PMA, used as a control. Under the same conditions, benzene induces differentiation of the promyelocytes into granulocytes as measured by the acquisition of superoxide production and granulocyte morphology. Preincubation with 40 {mu}M sphinganine, a PKC inhibitor, prevents the benzene-induced increase in cellular protein phosphorylation and the differentiation to granulocytes. These results indicate that benzene, by activation of PKC, can affect myeloid differentiation which may play a role in the ability of benzene to cause acute myelogenous leukemia.

  5. Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation agents.

    Science.gov (United States)

    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.

  6. Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium

    Energy Technology Data Exchange (ETDEWEB)

    Chitambar, C.R.; Seligman, P.A.

    1986-12-01

    We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular /sup 59/Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of /sup 59/Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation.

  7. The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO2 Nanoparticles

    Directory of Open Access Journals (Sweden)

    Kaiqi Lu

    2015-01-01

    Full Text Available Four samples of modified titanium dioxide (TiO2, Fe/TiO2 (2 wt%, Fe/TiO2 (5 wt%, and 5-ALA/TiO2, were experimented in photodynamic therapy (PDT on leukemia cells HL60, performing promising photocatalytic inactivation effect. Fe/TiO2 and 5-ALA/TiO2 were synthesized in methods of precipitation and ultrasonic methods, respectively. X-ray diffraction spectra and UV-Vis spectra were studied for the samples’ crystalline phase and redshift of absorption peak. Further, FTIR spectra and Raman spectra were obtained to examine the combination of 5-aminolevulinic (5-ALA and TiO2 nanoparticles. The toxicity of these four kinds of nanoparticles was studied through darkroom experiments. And based on the concentration which caused the same toxic effect (90% on HL60, PDT experiments of TiO2, Fe/TiO2 (2%, Fe/TiO2 (5%, and ALA/TiO2 were done, resulting in the fact that the photokilling efficiency was 69.7%, 71.6%, 72%, and 80.6%, respectively. Scanning electron microscope (SEM images of the samples were also taken to study the morphology of HL60 cells before and after PDT, resulting in the fact the activation of the modified TiO2 from PDT was the main cause of cell apoptosis.

  8. Isolation and reconstitution of the N-formylpeptide receptor from HL-60 derived neutrophils.

    Science.gov (United States)

    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.

  9. 4H-Chromene-based anticancer agents towards multi-drug resistant HL60/MX2 human leukemia: SAR at the 4th and 6th positions.

    Science.gov (United States)

    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.

  10. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

    Science.gov (United States)

    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.

  11. m iR-21反义寡核苷酸增强地西他滨体外抗白血病效应%Enhanced anti-leukemic activity of decitabine to leukemia HL-60 cells by anti-miR-21 oligonucleotide

    Institute of Scientific and Technical Information of China (English)

    王晔恺; 于倩; 林奇龙; 姚燕珍; 梅佩玉; 李翊卫

    2015-01-01

    目的:研究 miR-21反义寡核苷酸(anti-miR-21 oligonucleotide, AMO)对地西他滨(decitabine, DCA)抗白血病效应的影响及可能机制。方法:将AMO和无义寡核苷酸( scramble oligonucleotide , SCR)通过脂质体转染导入HL-60细胞,实时荧光定量PCR( real-time PCR)验证转染效率,再分别与DCA 0.5、2.0和4.0μmol/L作用48 h。 Real-time PCR分别检测人周期节律蛋白3(hPer3) mRNA表达,Annexin V/PI法检测凋亡,流式细胞术检测CD117和CD11b平均荧光强度(MFI)。结果: AMO转染组miR-21表达(0.35±0.07)低于空白组(0.71±0.07)和SCR转染组(0.66±0.05),差异有统计学意义(P<0.05)。 AMO转染组的HL-60细胞DCA的IC50低于空白组和SCR转染组(P<0.01)。同一浓度下,AMO组的早期凋亡率、CD11b的MFI和hPer3 mRNA均高于同一浓度药物作用的空白组和SCR组,CD117 MFI均低于同一浓度药物作用的空白组和SCR组,差异均具有统计学意义(P<0.01)。结论:AMO能显著促进DCA体外抗白血病效应,其机制可能与其协助激活hPer3的表达有关。%AIM:To investigate the role of anti-miR-21 oligonucleotide ( AMO) in the anti-leukemic activity of decitabine (DCA) in vitro.METHODS:AMO and scramble oligonucleotide (SCR) were constructed and transfected into HL-60 cells.The miR-21 expression was analyzed by real-time PCR to identify the transfection efficiency .The cells were treated with DCA at gradient concentrations (0.5, 2.0 and 4.0 μmol/L) for 48 h.The mRNA expression of human period circadian protein 3 (hPer3) was detected by real-time PCR.The early apoptotic rates were determined by flow cy-tometry with Annexin V/PI staining.Mean fluorescence intensities ( MFI) of CD117 and CD11b were also measured by flow cytometry.RESULTS:The miR-21 relative expression level in AMO group was significantly lower than that in blank group and SCR group (P<0.01).IC50 of DCA in AMO group was

  12. The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO2 Nanoparticles

    OpenAIRE

    Kaiqi Lu; Qiyun He; Li Chen; Baoquan Ai; Jianwen Xiong

    2015-01-01

    Four samples of modified titanium dioxide (TiO2), Fe/TiO2 (2 wt%), Fe/TiO2 (5 wt%), and 5-ALA/TiO2, were experimented in photodynamic therapy (PDT) on leukemia cells HL60, performing promising photocatalytic inactivation effect. Fe/TiO2 and 5-ALA/TiO2 were synthesized in methods of precipitation and ultrasonic methods, respectively. X-ray diffraction spectra and UV-Vis spectra were studied for the samples’ crystalline phase and redshift of absorption peak. Further, FTIR spectra and Raman spec...

  13. EGCG抑制HL-60细胞增殖作用的机制研究

    Institute of Scientific and Technical Information of China (English)

    欧阳琳; 姜浩

    2011-01-01

    目的探讨EGCG抑制白血病HL-60细胞增殖的作用机制。方法采用软琼脂集落形成实验和绘制细胞生长曲线,检测EGCG对HL-60细胞增殖的影响;FCM及DNA凝胶电泳法检测EGCG处理HL-60细胞后的凋亡情况;实时荧光定量RT-PCR和Wester blotting检测AKT1的表达。结果细胞生长曲线和软琼脂集落形成实验结果均显示EGCG呈浓度依赖性抑制HL-60细胞的增殖(P〈0.05);FCM及DNA凝胶电泳法结果显示EGCG呈浓度依赖性诱导HL-60细胞的凋亡(P〈0.05)。实时荧光定量RT-PCR和Wester blotting结果显示EGCG浓度升高,HL-60细胞中AKT1的表达降低。结论 EGCG可能通过下调AKT1的表达,促进细胞凋亡,发挥其对HL-60细胞增殖的抑制作用。

  14. Regulation of CD11b transcription by decreasing PRC2 and increased acH4 level during ATRA-induced HL-60 differentiation

    Institute of Scientific and Technical Information of China (English)

    Huarong Tang; Fangping Chen; Qian Tan; Sanqin Tan; Linxin Liu; Fan Zhang

    2009-01-01

    Polycomb repressive complex 2 (PRC2),which mediates trimethylation of lysine 27 on histone H3 (K27me3),plays an important role in many types of stem cell differentiation.Here,we try to reveal how PRC2,PRC2-mediated repressive histone marker H3K27me3,and active histone marker histone H4 acetylation (acH4) regulate the CD11b transcription during all-trans retinoic acid (ATRA)-induced HL-60 leukemia cell differentiation.By using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis,we found that the mRNA and protein expression levels of two members of PRC2 were decreased during ATRA-induced HL-60 differentiation,respectively.When treated with ATRA for 72 h,the EZH2 and SUZ12 mRNA levels were decreased to 35% and 38% of the control group,respectively.At the same time,the granulocytic mature surface marker CD11b expression was increased significantly at mRNA level detected by qPCR and protein level detected by flow cytometry.By using chromatin immunoprecipita-tion assay,we compared the local changes in SUZ12 binding and PRC2-mediated H3K27me3 at the promo-ter of CD11b during ATRA-induced HL-60 differ-entiation.Both the levels of SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b were decreased for 4.1 and 3.8 folds,respectively.And we also found the increase in the acH4 level up to 4 folds after 72 h of ATRA treatment.These results suggested that the histone modification including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4 may involve in CD11b transcription during HL-60 leukemia cells reprogram-ming to terminal differentiation.

  15. Histamine increases cytosolic Ca2+ in HL-60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist profile and induces functional differentiation.

    Science.gov (United States)

    Seifert, R; Höer, A; Schwaner, I; Buschauer, A

    1992-08-01

    Histamine H1 receptors mediate activation of phospholipase C, with subsequent increases in cytosolic Ca2+ concentration ([Ca2+]i), and H2 receptors mediate accumulation of cAMP. HL-60 promyelocytes possess H2 receptors, but it is not known whether these cells also possess H1 receptors. We studied the effects of histamine on [Ca2+]i and the functional importance of histamine receptors in HL-60 promyelocytes. In these cells, histamine and dimaprit increased [Ca2+]i with EC50 values of 15 microM and 30 microM, respectively. Diphenhydramine inhibited the effect of histamine (100 microM) on [Ca2+]i up to 40%, with an IC50 of 100 nM. Famotidine and cimetidine diminished the effect of histamine (100 microM) up to 75%, with IC50 values of 85 nM and 300 nM, respectively. Diphenhydramine plus famotidine abolished histamine-induced rises in [Ca2+]i. Impromidine, with an IC50 of 100 nM, abolished the effect of histamine (100 microM) on [Ca2+]i. Diphenhydramine, famotidine, cimetidine, and impromidine showed marked noncompetitive antagonism with histamine. Histamine-induced increases in [Ca2+]i were largely due to influx of Ca2+ from the extracellular space. Ca2+ influx was inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). Histamine activated phospholipase C. Histamine induced expression of formyl peptide receptors, which effect was abolished by famotidine. In U-937 promonocytes and in the human erythroleukemia cell lines HEL and K-562, histamine did not induce rises in [Ca2+]i. Our data suggest the following. (i) In HL-60 promyelocytes, histamine increases [Ca2+]i predominantly via H2 receptors and to a lesser extent via H1 receptors. (ii) The agonist/antagonist profile of the H2 receptor-mediated increases in [Ca2+]i differs markedly from that for cAMP accumulation, suggesting the involvement of different H2 receptor subtypes. (iii) In HL-60 promyelocytes, histamine activates nonselective cation channels and

  16. Purification of a 53kD pI 4. 8 cytosolic phosphoprotein from HL60

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. OPTIMIZATIONS FOR 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY IN PURGING LEUKEMIA CELL HL60

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Photodynamictherapy(PDT),whichinvolves administrationofatumorlocalizing photosensiti zingagentthatmayrequiremetabolizingsynthesis(i.e.,aprodrug),followedbyactivationofthea gentbylightofaspecificwavelength.Thistherapy resultsinasequenceofphotochemicalandphotobio logicprocessthatcausesirreversiblephotodamageto tumortissue.SeveralphotosensitizersinPDThavesofar beenappliedtoeradicateresidualtumorcellsinau tograftsandtoreducecontaminationoftumorcells inhemopathy[1-3].However,alltheexogenous photosensitizershavea...

  18. Basic Apoptotic Mechanisms of Lead Toxicity in Human Leukemia (Hl-60) Cells

    OpenAIRE

    Tchounwou, Paul B.; Carolyn B. Howard; Milner, Jessica N.; Yedjou, Clement G.

    2010-01-01

    Lead exposure represents a medical and public health emergency, especially in children consuming high amounts of lead-contaminated flake paints. It may also cause hematological effects to people of all ages. Recent studies in our laboratory have indicated that apoptosis may be associated with the lead-induced oxidative stress and DNA damage. However, the mechanisms underlying its effect on lymphocytes are still largely unknown. Therefore, the aim of the present study was to investigate the ap...

  19. Ni(II) and Cu(II) N(4)-ethylmorpholine citronellalthiosemicarbazonate: a comparative analysis of cytotoxic effects in malignant human cancer cell lines.

    Science.gov (United States)

    Bisceglie, Franco; Alinovi, Rossella; Pinelli, Silvana; Goldoni, Matteo; Buschini, Annamaria; Franzoni, Susanna; Mutti, Antonio; Tarasconi, Pieralberto; Pelosi, Giorgio

    2013-11-01

    In this paper we report a study conducted with two analogous complexes, bis(N(4)-ethylmorpholine citronellalthiosemicarbazonate) nickel(II) and -copper(II) on four tumour cell lines (U937, HL60, SK-N-MC and HT29). All cell lines appear to be sensitive to both metal complexes, but while in U937, HL60 and SK-N-MC, apoptosis is the main mode through which cell death occurs, HT29 cells undergo necrosis. Among the cell lines which undergo apoptosis, SK-N-MC response is characterized by the intrinsic pathway, whereas U937 and HL60 involve both the intrinsic and the extrinsic pathways. The redox activity of the two complexes provides experimental evidence that they can modulate reactive oxygen species (ROS) production as a function of both the metal and the cell line used. Among the four cell lines, HL60 does not seem to give a significant response to exposure to both compounds. In the case of the nickel derivative, ROS generation is a relatively early event, and ROS could be the mediator leading to cellular damage. HT29 shows a remarkable and rapid ROS increase and a significant induction of membrane peroxidation that could be correlated to the onset of necrosis.

  20. Lipopolysaccharides and the Enhanced Expression of TACE Mrna in HL-60 Cells and Adhesive Cells from Human Spleen

    Institute of Scientific and Technical Information of China (English)

    DING; Tingbo(

    2001-01-01

    [1]Flad H D Loppnow H.Agonists and antagonists for LPS-induced cytokines.Immunology 1993 187:303[2]Robache-Gallea S Morand V Bruneau J H et al.In vitro processing of human tumor necrosis factor-α.J Bio Chem 1995 270(40):23 688[3]Kim K U Kwon O J Jue D M.Pro-tumor necrosis factor cleaving enzyme in macrophage membrane/particulate.Immunology 1993 80(2) :134[4]Moss M L.Cloning of a disintegrin metalloproteinase that processes precursor tumor necrosis factor-α.Nature 1997 385 (6):733[5]Flick D A Gilford G E.The detection of biological activity of tumor necrosis factor-α.J Immunol Methods 1984 68:167[6]Sambrook J Fritsch E F Maniatis F.In Vitro Amplification of DNA by the Polymerase Chain Reaction.In:Molecular Cloning A Laboratory Manual.2nd edition.New York:Cold Spring Harbor Laboratory Press 1989.10.1-10.70[7]吴朝栋 李明珍 张艳萍等.中药热毒清对内毒素DIC家兔血浆肿瘤坏死因子α白细胞介素水平影响的研究.中国中西医结合杂志 1995 15:365

  1. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  2. Cell Response to Biological Regulators and Noxious Agents: Attractor States and Constrained Dynamics of Gene Expression Profiles

    Science.gov (United States)

    2008-09-30

    EML cells. HL60 cells are a human promyelocytic cell line derived from acute myeloid leukemia . Its ability to terminally differentiate into mature...states are exposed by sub-maximal stimulation of differentiation which places cells in such states (FIG. 1). The neutrophil state in HL60 promyelocytic

  3. Antisense RNA of Survivin Gene Inhibits the Proliferation of Leukemia Cells and Sensitizes Leukemia Cell Line to Taxol-induced Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wenhan LI; Xiaojuan WANG; Ping LEI; Qing YE; Huifen ZHU; Yue ZHANG; Jinfang SHAO; Jing YANG; Guanxin SHEN

    2008-01-01

    The effectS of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol.induced chemotherapy was explorcd.A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction.The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing.The recombi-nant plasmid was delivered into HL-60 cells by electroporation.Growth curves were plotted based on cell counting.Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol.DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay.The correct construction of the recombinant plasmid has been identificd bv restriction enzy.me digestion and DNA sequencing.A stable down.regulation has been achieved in HL-60 SVVas cells after G418 selection.Compared tO HL-60 cells.the proliferation of HL-60 SVVaS cells was signifi.cantly inhibited(P<0.05).Cytotoxicity assays indicated that IC50 of HL-60 SVVas for taxol was rela-tively lower than controls(P<0.01).Apoptosis assays revealed that taxol-induced apoptosis was de-tected in HL-60 sVVas cells incubated with 50 ng/ml taxol for 12 h,while in HL-60 cells incubated with 100 ng/ml taxol for 72 h.It was suggested that Survivin antisense RNA could inhibit the prolif-eration of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells.which may lay an ex-perimental foundation for further research on gene therapy in leukemia.

  4. Oral changes in individuals undergoing hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Regina Haddad Barrach

    2015-04-01

    Full Text Available INTRODUCTION: Patients undergoing hematopoietic stem cell transplantation receive high doses of chemotherapy and radiotherapy, which cause severe immunosuppression.OBJECTIVE: To report an oral disease management protocol before and after hematopoietic stem cell transplantation.METHODS: A prospective study was carried out with 65 patients aged > 18 years, with hematological diseases, who were allocated into two groups: A (allogeneic transplant, 34 patients; B (autologous transplant, 31 patients. A total of three dental status assessments were performed: in the pre-transplantation period (moment 1, one week after stem cell infusion (moment 2, and 100 days after transplantation (moment 3. In each moment, oral changes were assigned scores and classified as mild, moderate, and severe risks.RESULTS: The most frequent pathological conditions were gingivitis, pericoronitis in the third molar region, and ulcers at the third moment assessments. However, at moments 2 and 3, the most common disease was mucositis associated with toxicity from the drugs used in the immunosuppression.CONCLUSION: Mucositis accounted for the increased score and potential risk of clinical complications. Gingivitis, ulcers, and pericoronitis were other changes identified as potential risk factors for clinical complications.

  5. Telecommunication system for children undergoing stem cell transplantation.

    Science.gov (United States)

    Higuchi, Kazumi; Nakazawa, Yozo; Sakata, Nobuhiro; Takizawa, Masaomi; Ohso, Keiko; Tanaka, Miyuki; Yanagisawa, Ryu; Koike, Kenichi

    2011-12-01

    Isolation in a germ-free unit is a stressful experience for pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). To reduce the psychological distress of such children, a Web-based telecommunications system was developed. The authors developed a telecommunication system that linked a laminar air flow (LAF) room that had a high efficiency particulate air filter with the hospital school/patients' homes via the Internet. Fifteen children isolated in the LAF room for allogeneic HSCT were enrolled in this study. The present study evaluated whether the system was feasible for the patients during the acute phase of HSCT. In 10 patients, the proportion of days when they telecommunicated with teachers and/or other patients in the hospital school was 64.6 ± 32.3%. The telecommunication with the hospital school facilitated the continuation of school study under teachers' guidance, reducing the problem of lost schooling. In 13 patients, the proportion of days when they telecommunicated with their homes was 68.0 ± 34.8%. Ten of them frequently telecommunicated with their family members (especially siblings), and three patients called out to their pets at home. The incidence of telecommunication on the days when the patients had HSCT-related symptoms including vomiting did not differ from that of telecommunication on the days when no symptoms were evident. A telecommunication system linked to a hospital school and/or the patients' homes is feasible for children undergoing HSCT, and may improve their health-related quality of life. A larger, prospective study is required to evaluate whether the telecommunication system can reduce HSCT-associated psychological and psychiatric symptoms. © 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.

  6. EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To understand the mechanism of the hypertherjia and harringtonine in purging of leukemia cells In vitro.telomerase activity of HL-60 cells treated by hyperthermia(42℃ for one hour) and different concentrations of Harringtonine were investigated .Methods Using telomeric Repeats Amplification Protocol(TRAP)and ELISA techniques to analyze the telomerase activity of HL-60 cells,Results:Our results showed that harringtonine inhibited the telomerase activity of HL-60 cells in a dosage related manner,Moreover,the telomerase activity of HL-60 cells was significantly decreased after the hyperthermia treatment as compared with untreated cells.Conclusion:The effect of the hyperthermia and Harringtonine on purging leukemia cellsIn vitro may be mediated by down regulation of telomerase activity of tumor cells.

  7. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    National Research Council Canada - National Science Library

    Lizard, G; Fournel, S; Genestier, L; Dhedin, N; Chaput, C; Flacher, M; Mutin, M; Panaye, G; Revillard, J P

    1995-01-01

    ... of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria...

  8. Plasma membrane ubiquinone controls ceramide production and prevents cell death induced by serum withdrawal.

    Science.gov (United States)

    Barroso, M P; Gómez-Díaz, C; Villalba, J M; Burón, M I; López-Lluch, G; Navas, P

    1997-06-01

    Serum provides cultured cells with survival factors required to maintain growth. Its withdrawal induces the development of programmed cell death. HL-60 cells were sensitive to serum removal, and an increase of lipid peroxidation and apoptosis was observed. Long-term treatment with ethidium bromide induced the mitochondria-deficient rho(o)HL-60 cell line. These cells were surprisingly more resistant to serum removal, displaying fewer apoptotic cells and lower lipid peroxidation. HL-60 cells contained less ubiquinone at the plasma membrane than rho(o)HL-60 cells. Both cell types increased plasma membrane ubiquinone in response to serum removal, although this increase was much higher in rho(o) cells. Addition of ubiquinone to both cell cultures in the absence of serum improved cell survival with decreasing lipid peroxidation and apoptosis. Ceramide was accumulated after serum removal in HL-60 but not in rho(o)HL-60 cells, and exogenous ubiquinone reduced this accumulation. These results demonstrate a relationship between ubiquinone levels in the plasma membrane and the induction of serum withdrawal-induced apoptosis, and ceramide accumulation. Thus, ubiquinone, which is a central component of the plasma membrane electron transport system, can represent a first level of protection against oxidative damage caused by serum withdrawal.

  9. Wnt and the cancer niche: paracrine interactions with gastrointestinal cancer cells undergoing asymmetric cell division.

    Science.gov (United States)

    Xin, Hong-Wu; Ambe, Chenwi M; Ray, Satyajit; Kim, Bo-Kyu; Koizumi, Tomotake; Wiegand, Gordon W; Hari, Danielle; Mullinax, John E; Jaiswal, Kshama R; Garfield, Susan H; Stojadinovic, Alexander; Rudloff, Udo; Thorgeirsson, Snorri S; Avital, Itzhak

    2013-01-01

    Stem-like cancer cells contribute to cancer initiation and maintenance. Stem cells can self-renew by asymmetric cell division (ACD). ACD with non-random chromosomal cosegregation (ACD-NRCC) is one possible self-renewal mechanism. There is a paucity of evidence supporting ACD-NRCC in human cancer. Our aim was to investigate ACD-NRCC and its potential interactions with the cancer niche (microenvironment) in gastrointestinal cancers. We used DNA double and single labeling approaches with FACS to isolate live cells undergoing ACD-NRCC. Gastrointestinal cancers contain rare subpopulations of cells capable of ACD-NRCC. ACD-NRCC was detected preferentially in subpopulations of cells previously suggested to be stem-like/tumor-initiating cancer cells. ACD-NRCC was independent of cell-to-cell contact, and was regulated by the cancer niche in a heat-sensitive paracrine fashion. Wnt pathway genes and proteins are differentially expressed in cells undergoing ACD-NRCC vs. symmetric cell division. Blocking the Wnt pathway with IWP2 (WNT antagonist) or siRNA-TCF4 resulted in suppression of ACD-NRCC. However, using a Wnt-agonist did not increase the relative proportion of cells undergoing ACD-NRCC. Gastrointestinal cancers contain subpopulations of cells capable of ACD-NRCC. Here we show for the first time that ACD-NRCC can be regulated by the Wnt pathway, and by the cancer niche in a paracrine fashion. However, whether ACD-NRCC is exclusively associated with stem-like cancer cells remains to be determined. Further study of these findings might generate novel insights into stem cell and cancer biology. Targeting the mechanism of ACD-NRCC might engender novel approaches for cancer therapy.

  10. Effects of human promyelocytic leukemia HL-60 cel supernatant on differetiation of mononulear cel s from human umbilical cord blood%急性早幼粒白血病细胞培养上清液对人脐带血单个核细胞分化的影响

    Institute of Scientific and Technical Information of China (English)

    郑毅; 方宁; 陈代雄

    2013-01-01

    目的白血病细胞能够通过自分泌方式分泌细胞因子而作用于自身,使细胞大量的增殖而不分化;白血病细胞所分泌的细胞因子也能作用于人脐带血(UCB)单个核细胞(MNCs)中的CD34+/CD133+细胞亚群使之增殖而抑制其分化。关于急性早幼粒白血病细胞(HL-60细胞)培养上清液能否使UCB- MNCs分化尚未见报道,本实验主要目的是观察HL-60细胞培养上清液对人UCB-MNCs分化的影响。方法实验分为①有HL-60细胞培养上清液+StemSpan® SFEM组;②StemSpan® SFEM组;③HL-60细胞培养上清液+高糖(HG)-DMEM组;④HG-DMEM组;⑤HL-60细胞培养上清液+低糖(LG)-DMEM组;⑥LG-DMEM组。收分离的人UCB-MNCs按2.5×106/瓶分别接种于6组培养液中,37℃,5%CO2,饱和湿度培养,于培养前和培养12天用流式细胞仪(FCM)分析表型CD34,CD90和CD166的变化。结果各实验组CD34,CD34/CD90和CD166阳性细胞百分比均较培养前升高。结论%Objective:Leukemic cel s can excrete cytokines by an autocrine way, which contribute to its large-scale proliferation, and the cytokines may also contribute to the proliferations of CD34+/CD133+ subpopulation of UCB-MNCs too. To the effect of human promyelocytic leukemia HL-60 cel supernatant on the differentiation of UCB-MNCs, there is not related report so far. The aim of this study is to investigate effects of HL-60 cel supernatant on differentiations of mononuclear cel s from human umbilical cord blood. Methods:Six various cultural system are listed as fol ows: ① HL-60 cel supernatant+ StemSpan–SFEM; ② StemSpan–SFEM; ③ HL-60 cel supernatant+ HG-DMEM; ④ HG-DMEM; ⑤HL-60 cel supernatant LG-DMEM; ⑥ LG-DMEM. 2.50×106 of UCB-MNCs per bottle with the above each cultural system is incubated at 37℃ in a 5%CO2, saturation humidity of incubator, the changes of phenotype of CD34,CD90 and CD166 analyzed by FCM after 12 days. Results: Positive cel s with CD34+, CD166+ and CD34+/CD

  11. Effects of Dioscin Extracted from Polygonatum Zanlanscianense Pamp on Several Human Tumor Cell Lines

    Institute of Scientific and Technical Information of China (English)

    王钊; 周江兵; 巨勇; 姚沈勤; 张洪钧

    2001-01-01

    Dioscin was extracted and isolated from Polygonatum Zanlanscianense Pamp. The effects of dioscin on HL60, HeLa, H14, and MDA-MB-435 cell lines were studied with the results showing that dioscin dramatically inhibited the growth of the MDA-MB-435, H14, HL60, and HeLa cell lines. The IC50 of dioscin on these cell lines were 2.6, 0.8, 7. 5, and 4.5 μ mol/L respectively.

  12. The use of ovarian cancer cells from patients undergoing surgery to generate primary cultures capable of undergoing functional analysis.

    Science.gov (United States)

    O Donnell, Rachel L; McCormick, Aiste; Mukhopadhyay, Asima; Woodhouse, Laura C; Moat, Madeleine; Grundy, Anna; Dixon, Michelle; Kaufman, Angelika; Soohoo, San; Elattar, Ahmed; Curtin, Nicola J; Edmondson, Richard J

    2014-01-01

    The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

  13. The use of ovarian cancer cells from patients undergoing surgery to generate primary cultures capable of undergoing functional analysis.

    Directory of Open Access Journals (Sweden)

    Rachel L O Donnell

    Full Text Available The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91% cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

  14. Oncogene Regulation during the Growth and Differentiation of a Human Promyelocytic Leukemia Cell Line.

    Science.gov (United States)

    Ely, Constance Marie

    To determine the significance of the regulation of the cellular oncogenes c-myc and c-myb during myeloid and monocytic differentiation, we analyzed oncogene expression concurrent with functional and morphological differences in HL-60 cells and in a partial differentiation resistant HL-60 clone (HL-60-1E3). Although HL-60-1E3 cells are unable to develop mature terminally differentiated features with PDBu or DMSO stimulation, they do exhibit partial differentiation features with these conditions. Treatments of HL-60-1E3 cells with PDBu preceded by treatment with dimethylsulfoxide (DMSO), results in complete maturation to macrophage-like cells. Using parallel PDBu-induction studies, we analyzed the kinetics of expression of c-myc, c-myb, c-fms, c-fos, c-raf, and histone H4, together with cell cycle frequency distribution, cytotoxic effector activity and clonogenic potential in HL-60 and HL-60-1E3 cells. The results of these studies revealed altered c-myc and c-myb regulation in resistant cells corresponding to a lack of terminal commitment as assessed by an increase in clonogenic potential and the inability to acquire cytotoxic function. These data suggest that maintenance of the suppressed state of c-myc and c-myb gene expression may be an important component of the regulatory mechanisms which allow HL-60 cells to complete macrophage-like terminal differentiation. A similar series of experiments examining the DMSO-induced granulocyte pathway revealed that differentiation resistance of HL-60-1E3 cells corresponded to altered regulation of both c-myc and c-myb, strengthening the hypothesis that regulation of both of these genes is integral to HL-60 differentiation. Biphasic c-myb expression was observed in both cell populations in the presence of DMSO where maximal expression took place at approximately 72 hours post-induction and was not linked to proliferation. Introduction of SV40:c-myc recombinant plasmids into HL-60 cells resulted in altered nuclear morphology

  15. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    Full Text Available BACKGROUND: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. CONCLUSIONS/SIGNIFICANCE: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  16. Staphylococcal SSL5 Binding to Human Leukemia Cells Inhibits Cell Adhesion to Endothelial Cells and Platelets

    Directory of Open Access Journals (Sweden)

    Annemiek M. E. Walenkamp

    2010-01-01

    Full Text Available Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5 was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies.

  17. Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora.

    Science.gov (United States)

    Hiratsuka, Rie; Terasaka, Osamu

    2011-04-01

    Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.

  18. Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

    Science.gov (United States)

    Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E; Ambe, Chenwi M; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J; Wiegand, Gordon W; Garfield, Susan H; Thorgeirsson, Snorri S; Avital, Itzhak

    2012-04-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. Copyright © 2012 AlphaMed Press.

  19. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N. [Hospital Edouard Herriot, Lyon (France)] [and others

    1995-11-01

    Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display and early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90{degrees} light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly, a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. 33 refs., 5 figs., 1 tab.

  20. Immature hematopoietic stem cells undergo maturation in the fetal liver.

    Science.gov (United States)

    Kieusseian, Aurelie; Brunet de la Grange, Philippe; Burlen-Defranoux, Odile; Godin, Isabelle; Cumano, Ana

    2012-10-01

    Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.

  1. Iron Overload in Patients Undergoing Hematopoietic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Vinod Pullarkat

    2010-01-01

    Full Text Available Recipients of hematopoietic stem cell transplantation (HSCT frequently have iron overload resulting from chronic transfusion therapy for anemia. In some cases, for example, in patients with myelodysplastic syndromes and thalassemia, this can be further exacerbated by increased absorption of iron from the gut as a result of ineffective erythropoiesis. Accumulating evidence has established the negative impact of elevated pretransplantation serum ferritin, a surrogate marker of iron overload, on overall survival and nonrelapse mortality after HSCT. Complications of HSCT associated with iron overload include increased bacterial and fungal infections as well as sinusoidal obstruction syndrome and possibly other regimen-related toxicities. Based on current evidence, particular attention should be paid to prevention and management of iron overload in allogeneic HSCT candidates, especially in patients with thalassemia and myelodysplastic syndromes. The pathophysiology of iron overload in the HSCT patient and optimum strategies to deal with iron overload during and after HSCT require further study.

  2. Modelling a tethered mammalian sperm cell undergoing hyperactivation

    KAUST Repository

    Curtis, M.P.

    2012-09-01

    The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions. © 2012 Elsevier Ltd.

  3. Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells

    Directory of Open Access Journals (Sweden)

    Naranjo Victoria

    2007-09-01

    Full Text Available Abstract Background The tick-borne intracellular pathogen, Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with A. phagocytophilum. Methods The human Sp110 and A. phagocytophilum msp4 mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on A. phagocytophilum infection was determined by RNA interference (RNAi. The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA. The A. phagocytophilum infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of msp4 and normalizing against human Alu sequences. Results While Sp110 mRNA levels increased concurrently with A. phagocytophilum infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels. Conclusion These results demonstrated that Sp110 expression is required for A. phagocytophilum infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which A. phagocytophilum modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.

  4. Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

    Science.gov (United States)

    Ebeling, J G; Vandenbark, G R; Kuhn, L J; Ganong, B R; Bell, R M; Niedel, J E

    1985-01-01

    Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation. Images PMID:3156372

  5. Mangiferin activates Nrf2-antioxidant response element signaling without reducing the sensitivity to etoposide of human myeloid leukemia cells in vitro

    Science.gov (United States)

    Zhang, Ben-ping; Zhao, Jie; Li, Shan-shan; Yang, Li-jing; Zeng, Ling-lan; Chen, Yan; Fang, Jun

    2014-01-01

    Aim: Mangiferin is glucosylxanthone extracted from plants of the Anacardiaceae and Gentianaceae families. The aim of this study was to investigate the effects of mangiferin on Nrf2-antioxidant response element (ARE) signaling and the sensitivity to etoposide of human myeloid leukemia cells in vitro. Methods: Human HL-60 myeloid leukemia cells and mononuclear human umbilical cord blood cells (MNCs) were examined. Nrf2 protein was detected using immunofluorescence staining and Western blotting. Binding of Nrf2 to ARE was examined with electrophoretic mobility shift assay. The level of NQO1 was assessed with real-time RT-PCR and Western blotting. DCFH-DA was used to evaluate intracellular ROS level. Cell proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. Results: Mangiferin (50 μmol/L) significantly increased Nrf2 protein accumulation in HL-60 cells, particularly in the nucleus. Mangiferin also enhanced the binding of Nrf2 to an ARE, significantly up-regulated NQO1 expression and reduced intracellular ROS in HL60 cells. Mangiferin alone dose-dependently inhibited the proliferation of HL-60 cells. Mangiferin (50 mol/L) did not attenuate etoposide-induced cytotoxicity in HL-60 cells, and combined treatment of mangiferin with low concentration of etoposide (0.8 μg/mL) even increased the cell inhibition rate. Nor did mangiferin change the rate of etoposide-induced apoptosis in HL-60 cells. In MNCs, mangiferin significantly relieved oxidative stress, but attenuated etoposide-induced cytotoxicity. Conclusion: Mangiferin is a novel Nrf2 activator that reduces oxidative stress and protects normal cells without reducing the sensitivity to etoposide of HL-60 leukemia cells in vitro. Mangiferin may be a potential chemotherapy adjuvant. PMID:24374812

  6. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  7. [Experimental study on induction of apoptosis of leukemic cells by Boswellia carterii Birdw extractive].

    Science.gov (United States)

    Qi, Z; Zhang, G; Zhu, W

    1999-01-01

    The purpose of the study was to investigate the apoptosis of leukemic cells induced by Boswellia Carterii Birdw(BCB). The target leukemia cell line HL60 and bone marrow leukemic cells from 30 acute non-lymphocytic leukemic(ANLL) patients (3 M1 11 M2a 10 M3 1 M4a 5 M5b) were studied. Apoptosis was detected by morphological observation, DNA electrophoresis, percentage of DNA fragmentation test and flow cytometric cell cycle analysis. It is concluded that BCB can induce apoptosis in ANLL cells and HL60 cells.

  8. Postoperative infection and natural killer cell function following blood transfusion in patients undergoing elective colorectal surgery

    DEFF Research Database (Denmark)

    Jensen, L S; Andersen, A J; Christiansen, P M

    1992-01-01

    The frequency of infection in 197 patients undergoing elective colorectal surgery and having either no blood transfusion, transfusion with whole blood, or filtered blood free from leucocytes and platelets was investigated in a prospective randomized trial. Natural killer cell function was measured...... before operation and 3, 7 and 30 days after surgery in 60 consecutive patients. Of the patients 104 required blood transfusion; 48 received filtered blood and 56 underwent whole blood transfusion. Postoperative infections developed in 13 patients transfused with whole blood (23 per cent, 95 per cent...... function was significantly (P less than 0.001) impaired up to 30 days after surgery in patients transfused with whole blood. These data provide a strong case against the use of whole blood transfusion in patients undergoing elective colorectal surgery. Udgivelsesdato: 1992-Jun...

  9. Modelling the damage potential of fluid flows for animal cells undergoing cultivation in bioreactors.

    Science.gov (United States)

    Stanford Keen, Giles

    1996-11-01

    Mechanical disruption and injury sustained by animal cells undergoing cultivation in bioreactors is an important problem in biotechnology. Damage to cells is thought to be caused primarily by bubbles bursting at the free surface of the culture medium. Here we present computational studies applying a mathematical model for the cell damage rates experienced by cells in laminar flow. Two fluid dynamical systems are considered - namely a converging channel and a single bursting bubble. The flows are calculated using a fourth-order finite difference technique on a stretched grid, and a boundary integral method respectively. It is possible to obtain an estimate for the number of cells in a particular population which are likely to be disrupted by the forces they experience in the flow. This is done by calculating the maximum rate of strain experienced by fluid particles, and combining this with experimental data on the strength and size of cells, obtained by micromanipulation techniques. The resulting information is then used together with the cell damage model to produce a cell damage prediction. The computational results are compared with experimental measurements of cell death, to validate the model for cell damage.

  10. Label-Free Discrimination of Cells Undergoing Apoptosis by Hyperspectral Confocual Reflectance Imaging

    Science.gov (United States)

    Bertani, F. R.; Botti, E.; Costanzo, A.; Ferrari, L.; Mussi, V.; D'Alessandro, M.; Selci, S.

    2013-12-01

    Among the optical techniques used for exploring the properties of cells and tissues, those based on hyperspectral label-free analysis are particularly interesting due to their non-invasive character and their ability to fast collect a huge number of information on the different sample constituents and their spatial distribution. Here we present results obtained with a novel hyperspectral reflectance confocal microscope of label-free discrimination of cells undergoing apoptosis. Our data, analyzed by means of a powerful statistical method, enable to obtain information on the biological status at a single cell level through the local measurement of reflectivity. Furthermore, an optical model of the local dielectric response gives an additional insight of the parameters linking the optical responsivity to the biological status.

  11. Apoptosis of human leukemic HL—60 cells induced to differentiate by treatment with RA or DMSO

    Institute of Scientific and Technical Information of China (English)

    SUNHONG; YONGCHAOWANG

    1995-01-01

    In present study,we studied the effect of all-trans retinoic acid(ATRA)and dimethylsulfoxide(DMSO)on the induction of apoptosis in HL-60 cell line.Based on morphological changes by Hochest 33342 staining and identification of internuclesomal NDA celeavage by gel electrophoresis,we observed aberrant nuclear chromatin condensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method.We found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6d after the initiation of the treatment However,Such an obvious apoptotic peak was not identified in DMSO-differentiated cells.Combining the research accomplished before.our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.

  12. Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation.

    Directory of Open Access Journals (Sweden)

    Rafaela C Sartore

    Full Text Available The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC cells, embryonic stem (ES cells and induced pluripotent stem (iPS cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal

  13. Cell-free activation of phagocyte NADPH-oxidase: tissue and differentiation-specific expression of cytosolic cofactor activity.

    Science.gov (United States)

    Parkinson, J F; Akard, L P; Schell, M J; Gabig, T G

    1987-06-30

    We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.

  14. Induction of leukemia cell apoptosis by cheliensisin A involves down-regulation of Bcl-2 expression

    Institute of Scientific and Technical Information of China (English)

    Li ZHONG; Chao-ming LI; Xiao-jiang HAO; Li-guang LOU

    2005-01-01

    Aim: To investigate the apoptosis-inducing effect of cheliensisin A (GC-51), a novel styryl-lactone isolated from Goniothalamus cheliensis, on human promyelocytic leukemia HL-60 cells and the mechanism of action involved.Methods: Apoptotic cell death was determined by morphological examination and DNA agarose gel electrophoresis. The activity of caspase-3 was assessed using Western blotting and the expression of Bcl-2 and Bax genes was analyzed using the reverse transcription-polymerase chain reaction (RT-PCR) method. Results:GC-51 significantly inhibited the proliferation of HL-60 cells with an IC50 of 2.4±0.2 μmol/L and effectively induced apoptosis in HL-60 cells. Exposure of HL-60cells to 10 μmol/L GC-51 for 8 h resulted in approximately 53% of the cells under going apoptosis. Caspase-3 was activated in GC-51-treated cells, which was manifested by the appearance of the 17 kDa active form of caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP). Meanwhile, GC-51 markedly reduced the expression of the anti-apoptotic gene Bcl-2 and increased the expression of the pro-apoptotic gene Bax. The apoptosis-inducing effect of GC-51 was cAMP dependent protein kinase (PKA) dependent because PKA, but not the protein kinase C, specific inhibitor H-89, blocked the induction of apoptosis by GC-51 in HL-60 cells. Conclusion: The results demonstrate that GC-51 effectively induces apoptosis in HL-60 cells and that this effect is PKA-dependent and involves the downregulation of Bcl-2 expression and the activation of caspase-3.

  15. Mitochondria released by cells undergoing TNF-α-induced necroptosis act as danger signals.

    Science.gov (United States)

    Maeda, A; Fadeel, B

    2014-07-03

    Necrosis leads to the release of so-called damage-associated molecular patterns (DAMPs), which may provoke inflammatory responses. However, the release of organelles from dying cells, and the consequences thereof have not been documented before. We demonstrate here that mitochondria are released from cells undergoing tumor necrosis factor-α (TNF-α)-induced, receptor-interacting protein (RIP)1-dependent necroptosis, a form of programmed necrosis. The released, purified mitochondria were determined to be intact as they did not emit appreciable amounts of mitochondrial DNA (mtDNA). Pharmacological inhibition of dynamin-related protein 1 (Drp1) prevented mitochondrial fission in TNF-α-triggered cells, but this did not block necroptosis nor the concomitant release of mitochondria. Importantly, primary human macrophages and dendritic cells engulfed mitochondria from necroptotic cells leading to modulation of macrophage secretion of cytokines and induction of dendritic cell maturation. Our results show that intact mitochondria are released from necroptotic cells and suggest that these organelles act as bona fide danger signals.

  16. Retinoic acid improves morphology of cultured peritoneal mesothelial cells from patients undergoing dialysis.

    Directory of Open Access Journals (Sweden)

    Carmen Retana

    Full Text Available Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability or High transporter (high solute permeability. Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite, as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM. We studied length and distribution of microvilli and cilia (scanning electron microscopy, epithelial (cytokeratin, claudin-1, ZO-1 and occludin and mesenchymal (vimentin and α-smooth muscle actin transition markers by immunofluorescence and Western blot, and transforming growth factor β1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor-β1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-β1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter

  17. Successful rescue of pure red cell aplasia in two aged patients undergoing pancrease-kindey transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yin-fu; YANG Tong-han; WANG Qing-yu; Wang Ping-xian; Fan Ming-qi; Feng Jia-yu

    2001-01-01

    To explore the correlation between hypoimmunity and the occurrence of pure red cell aplasia (PRCA) in senile patients undergoing combined pancrease-kindey transplantation. Methods: PRCA occurred in 2 patients out of 5 who were performed combined pancrease-kindey transplantation.The general scheme of treatment mainly included selective administration of immunosuppressants and antivirus drugs, infusion of red blood cells on the basis of surveillance of parvorirus B19 and the ratio of T4 and T8 as well as the changes of the myelogram.Results: The myelogram of the patients returned to normal in 2 and 3 weeks after operation, respectively, and subsequent follow-up revealed no recurrence. Conclusion: This series illustrate the point that advanced age, hypo immunity, parvorirus B19 and immunosuppressants are vulnerable to PRCA. Combined treatment is an effectiveremedy for these patients.

  18. Inhibiting effect of CaMK Ⅱ N up-regulation on leukemia cells growth and its mechanism

    Institute of Scientific and Technical Information of China (English)

    侯军

    2014-01-01

    Objective To investigate the inhibitory effects of CaMKⅡN on acute myeloid leukemia cell line HL-60to explore a novel therapeutic target of leukemia.Methods Human CaMKⅡN gene expression vector pcDNA3.1/hCaMKⅡN or empty vector pcDNA3.1/myc-His(-)B was transfected into HL-60 cells by Lipofectamine2000.Human CaMKⅡN proteins of transfected cells were detected by Westem blot.Cell proliferation affected by human CaMKⅡN was determined by MTT.Colonyforming assay was performed by soft agar

  19. Retrospective analysis of fluoroquinolone prophylaxis in patients undergoing allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Simondsen, Katherine A; Reed, Michael P; Mably, Mary S; Zhang, Yang; Longo, Walter L

    2013-12-01

    Patients undergoing allogeneic hematopoietic stem cell transplant are at a high risk for infection-related mortality in the immediate post-transplantation phase. Prophylaxis with a fluoroquinolone is now recommended to reduce this risk with the stipulation that surveillance for increased fluoroquinolone resistance Clostridium difficile associated diarrhea be conducted. We conducted a retrospective chart review of 48 patients who underwent an allogeneic hematopoietic stem cell transplant and received a fluoroquinolone for prophylaxis and 48 patients who underwent an allogeneic hematopoietic stem cell transplant who did not receive a fluoroquinolone for prophylaxis. All patients received the same standard antifungal, antiviral and anti-pneumocystis prophylaxis. Patients receiving fluoroquinolone prophylaxis had a lower incidence of febrile neutropenia than those not receiving prophylaxis, though the difference was not found to be statistically significant (83% vs. 67%, p = 0.098). Similar non-significant improvements in the number of positive cultures recovered during an episode of febrile neutropenia and antimicrobial days were noted. No significant increase in fluoroquinolone resistance, Clostridium difficile associated diarrhea, or in methicillin resistant Staphylococcus aureus infections were noted. Our single institution experience with fluoroquinolone prophylaxis for allogeneic hematopoietic stem cell transplant patients supports continuation of this practice. Expansion to autologous hematopoietic stem cell transplant patients may be appropriate based on guideline recommendations and our institution-specific experience with fluoroquinolone prophylaxis.

  20. Activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress.

    Science.gov (United States)

    Lu, Jun; Chen, Jian; Xu, Nianjun; Wu, Jun; Kang, Yani; Shen, Tingting; Kong, Hualei; Ma, Chao; Cheng, Ming; Shao, Zhifeng; Xu, Ling; Zhao, Xiaodong

    2016-09-01

    Application of cisplatin (DDP) for treating lung cancer is restricted due to its toxicity and lung cancer's drug resistance. In this study, we examined the effect of Jinfukang (JFK), an effective herbal medicine against lung cancer, on DDP-induced cytotoxicity in lung cancer cells. Morphologically, we observed that JFK increases DDP-induced pro-apoptosis in A549 cells in a synergistic manner. Transcriptome profiling analysis indicated that the combination of JFK and DDP regulates genes involved in apoptosis-related signaling pathways. Moreover, we found that the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines, such as NCI-H1975, NCI-H1650, and NCI-H2228. Particularly, we demonstrated that AIFM2 is activated by the combined treatment of JFK and DDP and partially mediates the synergistic pro-apoptosis effect. Collectively, this study not only offered the first evidence that JFK promotes DDP-induced cytotoxicity, and activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress, but also provided a novel insight for improving cytotoxicity by combining JFK with DDP to treat lung cancer cells.

  1. Red cell distribution width in anemic patients undergoing transcatheter aortic valve implantation

    Institute of Scientific and Technical Information of China (English)

    Katharina Hellhammer; Tobias Zeus; Pablo E Verde; Verena Veulemanns; Lisa Kahlstadt; Georg Wolff; Ralf Erkens; Ralf Westenfeld; Eliano P Navarese; Marc W Merx; Tienush Rassaf; Malte Kelm

    2016-01-01

    AIM: To determine the impact of red blood cell distribution width on outcome in anemic patients undergoing transcatheter aortic valve implantation(TAVI).METHODS: In a retrospective single center cohort study we determined the impact of baseline red cell distribution width(RDW) and anemia on outcome in 376 patients with aortic stenosis undergoing TAVI. All patients were discussed in the institutional heart team and declined for surgical aortic valve replacement due to high operative risk. Collected data included patient characteristics, imaging findings, periprocedural in hospital data, laboratory results and follow up data. Blood samples for hematology and biochemistry analysis were taken from every patient before and at fixed intervals up to 72 h after TAVI including blood count and creatinine. Descriptive statistics were used for patient’s characteristics. KaplanMeier survival curves were used for time to event outcomes. A recursive partitioning regression and classification was used to investigate the association between potential risk factors and outcome variables.RESULTS: Mean age in our study population was 81 ± 6.1 years. Anemia was prevalent in 63.6%(n = 239) of our patients. Age and creatinine were identified as risk factors for anemia. In our study population, anemia per se did influence 30-d mortality but did not predict longterm mortality. In contrast, a RDW > 14% showed to be highly predictable for a reduced short- and longterm survival in patients with aortic valve disease after TAVI procedure.CONCLUSION: Age and kidney function determine the degree of anemia. The anisocytosis of red blood cells in anemic patients supplements prognostic information in addition to that derived from the WHO-based definition of anemia.

  2. Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Epstein, A.L.; Clevenger, C.V.; Huberman, E.

    1985-02-01

    The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin DT induced the cells to acquire a phenotype that resembled that of granulocytes and monocytesmacrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. The authors suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells. 40 references, 2 figures, 1 table.

  3. Narrative Based Medicine as a tool for needs assessment of patients undergoing hematopoietic stem cell transplantation.

    Science.gov (United States)

    Gargiulo, Gianpaolo; Sansone, Vincenza; Rea, Teresa; Artioli, Giovanna; Botti, Stefano; Continisio, Grazia Isabella; Ferri, Paola; Masi, Daniela; Risitano, Antonio Maria; Simeone, Silvio; La Sala, Rachele

    2017-03-14

    In the last years we have seen an ever increasing number of patients with haematologic disorders who need hematopoietic stem cell transplantation (HSCT). The whole sector of HSCT results, infact to be in a continous scientific and technological clinical progress, offering a very advanced care. Despite this, some aspects are underconsidered, some of which could be fundamental to determine the success of the care pathway, such as the experience of the illness by the patient. Using a Narrative Based Medicine approach we wanted to investigate clinical, psychosocial and organizational aspects of the patient's journey whilst undergoing HSCT. Various narrative interviews were conducted using non-structured approach. Results were analysed by thematic contents. Psycological dimension is the most compromised: above all emerged sentiments of oppression linked to the isolation period in the Low Bacterial Load (LBL) room. To note are also the different dynamics with which the patients perceive the organisation and hospital structures, and how much these factors can influence their care experience. Results suggest the need in clinical practice of an integration between qualitative and clinical approach, so as to permit the psychosocial and relational necessities to emerge, often unexpressed by patients undergoing HSCT.

  4. Iron overload in patients with acute leukemia or MDS undergoing myeloablative stem cell transplantation.

    Science.gov (United States)

    Armand, Philippe; Kim, Haesook T; Rhodes, Joanna; Sainvil, Marie-Michele; Cutler, Corey; Ho, Vincent T; Koreth, John; Alyea, Edwin P; Hearsey, Doreen; Neufeld, Ellis J; Fleming, Mark D; Steen, Hanno; Anderson, Damon; Kwong, Raymond Y; Soiffer, Robert J; Antin, Joseph H

    2011-06-01

    Patients with hematologic malignancies undergoing allogeneic stem cell transplantation (HSCT) commonly have an elevated serum ferritin prior to HSCT, which has been associated with increased mortality after transplantation. This has led to the suggestion that iron overload is common and deleterious in this patient population. However, the relationship between serum ferritin and parenchymal iron overload in such patients is unknown. We report a prospective study of 48 patients with acute leukemia (AL) or myelodysplastic syndromes (MDS) undergoing myeloablative HSCT, using magnetic resonance imaging (MRI) to estimate liver iron content (LIC) and cardiac iron. The median (and range) pre-HSCT value of serum ferritin was 1549 ng/mL (20-6989); serum hepcidin, 59 ng/mL (10-468); labile plasma iron, 0 LPI units (0.0-0.9). Eighty-five percent of patients had hepatic iron overload (HIO), and 42% had significant HIO (LIC ≥5.0 mg/gdw). Only 1 patient had cardiac iron overload. There was a strong correlation between pre-HSCT serum ferritin and estimated LIC (r = .75), which was mostly dependent on prior transfusion history. Serum hepcidin was appropriately elevated in patients with HIO. Labile plasma iron elevation was rare. A regression calibration analysis supported the hypothesis that elevated pre-HSCT LIC is significantly associated with inferior post-HSCT survival. These results contribute to our understanding of the prevalence, mechanism, and consequences of iron overload in HSCT.

  5. Modeling and analysis of retinoic acid induced differentiation of uncommitted precursor cells.

    Science.gov (United States)

    Tasseff, Ryan; Nayak, Satyaprakash; Song, Sang Ok; Yen, Andrew; Varner, Jeffrey D

    2011-05-01

    Manipulation of differentiation programs has therapeutic potential in a spectrum of human cancers and neurodegenerative disorders. In this study, we integrated computational and experimental methods to unravel the response of a lineage uncommitted precursor cell-line, HL-60, to Retinoic Acid (RA). HL-60 is a human myeloblastic leukemia cell-line used extensively to study human differentiation programs. Initially, we focused on the role of the BLR1 receptor in RA-induced differentiation and G1/0-arrest in HL-60. BLR1, a putative G protein-coupled receptor expressed following RA exposure, is required for RA-induced cell-cycle arrest and differentiation and causes persistent MAPK signaling. A mathematical model of RA-induced cell-cycle arrest and differentiation was formulated and tested against BLR1 wild-type (wt) knock-out and knock-in HL-60 cell-lines with and without RA. The current model described the dynamics of 729 proteins and protein complexes interconnected by 1356 interactions. An ensemble strategy was used to compensate for uncertain model parameters. The ensemble of HL-60 models recapitulated the positive feedback between BLR1 and MAPK signaling. The ensemble of models also correctly predicted Rb and p47phox regulation and the correlation between p21-CDK4-cyclin D formation and G1/0-arrest following exposure to RA. Finally, we investigated the robustness of the HL-60 network architecture to structural perturbations and generated experimentally testable hypotheses for future study. Taken together, the model presented here was a first step toward a systematic framework for analysis of programmed differentiation. These studies also demonstrated that mechanistic network modeling can help prioritize experimental directions by generating falsifiable hypotheses despite uncertainty.

  6. Kinetics of circulating endothelial progenitor cells in patients undergoing carotid artery surgery

    Directory of Open Access Journals (Sweden)

    Kalender G

    2016-12-01

    Full Text Available G Kalender,1 A Kornberger,2 M Lisy,1 Andres Beiras-Fernandez,2 UA Stock2 1Deparment of General, Thoracic and Vascular Surgery, Hoechst Hospital, 2Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt am Main, Germany Aim: Endothelial progenitor cells (EPCs are primitive cells found in the bone marrow and peripheral blood (PB. In particular, the potential of EPCs to differentiate into mature endothelial cells remains of high interest for clinical applications such as bio-functionalized patches for autologous seeding after implantation. The objective of this study was to determine EPCs’ kinetics in patients undergoing carotid artery thromboendarterectomy (CTEA and patch angioplasty. Methods: Twenty CTEA patients were included (15 male, mean age 76 years. PB samples were taken at 1 day preoperatively, and at 1, 3, and 5 days postoperatively. Flow cytometric analysis was performed for CD34, CD133, KDR, and CD45. Expression of KDR, SDF-1α, and G-CSF was analyzed by means of enzyme-linked immunosorbent assay. Results: Fluorescence-activated cell sorting analysis revealed 0.031%±0.016% (% of PB mononuclear cells KDR+ cells and 0.052%±0.022% CD45-/CD34+/CD133+ cells, preoperatively. A 33% decrease of CD45–/CD34+/CD133+ cells was observed at day 1 after surgery. However, a relative number (compared to initial preoperative values of CD45-/CD34+/CD133+ cells was found on day 3 (82% and on day 5 (94% postoperatively. More profound upregulated levels of CD45–CD34+/CD133+ cells were observed for diabetic (+47% compared to nondiabetic and male (+38% compared to female patients. No significant postoperative time-dependent differences were found in numbers of KDR+ cells and the concentrations of the cytokines KDR and G-CSF. However, the SDF-1α levels decreased significantly on day 1 postoperatively but returned to preoperative levels by day 3. Conclusion: CTEA results in short-term downregulation of circulating

  7. Abrupt decrease of c—myc expression by antisense transcripts induses terminal differentiation and apoptosis in human promyelocytic leukemia HL—60 cells

    Institute of Scientific and Technical Information of China (English)

    HAOXIUJUAN; PEIHSIENTANG; 等

    1996-01-01

    This study was designed using c-myc antisense transcripts to evaluate how alteration of c-myc expression in human myeloid leukemic HL-60 cells could influence the myelomonocytic differentiation and induction of apoptosis.The recombinant plasmid pDACx expressing antisense transcripts to c-myc fragment containing a part of intron 1 and 137 nt exon 2 was constructed.pDACx was transfected into HL-60 cell line by lipofectin reagent.Cytochemical stainings including NBT reduction,peroxidase and α-NAE as well as detection of CD13 and CD33 antigens by flow cytometric analysis indicated occurrence of myelomonocytic differentiation in cells expressing antisense transcripts to c-myc.DNA degradation measured by DNA gel electrophoresis and typical morphological changes observed under electron microscope proved the swith-on of apoptosis in terminally differentiating HL-60 cells.

  8. 5-Episinuleptolide Acetate, a Norcembranoidal Diterpene from the Formosan Soft Coral Sinularia sp., Induces Leukemia Cell Apoptosis through Hsp90 Inhibition

    Directory of Open Access Journals (Sweden)

    Yao-Tsung Yeh

    2013-03-01

    Full Text Available 5-Episinuleptolide acetate (5EPA, a cytotoxic norcembranoidal diterpene recently identified from the Formosan soft coral Sinularia sp., exhibited potent activity against the K562, Molt 4 and HL 60 cancer cell lines. The antiproliferative assay, as well as the annexin V-FITC/propidium iodide (PI apoptotic assay, indicated that the HL 60 cell line is the most sensitive one towards 5EPA. This diterpenoid led to caspases -3, -8, and -9 activation as well as PARP cleavage. It also induced ROS generation, calcium accumulation and disruption of mitochondrial membrane potential. Additionally, the expression levels of Hsp90 protein and several client proteins were downregulated in response to 5EPA treatment. These results suggest that 5EPA’s cytotoxic effect on HL 60 cells may be attributed to the inhibition of Hsp90 as well as the induction of mitochondrial stress which finally results in apoptotic cell death.

  9. Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF.

    Science.gov (United States)

    Frankel, A E; Hall, P D; McLain, C; Safa, A R; Tagge, E P; Kreitman, R J

    1998-01-01

    Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.

  10. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  11. Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification.

    Science.gov (United States)

    Dono, M; Hashimoto, S; Fais, F; Trejo, V; Allen, S L; Lichtman, S M; Schulman, P; Vinciguerra, V P; Sellars, B; Gregersen, P K; Ferrarini, M; Chiorazzi, N

    1996-02-15

    Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha-expressing cDNA were present in greater amounts that unrelated (non-CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

  12. Transfusion rate and prevalence of unexpected red blood cell alloantibodies in women undergoing hysterectomy for benign disease

    DEFF Research Database (Denmark)

    Thoestesen, Lisbeth M; Rasmussen, Kjeld L; Lauszus, Finn F;

    2011-01-01

    To determine transfusion rates, risk factors for transfusion and the prevalence of unexpected red blood cell alloantibodies in women undergoing hysterectomy for benign disease. In addition, we aimed to evaluate the necessity of the pretransfusion testing for red blood cell alloantibodies....

  13. Evaluation of febrile neutropenia in patients undergoing hematopoietic stem cell transplantation.

    Directory of Open Access Journals (Sweden)

    Shahideh Amini

    2014-01-01

    Full Text Available The aim of this study was to determine the incidence and causes of fever as a major problem contributing to transplantation related mortality among patients undergoing hematopoietic stem cell transplantation (HSCT and evaluation of antibiotic use, according to reliable guidelines.We retrospectively reviewed hospital records of 195 adult patients who underwent HSCT between 2009-2011 at hematology-oncology and bone marrow transplantation research center. Baseline information and also data related to fever and neutropenia, patient's outcomes, duration of hospitalization and antibiotic use pattern were documented.A total of 195 patients were analyzed and a total of 268 febrile episodes in 180 patients were recorded (mean 1.5 episodes per patient. About 222 episodes (82% were associated with neutropenia which one-fourth of them were without any documented infection sources. Microbiologic documents showed that the relative frequencies of gram positive and gram negative bacteria were 62.5% and 37.5%, respectively. The hospital stay duration was directly related to the numbers of fever episodes (P<0.0001.The rate of febrile episodes in autologous stem cell transplantation was significantly higher compared to allogeneic type (P<0.05.It is necessary to determine not only the local profile of microbiologic pattern, but also antibiotic sensitivities in febrile neutropenic patients following hematopoietic stem cell transplantation, and reassess response to antibiotic treatment to establish any necessity for modifications to treatment guidelines in order to prevent any fatal complications from infection.

  14. B cell repertoires in HLA-sensitized kidney transplant candidates undergoing desensitization therapy.

    Science.gov (United States)

    Beausang, John F; Fan, H Christina; Sit, Rene; Hutchins, Maria U; Jirage, Kshama; Curtis, Rachael; Hutchins, Edward; Quake, Stephen R; Yabu, Julie M

    2017-01-13

    Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy. We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA. Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells. Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative

  15. Caspofungin as antifungal prophylaxis in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective analysis

    Directory of Open Access Journals (Sweden)

    Döring Michaela

    2012-07-01

    Full Text Available Abstract Background Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT often receive intravenous liposomal amphotericin B (L-AmB as antifungal prophylaxis. There are no guidelines for antifungal prophylaxis in children in this situation. Caspofungin (CAS, a broad-spectrum echinocandin, could be an effective alternative with lower nephrotoxicity than L-AmB. Methods We retrospectively analyzed the safety, feasibility, and efficacy of CAS in our center, and compared the results with L-AmB as antifungal monoprophylaxis in pediatric patients undergoing HSCT. 60 pediatric patients received L-AmB (1 or 3 mg/kg bw/day and another 60 patients received CAS (50 mg/m2/day as antifungal monoprophylaxis starting on day one after HSCT. The median ages of patients receiving L-AmB and CAS were 7.5 years and 9.5 years, respectively. Results No proven breakthrough fungal infection occurred in either group during the median treatment period of 23 days in the L-AmB group and 24 days in the CAS group. One patient receiving CAS developed probable invasive aspergillosis. During L-AmB treatment, potassium levels significantly decreased below normal values. Patients treated with L-AmB had more drug-related side effects and an increased need for oral supplementation with potassium, sodium bicarbonate and calcium upon discharge as compared with the CAS group. CAS was well-tolerated and safe in this cohort of immunocompromised pediatric patients, who underwent high-dose chemotherapy and HSCT. Conclusion Prophylactic CAS and L-AmB showed similar efficacy in this biggest cohort of pediatric patients after allogeneic HSCT reported, so far. A prospective randomized trial in children is warranted to allow for standardized guidelines.

  16. Poor peripheral blood stem cell mobilization affects long-term outcomes in multiple myeloma patients undergoing autologous stem cell transplantation.

    Science.gov (United States)

    Moreb, Jan S; Byrne, Michael; Shugarman, Ilicia; Zou, Fei; Xiong, Sican; May, William S; Norkin, Maxim; Hiemenz, John; Brown, Randall; Cogle, Christopher; Wingard, John R; Hsu, Jack W

    2017-05-29

    Peripheral blood stem cell (PBSC) mobilization is routinely undertaken prior to autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM). A number of studies have identified risk factors for poor PBSC mobilization, however, little data exists to correlate mobilization with disease-specific outcomes in this patient population. Prospective work in MM has demonstrated similar outcomes in a homogenous patient population. In this single institution analysis, we retrospectively studied the impact of poor PBSC mobilization on progression free survival (PFS) and OS in MM patients undergoing PBSC mobilization. Poor mobilizers are defined as patients that collected  10 mcg/kg/day) and/or plerixafor can significantly improve mobilization and ASCT chances in this population. These data indicate that poor mobilization can be predictable and is associated with more aggressive disease biology and worse outcomes, warranting intensive post-ASCT management. © 2017 Wiley Periodicals, Inc.

  17. A possible receptor for β2 glycoprotein Ⅰ on the membrane of hepatoma cell line smmc7721

    Institute of Scientific and Technical Information of China (English)

    高普均; 朴云峰; 王小丛; 曲立科; 时阳; 杨翰仪

    2003-01-01

    Objectives To study the interaction of beta-2-glycoprotein Ⅰ (β2GP Ⅰ) with the membrane of hepatocytes and determine whether β2GP Ⅰ participates in HBV infection.Methods Ligand blotting, fluorescence microscopy, and fluorescence activated cell sorter (FACS) analysis were used to detect the specific interaction of β2GP Ⅰ with the hepatoma cell line smmc7721, the gastric carcinoma cell line SGC7901, and the lymphoma cell line HL-60.Results A specific 40 kDa β2GP Ⅰ band was observed by ligand blotting in the case of smmc7721 cells. No such band was observed in SGC7901 or HL-60 cells. Fluorescence microscopy also revealed specific binding of FITC-β2GP Ⅰ to smmc7721 cells, but neither to SGC7901 nor HL-60 cells. FACS analysis demonstrated that the binding rate of FITC-β2GP Ⅰ to smmc7721 cells was significantly higher than these in SGC7901 and HL-60 cells (P<0.01). The binding rate to smmc7721 cells did not increase with increasing amounts of FITC-β2GP Ⅰ.Conclusions There is a specific β2GP Ⅰ-binding protein on the membrane of hepatoma cells in cell line smmc7721 which as the β2GP Ⅰ receptor may participate in HBV infection of hepatocytes.

  18. Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

    Science.gov (United States)

    Bissenbaev, Amangeldy K; Ishchenko, Alexander A; Taipakova, Sabira M; Saparbaev, Murat K

    2011-10-01

    Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

  19. Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection.

    Science.gov (United States)

    Bayarri-Lara, Clara; Ortega, Francisco G; Cueto Ladrón de Guevara, Antonio; Puche, Jose L; Ruiz Zafra, Javier; de Miguel-Pérez, Diego; Ramos, Abel Sánchez-Palencia; Giraldo-Ospina, Carlos Fernando; Navajas Gómez, Juan A; Delgado-Rodriguez, Miguel; Lorente, Jose A; Serrano, María Jose

    2016-01-01

    Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC) stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs) is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC. 56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR) expression. 51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1%) (p = 0.035). The mean number of CTCs was 3.16 per 10 ml (range 0-84) preoperatively and 0.66 (range 0-3) in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018) and a shorter disease free survival (DFS) (p = .008). In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50-21.946, p = 0.010) and N status (HR = 0.296, 95% CI: 0.091-0.961, p = 0.043) were independent prognostic factors for DFS. CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence.

  20. Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection.

    Directory of Open Access Journals (Sweden)

    Clara Bayarri-Lara

    Full Text Available Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC.56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR expression.51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1% (p = 0.035. The mean number of CTCs was 3.16 per 10 ml (range 0-84 preoperatively and 0.66 (range 0-3 in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018 and a shorter disease free survival (DFS (p = .008. In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50-21.946, p = 0.010 and N status (HR = 0.296, 95% CI: 0.091-0.961, p = 0.043 were independent prognostic factors for DFS.CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence.

  1. Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection

    Science.gov (United States)

    Cueto Ladrón de Guevara, Antonio; Puche, Jose L.; Ruiz Zafra, Javier; de Miguel-Pérez, Diego; Ramos, Abel Sánchez-Palencia; Giraldo-Ospina, Carlos Fernando; Navajas Gómez, Juan A.; Delgado-Rodriguez, Miguel; Lorente, Jose A.; Serrano, María Jose

    2016-01-01

    Background Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC) stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs) is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC. Patients and Methods 56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR) expression. Results 51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1%) (p = 0.035). The mean number of CTCs was 3.16 per 10 ml (range 0–84) preoperatively and 0.66 (range 0–3) in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018) and a shorter disease free survival (DFS) (p = .008). In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50–21.946, p = 0.010) and N status (HR = 0.296, 95% CI: 0.091–0.961, p = 0.043) were independent prognostic factors for DFS. Conclusion CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence. PMID:26913536

  2. Stomatitis-related pain in women with breast cancer undergoing autologous hematopoietic stem cell transplant.

    Science.gov (United States)

    Fall-Dickson, Jane M; Mock, Victoria; Berk, Ronald A; Grimm, Patricia M; Davidson, Nancy; Gaston-Johansson, Fannie

    2008-01-01

    The purpose of this cross-sectional, correlational study was to describe stomatitis-related pain in women with breast cancer undergoing autologous hematopoietic stem cell transplant. The hypotheses that significant, positive relationships would exist between oral pain and stomatitis, state anxiety, depression, and alteration in swallowing were tested. Stomatitis, sensory dimension of oral pain, and state anxiety were hypothesized to most accurately predict oral pain overall intensity. Thirty-two women were recruited at 2 East Coast comprehensive cancer centers. Data were collected on bone marrow transplantation day +7 +/- 24 hours using Painometer, Oral Mucositis Index-20, Oral Assessment Guide, State-Trait Anxiety Inventory, and Beck Depression Inventory. Data analysis included descriptive statistics, correlations, and stepwise multiple regression. All participants had stomatitis; 47% had oral pain, with a subset reporting continuous moderate to severe oral pain despite pain management algorithms. Significant, positive associations were seen between oral pain, stomatitis, and alteration in swallowing and between oral pain with swallowing and alteration in swallowing. Oral pain was not significantly correlated with state anxiety and depression. Oral sensory and affective pain intensity most accurately predicted oral pain overall intensity. Future research needs to explore factors that affect perception and response to stomatitis-related oropharyngeal pain and individual patient response to opioid treatment.

  3. Ovarian tissue cryopreservation in girls undergoing haematopoietic stem cell transplant: experience of a single centre.

    Science.gov (United States)

    Biasin, E; Salvagno, F; Berger, M; Nesi, F; Quarello, P; Vassallo, E; Evangelista, F; Marchino, G L; Revelli, A; Benedetto, C; Fagioli, F

    2015-09-01

    Fertility after childhood haemopoietic stem cell transplant (HSCT) is a major concern. Conditioning regimens before HSCT present a high risk (>80%) of ovarian failure. Since 2000, we have proposed cryopreservation of ovarian tissue to female patients undergoing HSCT at our centre, to preserve future fertility. After clinical and haematological evaluation, the patients underwent ovarian tissue collection by laparoscopy. The tissue was analysed by histologic examination to detect any tumour contamination and then frozen following the slow freezing procedure and cryopreserved in liquid nitrogen. From August 2000 to September 2013, 47 patients planned to receive HSCT, underwent ovarian tissue cryopreservation. The median age at diagnosis was 11.1 years and at the time of procedure it was 13 years, respectively. Twenty-four patients were not pubertal at the time of storage, whereas 23 patients had already experienced menarche. The median time between laparoscopy and HSCT was 25 days. Twenty-six out of 28 evaluable patients (93%) developed hypergonadotropic hypogonadism at a median time of 23.3 months after HSCT. One patient required autologous orthotopic transplantation that resulted in one live birth. Results show a very high rate of iatrogenic hypergonadotropic hypogonadism, highlighting the need for fertility preservation in these patients.

  4. The impact of preparation and support procedures for children with sickle cell disease undergoing MRI

    Energy Technology Data Exchange (ETDEWEB)

    Cejda, Katherine R. [St. Jude Children' s Research Hospital, Child Life Program, Memphis, TN (United States); Smeltzer, Matthew P. [St. Jude Children' s Research Hospital, Department of Biostatistics, Memphis, TN (United States); Hansbury, Eileen N. [Baylor International Hematology Center of Excellence and the Texas Children' s Center for Global Health, Houston, TX (United States); McCarville, Mary Elizabeth; Helton, Kathleen J. [St. Jude Children' s Research Hospital, Department of Radiological Sciences, Memphis, TN (United States); Hankins, Jane S. [St. Jude Children' s Research Hospital, Department of Hematology, Memphis, TN (United States)

    2012-10-15

    Children with sickle cell disease (SCD) often undergo MRI studies to assess brain injury or to quantify hepatic iron. MRI requires the child to lie motionless for 30-60 min, thus sedation/anesthesia might be used to facilitate successful completion of exams, but this poses additional risks for SCD patients. To improve children's ability to cope with MRI examinations and avoid sedation, our institution established preparation and support procedures (PSP). To investigate the impact of PSP in reducing the need for sedation during MRI exams among children with SCD. Data on successful completion of MRI testing were compared among 5- to 12-year-olds who underwent brain MRI or liver R2*MRI with or without receiving PSP. Seventy-one children with SCD (median age 9.85 years, range 5.57-12.99 years) underwent a brain MRI (n = 60) or liver R2*MRI (n = 11). Children who received PSP were more likely to complete an interpretable MRI exam than those who did not 30 of 33; 91% vs. 27 of 38; 71%, unadjusted OR = 4.1 (P = 0.04) and OR = 8.5 (P < 0.01) when adjusting for age. PSP can help young children with SCD complete clinically interpretable, nonsedated MRI exams, avoiding the risks of sedation/anesthesia. (orig.)

  5. Physical exercise for patients undergoing hematopoietic stem cell transplantation: systematic review and meta-analyses of randomized controlled trials

    NARCIS (Netherlands)

    Haren, I.E.P.M.; Timmerman, H.; Potting, C.M.J.; Blijlevens, N.M.A.; Staal, J.B.; Nijhuis-Van der Sanden, M.W.G.

    2013-01-01

    BACKGROUND: The treatment-related burden for patients undergoing hematopoietic stem cell transplantation (HSCT) may be relieved by physical exercises. PURPOSE: The purpose of this study was to summarize and analyze the evidence provided by randomized controlled trials (RCTs) on physical exercise int

  6. Perioperative iloprost and endothelial progenitor cells in uremic patients with severe limb ischemia undergoing peripheral revascularization.

    Science.gov (United States)

    Coppolino, Giuseppe; Buemi, Antoine; Bolignano, Davide; Lacquaniti, Antonio; La Spada, Michele; Stilo, Francesco; De Caridi, Giovanni; Benedetto, Francesco; Loddo, Saverio; Buemi, Michele; Spinelli, Francesco

    2009-11-01

    The incidence of severe limb ischemia (SLI) is high among haemodialysis (HD) patients. Limb rescue rate after surgical revascularization is relatively poor compared with patients with normal renal function. Prostanoids are an interesting category as adjuvants to revascularization. New vessel growth develops not exclusively by proliferation of endothelial cells in vascular extremities but also by cells mobilized from the bone marrow (HSC), transformed into endothelial progenitor cells (EPC) contributing to both re-endothelialization and neovascularization. Basal number of HSC and EPC is significantly reduced in HD patients and correlated with a subsequent defective neovascularization. The aim of this study was to evaluate the effects of perioperative treatment with iloprost in uremic patients with acute ischemia of lower limbs, undergoing surgical revascularization, on endothelial progenitor cells, hypothesizing a possible biological mechanism induced by the prostanoids. A search was also made for vascular remodeling processes through the analysis of the concentrations of soluble adhesion molecules (i-CAM, v-CAM, e-selectin), biochemical markers of endothelial activation. Thirty HD patients with SLI undergoing peripheral revascularization were enrolled (15 were treated with iloprost and 15 with a placebo). Iloprost was administered as an intra-arterial bolus of 3000 ng over 1 to 3 min immediately after revascularization and in the same affected artery. Serum samples were taken before revascularization (T0), at 6 (T6) and 24 h (T24) after infusion to measure sICAM-1, sE-selectin, and sVCAM-1, and for quantification of HSC and EPC. Progenitors were identified by specific surface markers CD34+, CD133+ and VEGFR2+. Count was conducted using PROCOUNT performed in a TRUCOUNT tube and with a FACSort flow cytometer. Before revascularization, all patients showed a decreased number of HSC and EPC. After 6 h, HSC augmented significantly compared with T0 in both groups. The

  7. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

    Science.gov (United States)

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

    2006-04-21

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

  8. Defibrotide for the management of sinusoidal obstruction syndrome in patients who undergo haemopoietic stem cell transplantation.

    Science.gov (United States)

    Coutsouvelis, John; Avery, Sharon; Dooley, Michael; Kirkpatrick, Carl; Spencer, Andrew

    2016-11-01

    Sinusoidal obstruction syndrome, previously known as veno-occlusive disease (VOD/SOS), is a complication in patients undergoing haemopoietic stem cell transplantation (HSCT). Severe VOD/SOS, including progression to multi-organ failure, has resulted in a mortality of greater than 80%. Defibrotide's varying pharmacological actions, particularly on endothelial cells, make it is a useful agent to consider for prophylaxis and treatment of VOD/SOS. Barriers to its routine use include the high acquisition cost and the fact that neither the oral or parenteral formulations are licensed products in many countries at this time. This review summarises available literature on the use of defibrotide in the management of VOD/SOS. Publications consist predominantly of single centre cohort studies and case series. Available evidence indicates that defibrotide is effective in the management of VOD/SOS. Using defibrotide prophylaxis should also be considered, especially in the paediatric setting, where there are available results from a large, open label, randomized controlled trial. Patient outcome data from the larger studies and compassionate programs can inform consensus recommendations on dosing regimen and criteria for the treatment of VOD/SOS with defibrotide in the adult population. The reviewed literature indicates an effective and safe dose for treatment is 25mg/kg/day, continued for at least 14days or until complete response is achieved. Further studies are required to determine the optimal dose and duration of treatment in both paediatric patients and adults. Recent recommendations and a phase 3 trial using historical controls indicate that defibrotide should be included as a pharmacotherapy option in protocols guiding management of VOD/SOS.

  9. Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis.

    Science.gov (United States)

    Jacobo, Patricia Verónica; Fass, Mónica; Pérez, Cecilia Valeria; Jarazo-Dietrich, Sabrina; Lustig, Livia; Theas, María Susana

    2012-11-01

    Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas-Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

  10. Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation

    Directory of Open Access Journals (Sweden)

    Papamentzelopoulou Myrto

    2012-11-01

    Full Text Available Abstract Background RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. Methods A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. Results Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0. Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52. From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013, higher number of retrieved oocytes (p = 0.016, higher basal LH serum levels (p = 0.016 and higher peak estradiol levels (p = 0.013, while the number of fertilized oocytes differed marginally between the two groups (p = 0.089. Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021 and E2 levels (OR = 0.25, p = 0.026. Conclusions Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.

  11. A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy.

    Science.gov (United States)

    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.

  12. 脱氧佛波醇乙酸酯联合阿糖胞苷对急性髓系白血病细胞分化的影响%Effect of 12-deoxyphorbol-13-acetate in combination with cytarabine on cell differentiation in acute myeloid leukemia cell lines

    Institute of Scientific and Technical Information of China (English)

    张艳锋; 郑旭; 善亚君; 柳晓兰; 余祖胤; 从玉文

    2013-01-01

    目的 研究脱氧佛波醇乙酸酯(12-deoxyphorbol 13-acetate,DPA)联合化疗药阿糖胞苷(cytarabine,Ara-C)对人急性髓系白血病(AML)细胞株HL-60、NB4和U937细胞分化的影响,并初步探讨其相关作用机制.方法 用DPA 250 nmol/1和Ara-C 100 nmol/L单独及联合处理HL-60细胞72 h,姬姆萨染色观察细胞形态学改变;不同剂量DPA和Ara-C单独及联合处理HL-60、NB4和U937细胞,将蛋白激酶C抑制剂bisindolymaleimide Ⅰ(GFX)和丝裂原活化蛋白激酶激酶抑制剂U0126分别预先加入DPA和Ara-C联合处理的HL-60细胞,流式细胞仪检测细胞CD11b表达;DPA和Ara-C单独及联合处理HL-60细胞24h后,Western印迹法检测C/EBP α、C/EBPβ和c-Myc蛋白表达的改变.结果 DPA 250 nmol/L与Ara-C 100 nmol/L联合处理HL-60细胞,可引起细胞体积增大、细胞浆增多及细胞核/浆比例减小等细胞分化的典型形态变化.流式检测结果表明,与Ara-C单独给药组相比,DPA和Ara-C联合处理HL-60细胞72 h后,细胞CD11b表达明显增加(P<0.01);DPA 250nmol/L联合Ara-C 50 nmol/L处理NB4和U937细胞48 h后,细胞CD11b表达明显增强(P<0.01).GFX或U0126分别预处理HL-60细胞能够完全阻断DPA联合Ara-C诱导的细胞分化.与空白对照组相比,DPA与Ara-C共处理明显降低HL-60细胞的c-Myc蛋白表达水平(P<0.01).结论 DPA可增强Ara-C诱导的AML细胞分化,其作用机制可能与PKC/ERK通路激活和c-Myc表达降低相关.%Objective To study the effects of the combination treatment of 12-deoxyphorbol 13-acetate (DPA) and chemotherapy agent cytarabine (Ara-C) on differentiation in human acute myeloid leukemia (AML) cell lines HL-60,NB4 and U937,and to explore the related mechanisms.Methods HL-60 cells were treated for 72 h with DPA (250 nmol/L) or Ara-C (100 nmol/L)alone or in combination,the morphological changes were then determined by Giemsa staining.Cell surface marker CD11b expression was detected by flow cytometry in the

  13. Differentiation-inducing activity of lupeol, a lupane-type triterpene from Chinese dandelion root (Hokouei-kon), on a mouse melanoma cell line.

    Science.gov (United States)

    Hata, K; Ishikawa, K; Hori, K; Konishi, T

    2000-08-01

    We examined the differentiation-inducing effects of extracts of 49 wild plants, 25 types of seaweed and 26 mushrooms in Akita on the human leukemia cell line HL60 and a B16 mouse melanoma-derived sub-clone with high differentiation capability (B16 2F2). Differentiation inducers of HL60 cells such as retinoic acid, showed no effects on the differentiation of B16 2F2 cells. Furthermore, chemical compounds known to be inducers of B16 cells, did not induce differentiation of HL60 cells. Screening tests showed that the differentiation of HL60 cells was induced by extracts of 28 wild plants, 10 types of seaweed and 2 mushrooms, and melanogenesis of B16 2F2 cells was increased by extracts of 21 wild plants, 8 types of seaweed and 7 mushrooms. All of the alcoholic extracts of plants belonging to the subfamily Cichorioideae of the family Compositae caused cell differentiation of the melanoma cell line. The extracts of Chinese dandelion root, also inhibited cell growth and induced melanogenesis of B16 2F2 cells. We isolated the active compound from ethanol extracts of the crude drug. Chemical and physical data for the active compound were identical with those for lupeol, a lupane-type triterpene.

  14. Reproductive issues in patients undergoing Hematopoietic Stem Cell Transplantation: an update.

    Science.gov (United States)

    Guida, Maurizio; Castaldi, Maria Antonietta; Rosamilio, Rosa; Giudice, Valentina; Orio, Francesco; Selleri, Carmine

    2016-11-01

    In 1963 George Mathé announced to the world that he had cured a patient of leukaemia by means of a bone-marrow transplant. Since than much progress has been made and nowadays Hematopoietic Stem Cell Transplantation (HSCT) is considered the most effective treatment of numerous severe haematological diseases. Gynaecological complications in HSCT women represent a serious concern for these patients, but often underestimated by clinicians in the view of Overall Survival. The main gynaecological complications of HSCT are represented by: premature ovarian failure (POF), thrombocytopenia-associated menorrhagia, genital symptoms or sexual problems in course of chronic GVHD (cGVHD), osteoporosis, secondary solid tumours due to immunosuppressive drugs to treat cGVHD and severity of cGVHD, and fertility and pregnancy issues. In particular fertility-related issues are always more relevant for patients, whose life expectation is constantly growing up after HSCT.Thus, taking care of a patient undergoing HSCT should primarily include gynaecological evaluation, even before conditioning regimen or chemotherapy for the underlying malignancy, as, in our opinion, it is of great importance to ensure a complete diagnostic work-up and intervention options to guarantee maximum reproductive health and a better quality of life in HSCT women.The present review aims at describing principal features of the aforementioned gynaecological complications of HSCT, and to define, on the basis of current international literature, a specific protocol for the prevention, diagnosis, management and follow-up of gynaecological complications of both autologous and heterologous transplantation, before and after the procedure.

  15. Extramedullary Relapse Following Total Marrow and Lymphoid Irradiation in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Hyun [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Stein, Anthony [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Tsai, Nicole [Department of Biostatistics, City of Hope National Medical Center, Duarte, California (United States); Schultheiss, Timothy E. [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Palmer, Joycelynne [Department of Biostatistics, City of Hope National Medical Center, Duarte, California (United States); Liu, An [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Rosenthal, Joseph [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Department of Pediatrics, City of Hope National Medical Center, Duarte, California (United States); Forman, Stephen J. [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Wong, Jeffrey Y.C., E-mail: jwong@coh.org [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States)

    2014-05-01

    Purpose: Approximately 5% to 20% of patients who undergo total body irradiation (TBI) in preparation for hematopoietic cell transplantation (HCT) can develop extramedullary (EM) relapse. Whereas total marrow and lymphoid irradiation (TMLI) provides a more conformally targeted radiation therapy for patients, organ sparing has the potential to place the patient at a higher risk for EM relapse than TBI. This study evaluated EM relapse in patients treated with TMLI at our institution. Methods and Materials: Patients eligible for analysis had been enrolled in 1 of 3 prospective TMLI trials between 2006 and 2012. The TMLI targeted bones, major lymph node chains, liver, spleen, testes, and brain, using image-guided tomotherapy with total dose ranging from 12 to 15 Gy. Results: A total of 101 patients with a median age of 47 years were studied. The median follow-up was 12.8 months. Incidence of EM relapse and bone marrow (BM) relapse were 12.9% and 25.7%, respectively. Of the 13 patients who had EM relapse, 4 also had BM relapse, and 7 had EM disease prior to HCT. There were a total of 19 EM relapse sites as the site of initial recurrence: 11 soft tissue, 6 lymph node, 2 skin. Nine of these sites were within the target region and received ≥12 Gy. Ten initial EM relapse sites were outside of the target region: 5 sites received 10.1 to 11.4 Gy while 5 sites received <10 Gy. Pretransplantation EM was the only significant predictor of subsequent EM relapse. The cumulative incidence of EM relapse was 4% at 1 year and 11.4% at 2 years. Conclusions: EM relapse incidence was as frequent in regions receiving ≥10 Gy as those receiving <10 Gy. EM relapse rates following TMLI that included HCT regimens were comparable to published results with regimens including TBI and suggest that TMLI is not associated with an increased EM relapse risk.

  16. Extraorally delivered photobiomodulation therapy for prevention of oropharyngeal mucositis in pediatric patients undergoing hematopoietic cell transplantation

    Science.gov (United States)

    Duncan, Christine N.; Yaroslavsky, Anna N.; London, Wendy B.; Juliano, Amy F.; Sonis, Stephen T.; Adnan, Ather; Treister, Nathaniel S.

    2017-02-01

    Background: Oral mucositis (OM) is a painful consequence of myeloablative hematopoietic cell transplantation (HCT). Extraorally delivered photobiomodulation therapy (PBMT) is a promising novel intervention for the prevention of OM in children. Objectives: With funding from an NIDCR R34 planning grant, the objectives of this study are 1) to model the dosimetry of external PBMT and the optimal device parameters for the planned clinical trial, and 2) to plan and design a placebo-controlled Phase 2 multicenter clinical trial to determine whether extraorally delivered PBMT can reduce the duration of severe OM in children, with intent for implementation under subsequent U01 funding. Methods: External PBMT dosimetry will be evaluated using pediatric head and neck MRI studies to obtain serial measurements of different tissues that will then be used to develop a sophisticated computational model. We plan to conduct a placebocontrolled Phase 2 multicenter clinical trial in which patients 4 to 21 years of age will be randomized 1:1:1 to receive external PBMT dose 1x, external PBMT dose 2x, or sham PBMT starting from conditioning, daily until day +20 post- HCT. Significance: Extraorally delivered PBMT is a feasible, potentially efficacious intervention that could improve the quality of life for all children undergoing myeloablative HCT. The planned Phase 2 study, based on rigorous dose modeling and with detailed attention to uniform delivery of therapy and OM assessments, will provide critical efficacy data and the potential basis for a subsequent definitive Phase 3 trial. Grant Support: NIDCR R34 DE025908-01

  17. Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation.

    Science.gov (United States)

    Liu, Lu; Ling, Junqi; Wei, Xi; Wu, Liping; Xiao, Yin

    2009-10-01

    During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

  18. Posaconazole-Loaded Leukocytes as a Novel Treatment Strategy Targeting Invasive Pulmonary Aspergillosis.

    Science.gov (United States)

    Baistrocchi, Shane R; Lee, Mark J; Lehoux, Melanie; Ralph, Benjamin; Snarr, Brendan D; Robitaille, Robert; Sheppard, Donald C

    2017-06-01

    Impaired delivery of antifungals to hyphae within necrotic lesions is thought to contribute to therapeutic failure in invasive pulmonary aspergillosis (IPA). We hypothesized that transfusion of leukocytes loaded ex vivo with the lipophilic antifungal posaconazole could improve delivery of antifungals to the sites of established infection and improve outcome in experimental IPA. The HL-60 leukemia cell line was differentiated to a neutrophil-like phenotype (differentiated HL-60 [dHL-60] cells) and then exposed to a range of posaconazole concentrations. The functional capacity and antifungal activity of these cells were assessed in vitro and in a mouse model of IPA. Posaconazole levels in dHL-60 cells were 265-fold greater than the exposure concentration. Posaconazole-loaded cells were viable and maintained their capacity to undergo active chemotaxis. Contact-dependent transfer of posaconazole from dHL-60 cells to hyphae was observed in vitro, resulting in decreased fungal viability. In a neutropenic mouse model of IPA, treatment with posaconazole-loaded dHL-60 cells resulted in significantly reduced fungal burden in comparison to treatment with dHL-60 cells alone. Posaconazole accumulates at high concentrations in dHL-60 cells and increases their antifungal activity in vitro and in vivo. These findings suggest that posaconazole-loading of leukocytes may hold promise for the therapy of IPA.

  19. Anticancer Potential of Aqueous Ethanol Seed Extract of Ziziphus mauritiana against Cancer Cell Lines and Ehrlich Ascites Carcinoma

    Directory of Open Access Journals (Sweden)

    Tulika Mishra

    2011-01-01

    Full Text Available Ziziphus mauritiana (Lamk. is a fruit tree that has folkloric implications against many ailments and diseases. In the present study, anticancer potential of seed extract of Ziziphus mauritiana in vitro against different cell lines (HL-60, Molt-4, HeLa, and normal cell line HGF by MTT assay as well as in vivo against Ehrich ascites carcinoma bearing Swiss albino mice was investigated. The extract was found to markedly inhibit the proliferation of HL-60 cells. Annexin and PI binding of treated HL-60 cells indicated apoptosis induction by extract in a dose-dependent manner. The cell cycle analysis revealed a prominent increase in sub Go population at concentration of 20 μg/ml and above. Agarose gel electrophoresis confirmed DNA fragmentation in HL-60 cells after 3 h incubation with extract. The extract also exhibited potent anticancer potential in vivo. Treatment of Ehrlich ascites carcinoma bearing Swiss albino mice with varied doses (100–800 mg/kg b.wt. of plant extract significantly reduced tumor volume and viable tumor cell count and improved haemoglobin content, RBC count, mean survival time, tumor inhibition, and percentage life span. The enhanced antioxidant status in extract-treated animals was evident from decline in levels of lipid peroxidation and increased levels of glutathione, catalase, and superoxide dismutase.

  20. KIR2DS2 and KIR2DS4 Promoter hypomethylation patterns in patients undergoing hematopoietic cell transplantation (HCT)

    OpenAIRE

    Gallez-Hawkins, Ghislaine M.; Li, Xiuli; Franck, Anne E.; Gendzekhadze, Ketevan; Nakamura, Ryotaro; Forman, Stephen J.; Senitzer, David; Zaia, John A.

    2012-01-01

    The killer cell Ig-like receptor (KIR)-MHC class I pathway is an integral part of natural killer cell immunity, and its role in host protection from both cancer and infection is important In addition, we have shown elevated KIR2DS2 and 2DS4 expression in PBMCs of patients undergoing hematopoietic cell transplantation (HCT) [1]. Since all inhibitory KIR promoters are known to be heavily methylated, the question asked here is how and when KIR2DS2 and 2DS4 promoters had changed their methylation...

  1. Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats

    Institute of Scientific and Technical Information of China (English)

    Ming-Qing Xu; Zhen-Xiang Yao

    2003-01-01

    AIM: To investigate functional change of dendritic cells (DCs)derived from allogeneic partial liver graft undergoing acuterejection in rats.METHODS: Allogeneic (SD rat to LEW rat) whole and 50 %partial liver transplantation were performed. DCs from livergrafts 0 hr and 4 days after transplantation were isolated andpropagated in the presence of GM-CSFin vitro. Morphologicalcharacteristics of DCs propagated for 4 days and 10 dayswere observed by electron rmicroscopy. Phenotypical featuresof DCs propagated for 10 days were analyzed by flowcytometry. Expression of IL-12 protein and IL-12 receptormRNA in DCs propagated for 10 days was also measured byWestern blotting and semiquantitative RT-PCR, respectively.Histological grading of rejection were determined.RESULTS: Allogeneic whole liver grafts showed no featuresof rejection at day 4 after transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate tosevere rejection at day 4 after transplantation. DCs derivedfrom allogeneic partial liver graft 4 days after transplantationexhibited typical morphological characteristics of DC after 4days' culture in the presence of GM-CSF. DCs from allogeneicwhole liver graft 0 hr and 4 days after transplantation didnot exhibit typical morphological characteristics of DC untilafter 10 days' culture in the presence of GM-CSF. After 10days' propagationin vitro, DCs derived from allogeneic wholeliver graft exhibited features of immature DC, with absenceof CD40, CD80 and CD86 surface expression, and low levelsof IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12receptor (IL-12Rβ1 and IL-12Rβ2) mRNA, whereas DCs fromallogeneic partial liver graft 4 days after transplantationdisplayed features of mature DC, with high levels of CD40,CD80 and CD86 surface expression, and as a consequence,higher expression of IL-12 proteins (IL-12 p35 and IL-12 p40)and IL-12 receptors (IL-12Rβ1 and IL-12Rβ2) mRNA thanthose of DCs both from partial liver graft 0 hr and whole livergraft

  2. The in-vitro antiproliferative effect of PRI-2191 and imatinib applied in combined treatment with cisplatin, idarubicin, or docetaxel on human leukemia cells.

    Science.gov (United States)

    Switalska, Marta; Nasulewicz-Goldeman, Anna; Opolska, Aleksandra; Maciejewska, Magdalena; Kutner, Andrzej; Wietrzyk, Joanna

    2012-01-01

    Imatinib mesylate (Gleevec, STI571) is a specific inhibitor of the Bcr/Abl fusion tyrosine kinase that exhibits potent antileukemic effects in chronic myelogenous leukemia. Bcr/Abl-positive K562 and Bcr/Abl-negative HL-60 human leukemia cells were used to investigate the effect of PRI-2191, a calcitriol analog, on the biological effects of imatinib combined with other anticancer drugs. The results show that PRI-2191 enhances the antiproliferative effect of imatinib on HL-60 cells. When these two agents together are applied with either docetaxel or cisplatin, but not with idarubicin, the antiproliferative effect could still be enhanced. Moreover, when the interaction between the chemotherapy agents was antagonistic or additive, PRI-2191 could even shift it to synergism. This effect correlated with an accumulation of HL-60 cells in the G0/G1 phase of the cell cycle and a decrease in the percentage of cells in the G2/M and S stage in the ternary combinations used. PRI-2191 did not influence apoptosis induced by imatinib alone or in ternary combinations with all the chemotherapy agents used. These results may suggest that the stronger antiproliferative effect of the combined treatment with PRI-2191 on HL-60 cells is related to cell cycle arrest rather than to the induction of apoptosis.

  3. Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.

    Science.gov (United States)

    Rogler, Charles E; Bebawee, Remon; Matarlo, Joe; Locker, Joseph; Pattamanuch, Nicole; Gupta, Sanjeev; Rogler, Leslie E

    2017-01-01

    Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

  4. Successful afatinib treatment of advanced non-small-cell lung cancer patients undergoing hemodialysis.

    Science.gov (United States)

    Imai, Hisao; Kaira, Kyoichi; Naruse, Ichiro; Hayashi, Hideki; Iihara, Hirotoshi; Kita, Yutaro; Mizusaki, Naoki; Asao, Takayuki; Itoh, Yoshinori; Sugiyama, Tadashi; Minato, Koichi; Yamada, Masanobu

    2017-01-01

    The treatment for patients with lung cancer undergoing hemodialysis, who are frequently elderly and have poor performance status, becomes a more important subject. However, the feasibility of afatinib in patients with chronic renal failure undergoing hemodialysis has not, so far, been reported. Here, afatinib was administered to three patients with NSCLC harboring EGFR mutation and chronic renal failure undergoing hemodialysis. Pharmacokinetic (PK) data of afatinib supported the safety of afatinib treatment. After receiving their written informed consent from all patients, they were administered 30 mg afatinib daily with HD three times a week. We performed PK analyses of afatinib on days 1, 2, 10, and 11 after initial administration of afatinib. All three patients exhibited a partial response without any serious adverse events during the administration of afatinib. These PK data were similar to those of patients with normal organ function, which were previously reported. Our findings may be particularly useful given the current opportunity to use afatinib as a first-line treatment for EGFR-mutated NSCLC patients, providing an additional option for patients with impaired renal function.

  5. Induction of apoptosis in human leukemia cells by naturally fermented sugar cane vinegar (kibizu) of Amami Ohshima Island.

    Science.gov (United States)

    Mimura, Akio; Suzuki, Yoshihiro; Toshima, Youhei; Yazaki, Shin-ichi; Ohtsuki, Takashi; Ui, Sadaharu; Hyodoh, Fuminori

    2004-01-01

    Naturally fermented vinegar such as Kibizu (sugar cane vinegar in Amami Ohshima, Japan), Kurozu (black rice vinegar in Kagoshima, Japan), Kouzu (black rice vinegar in China) and red wine vinegar in Italy had potent radical-scavenging activity analyzed by DPPH method. For the elucidation of food factor for cancer prevention contained in naturally fermented vinegar, the induction of apoptosis in human leukemia cell HL-60 was investigated with sugar cane vinegar Kibizu. Fraction eluted by 40% methanol from Amberlite XAD 2 chromatography of sugar cane vinegar showed potent radical scavenging activity. The fraction also showed the activity repressing growth of typical human leukemia cells such as HL-60, THP-1, Molt-4, U-937, Jurkat, Raji and K-562. On the other hand, the fraction did not have any growth inhibition activity against human fetal lung cell TIG-1. The most potent radical-scavenging activity and the growth repression activity of the leukemia cell were observed in the same chromatographic fraction of methanol 40%. From cell sorting FACS analyses, electron microscopic observations and cytochemical staining of chromatin and nuclear segments in human leukemia cell HL-60 treated with the active fraction, it was concluded that apoptosis was induced in the leukemia cell by the fraction of sugar cane vinegar and resulted in the repression of growth of the human leukemia cells. Chromatographic fraction of sugar cane juice eluted by 20% methanol showed potent activities of radical-scavenging and growth repression of HL-60. These results led us the consideration that active components in sugar cane juice could be converted to more lipophilic compounds with activity to induce apoptosis in HL-60 by microbial fermentation with yeast and acetic acid bacteria.

  6. Ginsenoside 20(S)-Rh2 Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells: Role of Orphan Nuclear Receptor Nur77.

    Science.gov (United States)

    Wang, Chengqiang; He, Hui; Dou, Guojun; Li, Juan; Zhang, Xiaomei; Jiang, Mingdong; Li, Pan; Huang, Xiaobo; Chen, Hongxi; Li, Li; Yang, Dajian; Qi, Hongyi

    2017-09-06

    Ginsenoside 20(S)-Rh2 has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. In our study, 20(S)-Rh2 induced the expression of orphan nuclear receptor Nur77 and death receptor proteins Fas, FasL, DR5, and TRAIL, as well as the cleavage of caspase 8 and caspase 3 in HL-60 cells. Importantly, shNur77 attenuated 20(S)-Rh2-induced apoptosis and Fas and DR5 expression. Meanwhile, 20(S)-Rh2 promoted Nur77 translocation from the nucleus to mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2 and activation of Bax. Furthermore, 20(S)-Rh2 promoted the differentiation of HL-60 cells as evidenced by Wright-Giemsa staining, NBT reduction assay, and detection of the myeloid differentiation marker CD11b by flow cytometry. Notably, shNur77 reversed 20(S)-Rh2-mediated HL-60 differentiation. Additionally, 20(S)-Rh2 also exhibited an antileukemic effect and induced Nur77 expression in NOD/SCID mice with the injection of HL-60 cells into the tail vein. Together, our studies suggest that the Nur77-mediated signaling pathway is highly involved in 20(S)-Rh2-induced apoptosis and differentiation of AML cells.

  7. Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation.

    Science.gov (United States)

    Fernández, Carlos; Lobo Md, María del Val T; Gómez-Coronado, Diego; Lasunción, Miguel A

    2004-10-15

    As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14alpha-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells.

  8. Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Sook-Kyoung Heo

    Full Text Available Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI, is approved for the second-line treatment of chronic myeloid leukemia (CML in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA. We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

  9. Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

    Science.gov (United States)

    Voss, Edward; Kuhl, Spencer; Buchele, Emma C.; Klemme, Michael R.; Russell, Kanoe B.; Ambrose, Joseph; Soll, Benjamin A.; Bossler, Aaron; Milhem, Mohammed; Goldman, Charles

    2017-01-01

    Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs. PMID:28264026

  10. Antioxidant Activities and Anti-Cancer Cell Proliferation Properties of Natsuhaze (Vaccinium oldhamii Miq., Shashanbo (V. bracteatum Thunb. and Blueberry Cultivars

    Directory of Open Access Journals (Sweden)

    Hirotoshi Tsuda

    2013-02-01

    Full Text Available Antioxidants are abundant in blueberries, and while there are many studies concerning the bioactive compound of fruit, it is only recently that the wild Vaccinium species has attracted attention for their diverse and abundant chemical components. The aim of this study was to investigate the bioactive compounds of blueberry cultivars and wild species found in Japan. Among the five extracts of the Vaccinium species, Natsuhaze (Vaccinium oldhamii Miq. was found to be the most effective at inhibiting the growth of HL-60 human leukemia cells in vitro. Although all ethanol extracts showed a growth inhibitory effect on HL-60 cells, the degree of the effects differed among the species. The extract of Natsuhaze induced apoptotic bodies and nucleosomal DNA fragmentation in the HL-60 cells. Of the extracts tested, that of Natsuhaze contained the largest amount of total polyphenols and showed the greatest antioxidant activity, but the anthocyanin content of Natsuhaze was similar to that of rabbiteye blueberry (V. virgatum Ait.. The results showed that total polyphenols contributed to the high antioxidant activity and growth inhibitory effect on HL-60 human leukemia cells of Natsuhaze extract.

  11. Antioxidant Activities and Anti-Cancer Cell Proliferation Properties of Natsuhaze (Vaccinium oldhamii Miq.), Shashanbo (V. bracteatum Thunb.) and Blueberry Cultivars.

    Science.gov (United States)

    Tsuda, Hirotoshi; Kunitake, Hisato; Kawasaki-Takaki, Ryoko; Nishiyama, Kazuo; Yamasaki, Masao; Komatsu, Haruki; Yukizaki, Chizuko

    2013-02-15

    Antioxidants are abundant in blueberries, and while there are many studies concerning the bioactive compound of fruit, it is only recently that the wild Vaccinium species has attracted attention for their diverse and abundant chemical components. The aim of this study was to investigate the bioactive compounds of blueberry cultivars and wild species found in Japan. Among the five extracts of the Vaccinium species, Natsuhaze (Vaccinium oldhamii Miq.) was found to be the most effective at inhibiting the growth of HL-60 human leukemia cells in vitro. Although all ethanol extracts showed a growth inhibitory effect on HL-60 cells, the degree of the effects differed among the species. The extract of Natsuhaze induced apoptotic bodies and nucleosomal DNA fragmentation in the HL-60 cells. Of the extracts tested, that of Natsuhaze contained the largest amount of total polyphenols and showed the greatest antioxidant activity, but the anthocyanin content of Natsuhaze was similar to that of rabbiteye blueberry (V. virgatum Ait.). The results showed that total polyphenols contributed to the high antioxidant activity and growth inhibitory effect on HL-60 human leukemia cells of Natsuhaze extract.

  12. Antioxidant Activities and Anti-Cancer Cell Proliferation Properties of Natsuhaze (Vaccinium oldhamii Miq.), Shashanbo (V. bracteatum Thunb.) and Blueberry Cultivars

    Science.gov (United States)

    Tsuda, Hirotoshi; Kunitake, Hisato; Kawasaki-Takaki, Ryoko; Nishiyama, Kazuo; Yamasaki, Masao; Komatsu, Haruki; Yukizaki, Chizuko

    2013-01-01

    Antioxidants are abundant in blueberries, and while there are many studies concerning the bioactive compound of fruit, it is only recently that the wild Vaccinium species has attracted attention for their diverse and abundant chemical components. The aim of this study was to investigate the bioactive compounds of blueberry cultivars and wild species found in Japan. Among the five extracts of the Vaccinium species, Natsuhaze (Vaccinium oldhamii Miq.) was found to be the most effective at inhibiting the growth of HL-60 human leukemia cells in vitro. Although all ethanol extracts showed a growth inhibitory effect on HL-60 cells, the degree of the effects differed among the species. The extract of Natsuhaze induced apoptotic bodies and nucleosomal DNA fragmentation in the HL-60 cells. Of the extracts tested, that of Natsuhaze contained the largest amount of total polyphenols and showed the greatest antioxidant activity, but the anthocyanin content of Natsuhaze was similar to that of rabbiteye blueberry (V. virgatum Ait.). The results showed that total polyphenols contributed to the high antioxidant activity and growth inhibitory effect on HL-60 human leukemia cells of Natsuhaze extract. PMID:27137366

  13. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.

    Science.gov (United States)

    Koopman, G; Reutelingsperger, C P; Kuijten, G A; Keehnen, R M; Pals, S T; van Oers, M H

    1994-09-01

    Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

  14. [Prophylaxis against tuberculosis in pediatric and adult patients undergoing solid organ and hematopoietic stem cells transplantation].

    Science.gov (United States)

    Lafourcade, Mónica

    2012-09-01

    Recipients of SOT and HSCT constitute a risk group for becoming ill with tuberculosis (TB). The prevalence of active TB in patients undergoing TOS is higher than in patients undergoing HSCT, probably for the shortest period of immunosuppression of the latter. Most TB cases occur in transplant patients by reactivation of latent infection after immunosuppression, which occurs most often within the first year post-transplant, causing graft loss and in some cases death. Relevant variables to assess the risk of TB infection in a transplant recipient are the medical history of donor and recipient, images, microbiology and tuberculin tests and interferon gamma levels. PPD is routinely performed in the donor and in the recipient before transplantation. If PPD is > 5 mm in the recipient or > 10 mm in the donor, it is neccesary to exclude active TB (pulmonary and renal) (A2). It is recommended che-moprophylaxis in recipients PPD (+) and in recipients with recent seroconversion (B3), if the donor has a history of untreated TB, there was contact to someone with active TB (B3), the radiological imeges are suspect (A2) and interferon gamma release assays is (+) (B2). The selected drug is isoniazid (C3).

  15. Effects of Electro-acupuncture on T Cell Subpopulations, NK Activity,Humoral Immunity and Leukocyte Count in Patients Undergoing Chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Ye Fang; Liu Deshan; Wang Shuli; Xu Lan; Wang Xinzhong

    2007-01-01

    Objective: To observe the effects of electro-acupuncture on T cell subpopulations, natural killer