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Sample records for k-562 human leukemia

  1. DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562-n

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    吕书晴; 许小平; 夏放; 居小萍; 李瑶; 应康; 毛裕民

    2003-01-01

    Objective: To study the molecular mechanism of different tumorigenicity in nude mice of human leukemia cell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells by using cDNA microarray technique. Results: Among the 12800 genes detected, some genes involved in material metabolism and material transport were differently expressed between K562-n and K562 cells. These genes include homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene, hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase, lysophosphatidic acid acyltransferase, alpha gene, argininosuccinate lyase gene, mitochondrial isocitrtate dehydrogenase, adhesion protein SQM1 gene, dimethylarginine dimethylamino-hydrolase gene, M1 subunit of ribonucleotide reductase and farnesyl pyrophosphate synthetase gene. Conclusion: The high tumorigenicity of K562-n cells is related to the different expression of some genes concerned with cell metabolism and material transpoert.

  2. Analysis of Gene Expression in the K562-n High Tumorigenitic Human Leukemia Cell Line

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    Shuqing Lü; Xiaoping Xu; Fang Xia; JianMin Wang

    2005-01-01

    OBJECTIVE The human leukemia K562-n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined.METHODS The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays.RESULTS Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562-n and K562 cells.Eighty-five of these genes have been registered in the GeneBank and 54are unknown. The genes accessible from the GeneBank include:1)oncogenes and tumor-supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions.CONCLUSION There are many genes differentially expressed between K562-n and K562 cells .The high tumorigenicity of the human leukemia K562-n cell line in nude mice might be related to its specific geneexpression profile.

  3. Antitumor Effect of Betulinic Acid on Human Acute Leukemia K562 Cells in vitro

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    吴秋玲; 何静; 方峻; 洪梅

    2010-01-01

    The effects of betulinic acid (BA), a pentacyclic lupane-type triterpene, on the cell viability, cell cycle and apoptosis in human leukemia K562 cells were investigated. The effects of BA on the growth of K562 cells were studied by MTT assay. Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry. The effects of BA on the cell cycle of K562 cells were studied by a PI method. The expression of Bax and capase-3 was detected by using Western blot. The results showed that BA was ...

  4. Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2.

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    Wang, C L; Ng, T B; Yuan, F; Liu, Z K; Liu, F

    2007-07-01

    A new cyclic lipopeptide (CLP) purified from Bacillus subtilis natto T-2 dose dependently inhibited growth in human leukemia K562 cells. The results of fluorescent staining indicated that CLP brought about apoptosis in K562 cells. Flow cytometric analysis also demonstrated that CLP caused dose-dependent apoptosis of K562 cells through cell arrest at G1 phase. Western blotting revealed that CLP-induced apoptosis in K562 cells was associated with caspase-3 and poly(ADP-ribose)polymerase (PARP) protein. It is estimated that CLP inhibited proliferation in K562 cells by inducing apoptosis.

  5. Analysis of the erythroid differentiation effect of flavonoid apigenin on K562 human chronic leukemia cells.

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    Isoda, Hiroko; Motojima, Hideko; Onaga, Shoko; Samet, Imen; Villareal, Myra O; Han, Junkyu

    2014-09-05

    The erythroid differentiation-inducing effect of apigenin and its derivatives on human chronic myeloid leukemia K562 has been reported but the functional group in its structure responsible for the effect has not yet been elucidated. Here, we determined the moiety responsible for the erythroid differentiation induction effect of apigenin by using different flavonoids to represent the functional groups in its structure. In addition, we compared apigenin and apigetrin, a flavonoid similar in structure to apigenin except for the glycoside in its structure. Morphological changes as well as expressions of specific markers in K562 cells treated with apigenin were compared with those treated with apigetrin, flavone, 7-hydroxyflavone, chrysin, luteolin, or naringenin. The anti-proliferative and erythroid differentiation-inducing effect of apigenin and the five flavonoids were then investigated and their effects on the α, β, and γ globin genes expressions were compared using real-time PCR. Results of the comparison between apigenin and apigetrin revealed that the glycoside part of apigetrin does not have a role in the induction of cell differentiation. Based on glycophorin A expression, the potency of the other flavonoids for induction of differentiation, was: apigenin>chrysin>flavone/7-hydroxyflavone>luteolin/naringenin. Results of the analysis of the relationship between the structure and function of the flavonoids suggest that the apigenin-induced K562 cell differentiation was due to the 2-3 double bond and hydroxyl groups in its structure. This is the first study that identified the specific functional group in apigenin that impact the erythroid differentiation effect in K562 cells.

  6. The effect of β-ionone on telomerase activity in the human leukemia cell line K562

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    Zohreh Faezizadeh

    2015-06-01

    Full Text Available Background: Telomerase is highly activated in most human cancer cells, therefore, its inhibition has been proposed as a novel and promising strategy for cancer therapy. Many plant-derived anticancer agents act through inhibition of telomerase activity and induction of apoptosis. β-ionone, a carotenoid compound isolated from Roseaceae, has been reported to possess anticancer properties. The present study was undertaken to examine the mechanism of β-ionone-induced apoptosis in human leukemia cell line K562 with special emphasis on its role in telomerase inhibition. Method: In this study the anti-proliferation effect of β-ionone on K562 cells was evaluated by MTT assay. Apoptosis rate was detected by Hoechst staining and flow cytometry analysis. Telomerase activity was measured by (TRAP ELISA assay. Results: Exposure of K562 cells to β-ionone caused a dose-dependent decrease in proliferation. Flow cytometry analysis and Hoechst staining showed that percentage of apoptotic cells markedly increased with an increase in β-ionone concentration. Compared to control cells, treatment of K562 cells with β-ionone resulted in a significant decrease of telomerase activity. Moreover, a positive correlation was detected between telomerase inhibition and apoptosis induction in the treated K562 cells. Conclusion: Based on these results, β-ionone is an appropriate candidate for inhibiting telomerase activity in K562 cells. Therefore, it may be utilized as a novel drug against some leukemia cell lines.

  7. Extracts of Medicinal Mushrooms Agaricus bisporus and Phellinus linteus Induce Proapoptotic Effects in the Human Leukemia Cell Line K562

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    Shnyreva, A.V.; Song, W.; Griensven, van L.J.L.D.

    2010-01-01

    We have studied the effects of Agaricus bisporus and Phellinus linteus ethanol extracts on transcriptional regulation of genes involved in cytokine release and apoptosis in the human leukemia cell line K562. In particular, we applied quantitative real-time PCR (Q-PCR) assays to monitor alterations o

  8. Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells.

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    Shi, Dan; Liu, Yan; Xi, Ronggang; Zou, Wei; Wu, Lijun; Zhang, Zhiran; Liu, Zhongyang; Qu, Chao; Xu, Baoli; Wang, Xiaobo

    Chronic myelogenous leukemia (CML) is characterized by the t(9;22) (q34;q11)-associated Bcr-Abl fusion gene, which is an essential element of clinical diagnosis. As a traditional Chinese medicine, realgar has been widely used for the treatment of various diseases for >1,500 years. Inspired by nano-drug, realgar nanoparticles (NPs) have been prepared with an average particle size of processes including tumorigenesis and tumor development. In previous studies, it was found that realgar NPs can inhibit several types of tumor cell proliferation. However, the therapeutic effect of realgar NPs on CML has not been fully elucidated. In the present paper, it was demonstrated that realgar NPs can inhibit the proliferation of K562 cells and degrade Bcr-Abl fusion protein effectively. Both apoptosis and autophagy were activated in a dose-dependent manner in realgar NPs treated cells, and the induction of autophagy was associated with class I phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Morphological analysis indicated that realgar NPs induced differentiation effectively in CML cells. Furthermore, it was identified that Cav-1 might play a crucial role in realgar NP therapy. In order to study the effects of Cav-1 on K562 cells during realgar NP treatment, a Cav-1 overexpression cell model was established by using transient transfection. The results indicated that Cav-1 overexpression inhibited K562 cell proliferation, promoted endogenic autophagy, and increased the sensitivity of K562 cells to realgar NPs. Therefore, the results demonstrated that realgar NPs degraded Bcr-Abl oncoprotein, while the underlying mechanism might be related to apoptosis and autophagy, and Cav-1 might be considered as a potential target for clinical comprehensive therapy of CML.

  9. Arecoline-induced death of human leukemia K562 cells is associated with surface up-modulation of TNFR2.

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    Chen, Ying-Jung; Chang, Long-Sen

    2012-05-01

    The goal of the present study is to explore the contribution of tumor necrosis factor-α (TNFα)-related pathway to the cytotoxicity of arecoline on human leukemia K562 cells. Arecoline treatment induced death of K562 cells and increased surface expression of TNFα, TNFR1, and TNFR2. Unlike that of TNFR1 mRNA, transcriptional levels of TNFα and TNFR2 mRNA increased in arecoline-treated cells. Moreover, arecoline-induced down-regulation of ADAM17 maturation was involved in surface up-modulation of TNFR1, TNFR2, and TNFα. Arecoline-elicited increase in intracellular Ca(2+) concentration was responsible for JNK/c-Jun pathway activation and ERK inactivation. Abolition of JNK/c-Jun pathway suppressed arecoline-induced increase in transcriptional level of TNFα and TNFR2 mRNA. TNFα and TNFR2 promoter luciferase activity and chromatin immunoprecipitating analyses revealed that c-Jun increasingly bound with TNFα and TNFR2 promoter upon arecoline treatment. Over-expression of constitutively active MEK1 abolished the effect of arecoline on suppressing ADAM17 maturation. Pretreatment with TNFR2 antibody abrogated arecoline-induced increased susceptibility of K562 cells for the cytotoxicity of TNFα and arecoline-induced cell death. Taken together, our data suggest that up-modulation of TNFR2 surface expression is associated with arecoline-induced death of K562 cells. Copyright © 2011 Wiley Periodicals, Inc.

  10. Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells

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    Shi D

    2016-11-01

    Full Text Available Dan Shi,1,* Yan Liu,1,* Ronggang Xi,1 Wei Zou,2 Lijun Wu,3 Zhiran Zhang,1 Zhongyang Liu,1 Chao Qu,1 Baoli Xu,1 Xiaobo Wang1 1Department of Pharmacy, The 210th Hospital of People’s Liberation Army, 2College of Life Science, Liaoning Normal University, Dalian, Liaoning, 3Department of Pharmaceutics, College of Pharmacy, Harbin Medical University, Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Chronic myelogenous leukemia (CML is characterized by the t(9;22 (q34;q11-associated Bcr-Abl fusion gene, which is an essential element of clinical diagnosis. As a traditional Chinese medicine, realgar has been widely used for the treatment of various diseases for >1,500 years. Inspired by nano-drug, realgar nanoparticles (NPs have been prepared with an average particle size of <100 nm in a previous work. Compared with coarse realgar, the realgar NPs have higher bioavailability. As a principal constituent protein of caveolae, caveolin-1 (Cav-1 participates in regulating various cellular physiological and pathological processes including tumorigenesis and tumor development. In previous studies, it was found that realgar NPs can inhibit several types of tumor cell proliferation. However, the therapeutic effect of realgar NPs on CML has not been fully elucidated. In the present paper, it was demonstrated that realgar NPs can inhibit the proliferation of K562 cells and degrade Bcr-Abl fusion protein effectively. Both apoptosis and autophagy were activated in a dose-dependent manner in realgar NPs treated cells, and the induction of autophagy was associated with class I phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Morphological analysis indicated that realgar NPs induced differentiation effectively in CML cells. Furthermore, it was identified that Cav-1 might play a crucial role in realgar NP therapy. In order to study the effects of Cav-1 on K562 cells during

  11. Mastic oil from Pistacia lentiscus var. chia inhibits growth and survival of human K562 leukemia cells and attenuates angiogenesis.

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    Loutrari, Heleni; Magkouta, Sophia; Pyriochou, Anastasia; Koika, Vasiliki; Kolisis, Fragiskos N; Papapetropoulos, Andreas; Roussos, Charis

    2006-01-01

    Mastic oil from Pistacia lentiscus var. chia, a natural plant extract traditionally used as a food additive, has been extensively studied for its antimicrobial activity attributed to the combination of its bioactive components. One of them, perillyl alcohol (POH), displays tumor chemopreventive, chemotherapeutic, and antiangiogenic properties. We investigated whether mastic oil would also suppress tumor cell growth and angiogenesis. We observed that mastic oil concentration and time dependently exerted an antiproliferative and proapoptotic effect on K562 human leukemia cells and inhibited the release of vascular endothelial growth factor (VEGF) from K562 and B16 mouse melanoma cells. Moreover, mastic oil caused a concentration-dependent inhibition of endothelial cell (EC) proliferation without affecting cell survival and a significant decrease of microvessel formation both in vitro and in vivo. Investigation of underlying mechanism(s) demonstrated that mastic oil reduced 1) in K562 cells the activation of extracellular signal-regulated kinases 1/2 (Erk1/2) known to control leukemia cell proliferation, survival, and VEGF secretion and 2) in EC the activation of RhoA, an essential regulator of neovessel organization. Overall, our results underscore that mastic oil, through its multiple effects on malignant cells and ECs, may be a useful natural dietary supplement for cancer prevention.

  12. Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

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    Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

    2005-10-01

    Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

  13. Induction of apoptosis by Cordyceps militaris fraction in human chronic myeloid leukemia K562 cells involved with mitochondrial dysfunction

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    Tian, Tian; Song, Liyan; Zheng, Qin; Hu, Xianjing; Yu, Rongmin

    2014-01-01

    Background: Cordyceps militaris is widely used for various ethno medical conditions including cancer and inflammation complications in traditional Chinese medicine. Objective: To investigate the in vitro antitumor activity of Cordyceps militaris fraction (CMF) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. Materials and Methods: CMF was prepared according to our previous report. Cell viability was assessed by MTT assay. The rate of apoptosis, distribution of cell cycle and loss of mitochondrial membrane potential were measured by flow cytometry. Caspase activities were analyzed by Western blot and oxygen consumption rate was recorded using the Oxytherm system. Results: The results demonstrated that CMF triggered growth inhibition in K562 cells with only minor toxicity on a normal human cell line and inhibited the proliferation of K562 cells in a dose- and time-dependent manner with IC50 value of 34.1 ± 2.0 μg/ml after 48 h incubation. This most likely resulted from cell cycle arrest at the S phase and the induction of apoptosis. In addition, CMF induced activation of caspase-3 and subsequent cleavage of poly ADP-ribose polymerase (PARP). The caspase signals may originate from mitochondrial dysfunction, which was supported by the finding of decreased mitochondria transmembrance potential and the lower oxygen consumption rate. Conclusion: CMF possessed the in vitro antitumor effect on K562 cells and CMF-induced apoptosis might be involved by the mitochondrial dysfunction and valuable to research and develop as a potential antitumor agency. PMID:25210321

  14. In vitro anti-telomerase activity of novel lycopene-loaded nanospheres in the human leukemia cell line K562

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    Gharib, Amir; Faezizadeh, Zohreh

    2014-01-01

    Background: Lycopene, a plant carotenoid, has potent effects against the various types of cancer cells. To date, the effect of lycopene in the free and encapsulated forms on the telomerase activity in human leukemia cell line K562 have not been investigated. The aim of the present study was to prepare a novel lycopene-loaded nanosphere and compare its anti-telomearse activity in K562 cell line with those of free lycopene. Materials and Methods: The lycopene-loaded nanospheres were prepared by nanoprecipitation method. The lycopene entrapment efficacy was measured by high-performance liquid chromatography (HPLC) method. The anti-proliferation effect of the lycopene in the free and encapsulated forms in the different times (0-72 h) and the different doses (0-100 μg/ml) on K562 cell line was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The changes of telomerase activity, following treatment with the lycopene in the free and encapsulated forms, were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay. Results: The entrapment efficacy of lycopene was 78.5% ± 2. Treatment of the K562 cell line with lycopene, in particular in encapsulated form, resulted in a significant inhibition of the cell growth and increasing of percentage of apoptotic cells. It has also been observed that the telomerase activity in the lycopene-loaded nanospheres-treated cells was significantly inhibited in a dose and time-dependent manner. Conclusion: Our data suggest a novel mechanism in the anti-cancer activity of the lycopene, in particular in encapsulated form, and could be provided a basis for the future development of anti-telomerase therapies. PMID:24914298

  15. Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

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    Yao, Jingyun; Wei, Xing [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China); Lu, Yanhua, E-mail: luyanhua@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China)

    2016-05-13

    Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line. - Highlights: • Chaetominine enhanced chemosensitivity of ADR against K562/Adr cells. • Chaetominine increased intracellular ADR levels via inhibiting MRP1. • Chaetominine induced apoptosis of K562/Adr cells through upregulation of ROS and modulation of Bax/Bcl-2. • Inhibition of MRP1 and Nrf2 by chaetominine treatment was correlative with blockade of PI3K/Akt signaling.

  16. Apoptotic Mechanism of Human Leukemia K562/A02 Cells Induced by Magnetic Ferroferric Oxide Nanoparticles Loaded with Wogonin

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    Miao-Xin Peng

    2016-01-01

    Conclusions: This study demonstrated that MNPs were the effective drug delivery vehicles to deliver wogonin to the leukemia cells. Through increasing cells arrested at G0/G1-phase and inducing apoptosis of K562/A02 cells, MNPs could enhance the therapeutic effects of wogonin on leukemia cells. These findings indicated that MNPs loaded with wogonin could provide a promising way for better leukemia treatment.

  17. 5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

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    Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Fan, Zhen-Chuan [Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457 (China); Obesita and Algaegen LLC, College Station, TX 77845 (United States); Zhang, Yong-Min [Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 Place Jussieu, 75005 Paris (France); Teng, Yu-Ou, E-mail: tyo201485@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Yu, Peng, E-mail: yupeng@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

    2014-08-08

    Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

  18. Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance.

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    Pytel, Dariusz; Wysocki, Tomasz; Majsterek, Ireneusz

    2006-09-01

    The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.

  19. Reversal Effect and Mechanisms of Recombinant Human Tumor Necrosis Factor-NC Against the Doxorubicin Resistance in Leukemia K562/Doxorubicin Cells

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    ZHOU Jing-hong; CHEN Bo-hua

    2015-01-01

    Objective: To explore the reversal effect and mechanisms of recombinant human tumor necrosis factor-NC (rhTNF-NC) against the doxorubicin (Dox) resistance in chronic myelogenous leukemia (CML) K562/Dox cells. Methods: The chemo-sensitivity of tumor cells dealt with different concentrations of rhTNF-NC to Dox was detected by tetrazolium dye assay (MTT). The intra-cellular Dox accumulation represented by lfuorescence intensity was determined by lfow cytometry (FCM) at the excitation wave length of 488 nm and emission wave length of 550 nm. The expression of multidrug resistance (MDR)-related genes and proteins was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Results:After being exposed to gradually increasing concentrations of Dox for 10 consecutive months, K562/Dox cells were more resistant to Dox (nearly 132 times) than Dox-sensitive K562 cells. The IC50 of Dox for K562 and K562/Dox cells were (0.04±0.01) and (5.55±0.08) μmol/L, respectively. When K562/Dox cells were treated with rhTNF-NC at 500, 2 500 or 5 000U/mL, the IC50 of Dox was decreased to (2.22±0.34), (1.41±0.13) and (1.04±0.09) μmol/L, respectively. The concentration-response curves were moved upward by the treatment of rhTNF-NC (P Conclusion: rhTNF-NC can effectively augment the drug accumulation in tumor cells. This is due to the up-regulation of TopoIIα and down-regulation of MDR1, MRP and GSTπ at mRNA expression as well as reduction of P-gp and PKCα expression.

  20. Carbenoxolone Induces Apoptosis and Inhibits Survivin and Survivin-ΔEx3 Genes Expression in Human Leukemia K562 Cells

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    Z. Sanaat

    2011-12-01

    Full Text Available Background and the purpose of the study: Leukemia is a malignant disorder of the blood progenitor/stem cells which is characterized by abnormal proliferation of white blood cells. Although anti-cancer drugs induce apoptosis in cancerous cells, drug resistance is the significant problem mainly due to over-expression of inhibitors of apoptosis proteins (IAPs such as survivin. In this content, it has been reported that an anti-inflammatory drug, Carbenoxolone (CBX, could induce apoptosis and growth inhibition in several types of cancerous cells. In the present study, effects of CBX on apoptosis and level of the expression of survivin gene and its ΔEx3 splicing variant have were evaluated in K562 cells.Methods: K562 cells were cultured and treated with different concentrations of CBX (50-300 μM at different time intervals (12-48 hrs. Trypan blue exclusion test was used to evaluate cell viability. Fluorescent microscopy (Acridine Orange/Ethidium Bromide double staining and DNA fragmentation assay were used to study apoptosis. The expression level of survivin and its ΔEx3 splice variant were studied by RT- PCR.Results and Major Conclusion: It was found that both growth inhibition and apoptosis occurred in K562 cells. In addition, down-regulation of survivin and survin-ΔEx3 were observed, after 2-4 hrs treatment with 150 μM of CBX. However, the expression level of survivin and its ΔEx3 splice variant increased in subsequent time (6-12 hrs nearly to the level of control cells. From the results of this study, it may be concluded that CBX can be considered as a candidate for further studies in CML treatment, especially in the case of drug- resistant leukemia cells.

  1. Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

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    Song Tusheng; Yang Ling; Huang Chen; Liu Liying; Ni Lei; Wang Aiying; Luo Yu

    2007-01-01

    Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P<0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

  2. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation.

  3. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    Science.gov (United States)

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

  4. Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562.

    Science.gov (United States)

    Sreekanth, Devalraju; Arunasree, M K; Roy, Karnati R; Chandramohan Reddy, T; Reddy, Gorla V; Reddanna, Pallu

    2007-11-01

    Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.

  5. Anti-proliferative and apoptotic effects of the derivatives from 4-aryl-4H-chromene family on human leukemia K562 cells.

    Science.gov (United States)

    Aryapour, Hassan; Mahdavi, Majid; Mohebbi, Seyed Reza; Zali, Mohammad Reza; Foroumadi, Alireza

    2012-09-01

    Previous studies suggest that 4-aryl-4H-chromenes are potent apoptosis-inducing agents in various cancer cell lines. In this study, anti-proliferative and apoptotic effects of the derivatives from 4-aryl-4H-chromene family were investigated in the human leukemia K562 cells using [3-(4,5)-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) growth inhibition assay. 3-NC was more active among these compounds with IC₅₀ of 65 nM and was selected for further studies. Apoptosis, as the mechanism of cell death, was investigated morphologically by Hoechst 33258 staining, cell surface expression assay of phosphatidylserine by Annexin V/PI technique, caspase-3 activation assay, as well as the formation of DNA ladder. The K562 cells underwent apoptosis upon a single dose (at IC₅₀ value) of the compound, and also increased caspase-3 activity by more than 2.3-fold, following a 72 h treatment. Caspase-9 was also activated which could be detected 48 hours post-treatment. Furthermore, Western blot analysis revealed that the treatment with the compound down-regulated the expression of certain IAP protein, including survivin. These data further suggest that these derivatives from 4-aryl-4H-chromene may provide a novel therapeutic approach for the treatment of leukemia.

  6. Natural and semi-synthetic clerodanes of Croton cajucara and their cytotoxic effects against ehrlich carcinoma and human K562 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Maciel, Maria Aparecida M. [Universidade Federal do Rio Grande do Norte, Natal, RN (Brazil). Dept. de Quimica; Martins, Jenilce R.; Pinto, Angelo C.; Kaiser, Carlos R. [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Quimica; Esteves-Souza, Andressa; Echevarria, Aurea [Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ (Brazil). Dept. de Quimica]. E-mail: echevarr@ufrrj.br

    2007-03-15

    The clerodane-type diterpene, trans-dehydrocrotonin (1) the major component of Croton cajucara has shown striking correlation with its therapeutic use in traditional folk medicine. Phytochemical investigations led to the isolation of the metabolites 1, cajucarinolide (6), isocajucarinolide (7), trans-crotonin (2), trans-cajucarin B (3), cis-cajucarin B (4), trans-cajucarin A (5), N-methyltyrosine, vanillic acid and 4-hydroxy-benzoic acid. 6 and 7 were synthesized in good yield by regiospecific oxidation of 1 using singlet-oxygen. All clerodanes were studied for their cytotoxic effects against human K562 leukemia and Ehrlich carcinoma cells. Ehrlich carcinoma assays with IC{sub 50} = 166 {mu}M (1), 164 {mu}M (2), 65 {mu}M (6) and 10 {mu}M (7) related to cell growth inhibitory effects were dose dependent. Furthermore, moderate cytotoxic activity against K562 leukemia cells was observed with IC{sub 50} = 38 {mu}M (3), 33 {mu}M (5), 36 {mu}M (6) and 43 {mu}M (7). The semi-synthetic 2, 6 and 7 showed similar results when compared to the corresponding natural clerodanes. (author)

  7. Effects of Thalidomide on the Apoptosis of Human Chronic Myeloid Leukemia K562 Cell Line%沙利度胺对人慢性粒细胞白血病细胞株K562细胞凋亡的影响研究

    Institute of Scientific and Technical Information of China (English)

    张志; 张玉高; 韩丽英; 陈枫

    2012-01-01

    OBJECTIVE: To study the effects of thalidomide on the apoptosis of human chronic myeloid leukemia K562 cell line. METHODS: K.562 cells were divided into control (non-treated) group and thalidomide low-dose, medium-dose and high-dose groups (0.5, 1.0, 2.0 mmol·L-1). After 24 h, 48 h, 72 h and 96 h of treatment, MTT assay was used to determine the cell growth inhibition rate. Morphology of the K562 cells was observed by the Wright-Giemsa staining method. The rate of apoptosis was analyzed by flow cytometry (FCM) with AnnexinV-FITC/PI double-staining method. RESULTS: The growth inhibition rate and apoptosis rate of K562 cells were positively related with the concentration of thalidomide and treatment duration. Compared with control group, the growth inhibition rate of K562 cells in thalidomide groups increased significantly after 72 h and 96 h of treatment (P<0.05), and apoptosis rate of K562 cells increased significantly at 4 time points (P<0.01). After exposure to thalidomide for 72 h, K562 cells underwent typical morphological changes of apoptosis such as dwindling in size. CONCLUSIONS: Thalidomide inhibits the proliferation of K.562 cells in a dose and time-dependent manner to some extent. Thalidomide obviously induces the apoptosis of the K562 cells.%目的:研究沙利度胺对人慢性粒细胞白血病细胞株K562细胞凋亡的影响.方法:将K562细胞分为对照(未加药)组和沙利度胺低、中、高剂量(0.5、1.0、2.0 mmol·L-1)组,加入相应药物分别作用24、48、72、96 h后,MTT法检测各组细胞生长抑制率,Wright-Giemsa染色观察各组细胞形态变化,Annexin V-FITC/PI双染法流式细胞仪检测各组细胞凋亡率.结果:K562细胞的生长抑制10率和凋亡率与沙利度胺浓度和作用时间均呈正相关;与对照组比较,沙利度胺3个剂量组作用72、96h后细胞的生长抑制率明显升高(P<0.05),4个时间点的细胞凋亡率均明显升高(P<0.01).沙利度胺3个剂量组作用72h后,K562

  8. Isorhamnetin 3-O-robinobioside from Nitraria retusa leaves enhance antioxidant and antigenotoxic activity in human chronic myelogenous leukemia cell line K562

    Directory of Open Access Journals (Sweden)

    Boubaker Jihed

    2012-08-01

    Full Text Available Abstract Background In this report, the isorhamnetin 3-o-robinobioside and its original extract, the ethyl acetate extract, from Nitraria retusa leaves, were evaluated for their ability to induce antioxidant and antigenotoxic effects in human chronic myelogenous leukemia cell line. Methods Nitraria retusa products properties were carried out by firstly evaluating their effects against lipid peroxidation induced by H2O2, using the thiobarbituric acid reactive substances species (TBARS assay, and proceeding to the assay of cellular antioxidant activity, then doing the comet assay. Results The isorhamnetin 3-o-robinobioside showed a protective effect against lipid peroxidation induced by H2O2. The same natural compound and ethyl acetate extract inhibited oxidation induced by 2,2′-azobis (2-amidinopropane dihydrochloride in human chronic myelogenous leukemia cells with respectively 50% inhibitory concentration values of 0.225 mg/ml and 0.31 mg/ml, reflecting a significant antioxidant potential. The same two products inhibited the genotoxicity induced by hydroxyl radicals in the same human cell line (by 77.77% at a concentration of 800 μg/ml and by 80.55% at a concentration of 1000 μg/ml respectively. Conclusions The isorhamnetin 3- o-robinobioside and its original extract, the ethyl acetate extract, from Nitraria retusa leaves, have a great antioxidant and antigenotoxic potential on human chronic myelogenous leukemia cell line K562.

  9. Cell proliferation inhibitory effect on human myeloid leukemia K562 of bauhinione from Bauhiniae variegata L.%洋紫荆中菲醌化合物bauhinione对K562细胞的增殖抑制作用

    Institute of Scientific and Technical Information of China (English)

    赵燕燕; 崔承彬; 孙启时

    2007-01-01

    目的 探讨从洋紫荆中分离得到的新的菲醌化合物bauhinione对体外培养的人类慢性骨髓性白血病K562细胞的增殖抑制作用.方法 应用罗丹明B法检测bauhinione对癌细胞K562和tsFT210的抑制作用;采用形态学观察以及流式细胞术(FCM)检测该化合物对K562细胞的细胞周期抑制和诱导凋亡作用;通过倍量稀释法考察该化合物的最小抑制质量浓度及其质量浓度为12.5 mg·L-1时的时效关系.结果 Bauhinione对两种癌细胞的IC50值分别为(24.9±2.1)mg·L-1和(111.36±3.5)mg·L-1,说明K562细胞对bauhinione的作用敏感;Bauhinione对K562细胞的最小抑制质量浓度为6.25 mg·L-1;对K562细胞的增殖抑制作用是通过G2-M期抑制和诱导凋亡实现的,并与时间呈依赖关系.结论 Bauhinione在体外有抑制K562细胞增殖的作用,为洋紫荆的抗癌活性成分之一.

  10. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Cheng, Tian-Lu [Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Shinne-Ren [Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  11. Changes of Proliferation and Apoptosis of K562 Cells after Co-culture with Leukemia Bone Marrow Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Katja Karjalainen; Carlos E Bueso-Ramos; Hagop M Kantarjian

    2014-01-01

    Objective:To compare the changes of proliferation and apoptosis of K562 cells after co-culture with human leukemia bone marrow mesenchymal stem cells (LMSC). Methods: The prepared cells were randomly divided into SCG group, SCG+0%FBS group, SCG+0%FBS group and CCG+0%FBS group. Cell counting kit-8 (CCK-8) analytic approach was adopted to detect the optical density (OD) of K562 cells in SCG and CCG groups, and the conditions of K562 cell proliferation under different cultured circumstances were compared. Flow cytometer (FCM) was used to detect the changes of K562 cell cycle after co-culture with LMSC, Annexin V/polyimide (PI) lfuorescence labeling method to detect the changes of K562 cell apoptosis after co-culture with LMSC and serum starvation. Results:After co-culture with LMSC, the proliferation of K562 cells was markedly inhibited, and OD in CCG group was conspicuously lower than that in SCG group. Flow cytometer (FCM) detection on cell cycles demonstrated that after co-culture with LMSC, the proportion of cells in gap phases 0~1 (G0~G1) went up notably, whereas that in phase S went down obviously. Besides, the proportion of cells in phases G2~M was on the rise. K562 cell apoptosis in CCG+0%FBS group was more than in SCG+10%FBS group, and less than in SCG+0%FBS group, indicating LMSC had the function of resisting leukemia cell apoptosis. Conclusion: LMSC exerts the effect of inhibiting the proliferation by blocking K562 cell cycles in phases G0~G1, and inhibiting K562 cell apoptosis induced by serum starvation.

  12. Changes of Proliferation and Apoptosis of K562 Cells after Co-culture with Leukemia Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Katja Karjalainen

    2014-06-01

    Full Text Available Objective: To compare the changes of proliferation and apoptosis of K562 cells after co-culture with human leukemia bone marrow mesenchymal stem cells (LMSC. Methods: The prepared cells were randomly divided into SCG group, SCG + 0%FBS group, SCG + 0%FBS group and CCG + 0%FBS group. Cell counting kit-8 (CCK-8 analytic approach was adopted to detect the optical density (OD of K562 cells in SCG and CCG groups, and the conditions of K562 cell proliferation under different cultured circumstances were compared. Flow cytometer (FCM was used to detect the changes of K562 cell cycle after co-culture with LMSC, Annexin V/polyimide (PI fluorescence labeling method to detect the changes of K562 cell apoptosis after co-culture with LMSC and serum starvation. Results: After co-culture with LMSC, the proliferation of K562 cells was markedly inhibited, and OD in CCG group was conspicuously lower than that in SCG group. Flow cytometer (FCM detection on cell cycles demonstrated that after co-culture with LMSC, the proportion of cells in gap phases 0 - 1 (G0 - G1 went up notably, whereas that in phase S went down obviously. Besides, the proportion of cells in phases G2 - M was on the rise. K562 cell apoptosis in CCG + 0%FBS group was more than in SCG + 10%FBS group, and less than in SCG + 0%FBS group, indicating LMSC had the function of resisting leukemia cell apoptosis. Conclusion: LMSC exerts the effect of inhibiting the proliferation by blocking K562 cell cycles in phases G0 - G1, and inhibiting K562 cell apoptosis induced by serum starvation.

  13. Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells.

    Directory of Open Access Journals (Sweden)

    Huey-Lan Huang

    Full Text Available Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6, ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.

  14. HJC, a new arylnaphthalene lignan isolated from Justicia procumbens, causes apoptosis and caspase activation in K562 leukemia cells.

    Science.gov (United States)

    Luo, Jiaoyang; Kong, Weijun; Yang, Meihua

    2014-01-01

    The aim of this study is to investigate whether HJC, isolated from Justicia procumbens for the first time, can suppress the proliferation and induce apoptosis of human leukemia K562 cells and finally clarify its related mechanism. The chemical structure of HJC was validated by LC-ESI-MS/MS, cytotoxicity was assayed using MTT, and apoptosis was investigated by flow cytometry. These assays indicated that HJC remarkably inhibited the growth in K562 cells by decreasing cell proliferation, reducing the SOD activity, enhancing ROS levels and inducing apoptosis. Activation of caspase-3 indicated that HJC may be inducing intrinsic and extrinsic apoptosis pathways and that HJC-induced apoptosis was caspase-dependent. This study suggests that HJC is a high-potency anti-tumor agent, and it induces apoptosis through a caspase-dependent pathway in human leukemia K562 cells. It also presents a potential alternative to leukemia therapy.

  15. Fangchinoline induces G0/G1 arrest by modulating the expression of CDKN1A and CCND2 in K562 human chronic myelogenous leukemia cells

    Science.gov (United States)

    WANG, YUPING; CHEN, JIE; WANG, LIN; HUANG, YUJI; LENG, YE; WANG, GUIYING

    2013-01-01

    Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the oncoprotein BCR-ABL, which exhibits a constitutive tyrosine kinase activity. Imatinib mesylate (IM), an inhibitor of the tyrosine kinase activity of BCR-ABL, has been used as a first-line therapy for CML. However, IM is less effective in the accelerated phase and blastic phases of CML and certain patients develop IM resistance due to the mutation and amplification of the BCR-ABL gene. Fangchinoline, an important chemical constituent from the dried roots of Stephaniae tetrandrae S. Moore, exhibits significant antitumor activity in various types of cancers, including breast, prostate and hepatocellular carcinoma. However, the effects and the underlying mechanisms of fangchinoline in CML remain unclear. In the present study, we identified that fangchinoline inhibits cell proliferation in a dose- and time-dependent manner in K562 cells derived from the blast crisis of CML. Additional experiments revealed that fangchinoline induces cell cycle arrest at the G0/G1 phase and has no effect on apoptosis, which is mediated through the upregulation of cyclin-dependent kinase (CDK)-N1A and MCL-1 mRNA levels, as well as the downregulation of cyclin D2 (CCND2) mRNA levels. These findings suggest the potential of fangchinoline as an effective antitumor agent in CML. PMID:23596478

  16. Pentamidine sensitizes chronic myelogenous leukemia K562 cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Qiu, Geng; Jiang, Jikai; Liu, Xiao-shan

    2012-11-01

    Pentamidine (PMD) is an anti-protozoa drug with potential anticancer activity. Here we show that PMD at clinically achievable plasma drug concentrations slightly inhibited the growth of human leukemia cell lines. PMD close to its therapeutic doses sensitized TRAIL-resistant K562 cells to the cytokine and potentiated TRAIL-induced apoptosis through activation of caspase-8 and -3. When we investigated the underlying mechanism, we observed that treatment with PMD increased DR5 expression at both mRNA and protein levels and down-regulated anti-apoptotic XIAP and Mcl-1 protein levels. This study provides a rationale for a more in-depth exploration into the combined treatment with PMD and TRAIL as a valuable strategy for leukemia therapy.

  17. In vitro effect of imatinib mesylate loaded on polybutylcyanoacrylate nanoparticles on leukemia cell line K562.

    Science.gov (United States)

    Hasandoost, Leyla; Akbarzadeh, Azim; Attar, Hossein; Heydarinasab, Amir

    2017-05-01

    The study aimed to prepare imatinib mesylate-loaded polybutylcyanoacrylate (PBCA) nanoparticles and evaluate their efficacy on leukemia cell line K562. The formulation was prepared by miniemulsion polymerization technique. Nanoparticles were characterized by dynamic light scattering (DLS), spectrophotometry, Fourier transform infrared spectroscopy (FTIR), dialysis membrane, and 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) techniques. Nanoscale particles with high encapsulation efficiency (86%) and physical entrapment of drug were observed. In addition, nanoparticles showed suitable drug retention capability and potentiate the cytotoxicity effects of imatinib mesylate. Findings of study suggested PBCA nanoparticles are promising carrier for imatinib mesylate delivery to leukemia cell line K562.

  18. Curcumin inhibits WT1 gene expression in human leukemic K562 cells

    Institute of Scientific and Technical Information of China (English)

    Songyot ANUCHAPREEDA; Pattra THANARATTANAKORN; Somjai SITTIPREECHACHARN; Prasit CHANARAT; Pornngarm LIMTRAKUL

    2006-01-01

    Aim: Wilms' tumorl (WT1) gene is highly expressed in leukemic blast cells of myeloid and lymphoid origin. Thus, WT1 mRNA and protein serve as promising tumor markers for the detection of leukemia and monitoring of disease progression. The purpose of this study was to investigate the modulating effects of curcumin on WT1 gene expression in the human leukemic cell line K562. Methods: The cytotoxicity of curcumin on the K562 cell line was evaluated by using 3-(4,5-dimethyl-2 thiazoyl)2,5-diphenyl-tetrazolium bromide (MTT) assay. The K562 cell line was treated with a non-cytotoxic dose of curcumin (5,10, or 15 umol/L)for 13 d. The expression levels of WT1 protein and WT1 mRNA were assessed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results: Curcumin had a cytotoxic effect on K562 leukemic cells with an inhibitory concentration at 50% (IC50) of approximately 20 ug/mL (54.3 umol/L). Non-cytotoxic doses of curcumin, at concentrations of 5,10, and 15 umol/L for 2 d, decreased the level of WT1 protein and WT1 mRNA in the K562 cell line in a dose-dependent manner. Similarly, curcumin at a concentration of 10 umol/L significantly decreased the level of WT1 protein and mRNA in a time-dependent manner. Conclusion: The inhibitory effects of curcumin are associated with a decrease in the levels of both WT1 protein and WT1 mRNA. The current study provides a molecular basis for future clinical trials in leukemic patients. Thus, curcumin could be a promising chemotherapeutic agent for human leukemia.

  19. The venom of the spider Macrothele raveni induces apoptosis in the myelogenous leukemia K562 cell line.

    Science.gov (United States)

    Liu, Zhonghua; Zhao, Yan; Li, Jing; Xu, Shiyan; Liu, Changjun; Zhu, Yanghui; Liang, Songping

    2012-08-01

    Spider venoms are a rich source of bioactive compounds with therapeutic potential. In traditional Chinese medicine, spiders and spider venoms have been used in the treatment of various ailments. In the present study, the venom of the spider Macrothele raveni potently suppressed cell growth in the myelogenous leukemia K562 cell line in a dose and time-dependent manner with an IC(50) of 5.1 μg/mL. The venom also had a low inhibitory effect on human lymphocytes with an IC(50) of approximately 36.4 μg/mL, indicating that the venom is relatively selective for leukemic cells. Venom treated K562 cells showed typical morphological indicators of apoptosis including condensation of nuclei and fragmentation of DNA. Annexin V-FITC and propidium iodide dual staining further demonstrated that the venom had potent apoptogenic activity. Venom treatment induced caspase 3 and caspase 8 activation in K562 cells and promoted PARP cleavage. The present results indicate that the venom of the spider M. raveni potently and selectively suppresses the growth of K562 cells by inducing apoptosis via caspase 3 and caspase 8 mediated signaling pathways.

  20. JNK1/c-Jun and p38 alpha MAPK/ATF-2 pathways are responsible for upregulation of Fas/FasL in human chronic myeloid leukemia K562 cells upon exposure to Taiwan cobra phospholipase A2.

    Science.gov (United States)

    Chen, Ku-Chung; Chiou, Yi-Ling; Chang, Long-Sen

    2009-10-15

    Fas and FasL expression upregulation was found in human leukemia K562 cells upon exposure to Naja naja atra phospholipase A(2) (PLA(2)). PLA(2) treatment induced an increase in intracellular Ca(2+) ([Ca(2+)]i) and ROS generation levels, leading to activation of p38 MAPK and JNK. Suppression of both p38 MAPK and JNK abrogated Fas and FasL upregulation. Unlike PLA(2), catalytically inactive PLA(2) treatment did not markedly increase Fas and FasL protein expression, and p38 MAPK activation was exclusively responsible for catalytically inactive PLA(2)-induced increase in Fas and FasL protein expression. Knockdown of p38 alpha MAPK and JNK1 by siRNA proved that p38 alpha MAPK and JNK1 were involved in ATF-2 and c-Jun phosphorylation, respectively. Compared with the p38 alpha MAPK/ATF-2 pathway, the JNK1/c-Jun pathway played a crucial role in Fas/FasL upregulation. Unlike arachidonic acid, lysophosphatidylcholine mimicked the PLA(2) action in inducing Fas/FasL upregulation. Together with the previous finding that c-Jun and ATF-2 are involved in transcriptional regulation of Fas and FasL, our data suggest that PLA(2) induces Fas and FasL upregulation through p38 alpha MAPK/ATF-2 and JNK1/c-Jun pathways in K562 cells, and PLA(2) catalytic activity is involved in this action. (c) 2009 Wiley-Liss, Inc.

  1. PEGylated dendritic nanoarchitechture improves mean survival time of BDF1mice bearing myelogenous k -562 leukemia

    Institute of Scientific and Technical Information of China (English)

    Ramadoss Karthikeyan; Pureti Madhu Kumar; Palanirajan Vijayaraj Kumar

    2013-01-01

    Objective:To developing and exploring the use ofPEGylated poly(propylene imine) dendritic architecture for the delivery of an anti leukemic activity ofPrednisolone.Methods:For this study,PEGylated poly(propylene imine) dendritic architecture was synthesized and loaded with Prednisolone and targeted to the ascetic form of myelogenous leukemia k-562 cellines in hybrid miceBDF1, was used as tumor model.Theantileukemic activity was assessed by use of the criterionT/C%, whereT was the mean survival time(MST, days) of the drug treated mice, bearing k-562 leukemia andC- the mean survival time(MST, days) of untreated control animals, bearing the same leukemia cellines.Results:An antileukemic activity of the studiedPrednisolone loaded PEGylatedPolypropyleneimine(PPI) dendrimer was found to have increasing the mean survival time of the k-562 myelogenous leukemia cellines bearingBDF1 mice.The criterion“increase of life span”(ILS%) reached maximally270.1% for the drug loaded dendrimer.Conclusion:The studied dendrimer withPrednisolone showed lower toxicity with improved antileukemic activity in comparison with freePrednisolone.The further experiments in this field are in progress, aiming to design better dendritic formulations, with potential clinical use.

  2. Proteomic analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in leukemia K562 cells

    Institute of Scientific and Technical Information of China (English)

    WANG Zi-hui; YU Ding; CHEN Yan; HAO Jian-zhong

    2005-01-01

    Background Arsenic trioxide (As2O3) has been identified as a very potent anti-acute leukemic agent. However its role in apoptosis needs to be elucidated. As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms. Drug-target interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3. This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics. Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations. While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells. Conclusions: As2O3 induces apoptosis in K562 cells in a dose and time-dependent manner. Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test. These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment.

  3. The fusarin analogue NG-391 impairs nucleic acid formation in K-562 leukemia cells

    Science.gov (United States)

    The clavicipitaceous fungus Metarhizium robertsii produces the fusarin-like mycotoxin NG-391. We report on the biological effects of NG-391 on K-562 human cancer cells, obtained with radionuclide incorporation assays, along with nucleosome release and caspase assays, respectively. Our data suggests ...

  4. BME, a novel compound of anthraquinone, down regulated P-glycoprotein expression in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells via generation of reactive oxygen species.

    Science.gov (United States)

    Wang, Jianhong; Liu, Lu; Cen, Juan; Ji, Biansheng

    2015-09-01

    P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in tumor cells is still a main obstacle for the chemotherapeutic treatment of cancers. Thus, development of effective MDR reversing agents is an important approach in the clinic. The present study revealed that BME, a novel compound of anthraquinone, elevated intracellular accumulation of the P-gp substrates and reduced concentration resulting in 50% inhibition of cell growth (IC50) values for doxorubicin (DOX) in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. Further more, BME was also reported to down regulated P-gp expression accompanying with generation of nontoxic low level of intracellular reactive oxygen species (iROS) and activation of extracellular signal-regulated kinase (ERK)1/2 as well as c-JUN N-terminal kinase (JNK). However, treatment with N-acetyl-cysteine (NAC), U0216 and SP600125 almost abolished actions of the BME mentioned above. These results indicated that the effect of the BME on the P-gp may be involved in generation of nontoxic low level of iROS and activation of ERK1/2 or JNK, which suggested valuable clues to screen and develop P-gp reversing agents.

  5. Comparison of microRNA expression profiles in K562-cells-derived microvesicles and parental cells, and analysis of their roles in leukemia.

    Science.gov (United States)

    Chen, Xiaomei; Xiong, Wei; Li, Huiyu

    2016-12-01

    Microvesicles (MVs) are 30-1,000-nm extracellular vesicles that are released from a multitude of cell types and perform diverse cellular functions, including intercellular communication, antigen presentation, and transfer of proteins, messenger RNA and microRNA (also known as miR). MicroRNAs have been demonstrated to be aberrantly expressed in leukemia, and the overall microRNA expression profile may differentiate normal blood cells vs. leukemia cells. MVs containing microRNAs may enable intercellular cross-talk in vivo. This prompted us to investigate specific variations of microRNA expression patterns in MVs derived from leukemia cells. The present study examined the microRNA expression profile of MVs from chronic myeloid leukemia K562 cells and that of MVs from normal human volunteers' peripheral blood cells. The potential targets of the differentially expressed microRNAs were predicted using computational searches. Bioinformatic analyses of the predicted target genes were performed for further evaluation. The present study analyzed microRNAs of MVs derived from leukemia and normal cells, and characterized specific microRNAs expression. The results revealed that MVs derived from K562 cells expressed 181 microRNAs of the 888 microRNAs assessed. Further analysis revealed that 16 microRNAs were downregulated, while 7 were upregulated in these MVs. In addition, significant differences in microRNA expression profiles between MVs derived from K562 cells and K562 cells were identified. The present results revealed that 77 and 122 microRNAs were only expressed in MVs derived from K562 cells and in K562 cells, respectively. There were 104 microRNAs co-expressed in MVs derived from K562 cells and in K562 cells. Target gene-related pathway analyses demonstrated that the majority of the dysregulated microRNAs were involved in pathways associated with leukemia, particularly the mitogen-activated protein kinase (MAPK) and the p53 signaling pathways. By further conducting

  6. Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

    Institute of Scientific and Technical Information of China (English)

    宋土生; 杨玲; 黄辰; 刘利英; 倪磊; 王爱英; 罗禹

    2007-01-01

    Indole-3-acetic acid(IAA)is an i mportant typeof the plant growth hor mone found in higherplants,and participate inthe regulation of plant celldivision,elongation and differentiation.It is pres-ent in human urine,blood plas ma and central nerv-ous system.IAAis well tolerated in human and isnot oxidized by mammalian peroxidase.Recent re-searches suggest that the combination of IAA andhorseradish peroxidase(HRP)is cytotoxic to mam-malian cells,and could be used as a novel cancertherapy[1-3],while neither IAAn...

  7. 白血病细胞K562来源的外体对人脐带间充质干细胞作用的研究%Effect of exosomes released from chronic myeloid leukemia K562 cells on human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    顾建美; 钱晖; 朱伟; 许文荣

    2012-01-01

    目的 探讨白血病细胞株(K562)来源的外体(exosomes)对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hucMSCs)的影响.方法 用离心超滤和蔗糖密度梯度超速离心法从K562细胞的培养上清液中分离并纯化exosomes.透射电子显微镜观察其形态;将exosomes与hucMSCs共育,细胞计数板法检测exosomes对hucMSCs增殖的影响;实时荧光定量PCR检测肿瘤相关成纤维细胞(CAFs)相关基因FAP、α-SMA和IL-6的表达;western blot检测exosomes标志性蛋白CD9、CD81及CAFs相关蛋白FAP,α-SMA的表达.结果 透射电镜下观察分离的exosomes呈椭圆或碟状的囊泡结构,直径在30~100 nm;细胞计数板法检测结果表明,不同浓度的exosomes均能抑制hucMSCs细胞增殖且呈剂量依赖性(P均<0.05);荧光定量PCR结果表明,不同浓度的exosomes作用于hucMSCs,其FAP、α-SMA和IL-6表达量明显增加(P均<0.05);western blot 结果表明,exosomes可表达标志性蛋白CD9、CD81,且exosomes作用hucMSCs后FAP、α-SMA蛋白表达量增加.结论 K562细胞来源的exosomes能抑制hucMSCs增殖,且能诱导hucMSCs向CAFs的分化.

  8. Screening and characterization of aptamers of chronic myelognous leukemia K562 cells%慢性髓细胞性白血病K562细胞适体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    刘玲玲; 韩跃武; 祝凯华; 李真真; 韩亚萍; 路艳; 王春霞

    2009-01-01

    目的:筛选并鉴定出慢性髓细胞性白血病(CML)K562细胞的寡核苷酸适体.方法:体外合成长度为88个碱基的随机单链DNA(ssDNA)文库,采用生物素-链霉亲和素磁珠法制备次级文库,以正常人血液中提取的中性粒细胞为反筛细胞,利用指数富集的配基系统进化(SELEX)技术筛选出与CML K562细胞特异结合的适体.将筛选得到的适体回收纯化后连接pGEM-T质粒载体,经蓝白筛选后,随机挑选24个克隆子进行序列测定.采用荧光标记引物法检测ssDNA文库与K562细胞的亲和力,并用Clustal 2.05和DNA sis V 2.5软件对适体序列进行一级结构同源性分析和二级结构预测.结果:经过13轮循环筛选,CML K562细胞适体的A值从0.12上升到1.25,至第13轮A值无明显增高.一级结构分析无同源序列,但可分为6个家族,其中5个家族各自具有保守序列,家族6无保守序列.二级结构分析表明,适体形成的茎环、凸环结构可能是与K562细胞特异性结合的结构基础.结论:利用SELEX技术成功筛选出高亲和性的CML K562细胞适体.%AIM: To screen and characterize oligonucleotide aptamers of chronic myelognous leukemia K562 cells. METHODS: Oligonucleotide aptamers specifically binding to chronic myelog-nous leukemia K562 cells were screened from 88 nt random ssDNA library in vitro syntbesis by SELEX method, sub-library was prepared by biotin-streptavidin magnetic beads and neutro-phils from blood of normal humans were used as anti-sieve cells. The screened aptamters were purified and connected to pGEM-T plasmid vector and 24 clones of random aelection were sequenced after screening by the blue and white. The affinities of the screened aptamers binding to chronic myelognous leukemia K562 cells were detected by fluorescent primers. Homology analyses of the primary structure and secondary structure prediction to the screened aptamters were conducted with Clustal 2.05 and DNA sis V 2.5 software. RESULTS: After

  9. 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

  10. Separation and purification and in vitro anti-proliferative activity of leukemia cell K562 of Galium aparine L. petroleum

    Directory of Open Access Journals (Sweden)

    Guoqing Shi

    2016-05-01

    Full Text Available To explore material basis of in vitro anti-proliferative activity of leukemia cell K562 of petroleum ether phase of product resulting from Galium aparine L. 60% ethanol extraction, the experiment adopts column chromatography combined with thin layer preparation, isolates and purifies petroleum ether, conducts structural identification of obtained single compound and applies MTT method for viability assay of in vitro anti-proliferative activity of leukemia cell K562. Experimental results show that G. aparine L. petroleum ether contains mainly β-sitosterol, daucosterol and dibutyl phthalate and other substances. Under experimental conditions, the three could inhibit the proliferation of leukemia cell K562 with dose-effect and time-effect relationship, of which dibutyl phthalate has strongest activity. Dibutyl phthalate with excellent activity, β-sitosterol with rich content and moderate effect should be the main contributor to its biological activity.

  11. The involvement of human-nuc gene in polyploidization of K562 cell line.

    Science.gov (United States)

    Cavalloni, G; Danè, A; Piacibello, W; Bruno, S; Lamas, E; Bréchot, C; Aglietta, M

    2000-12-01

    During megakaryocyte differentiation, the immature megakaryocyte increases its ploidy to a 2(x) DNA content by a process called endomitosis. This leads to the formation of a giant cell, the mature megakaryocyte, which gives rise to platelets. We investigated the role of human-nuc (h-nuc), a gene involved in septum formation in karyokynesis in yeast, during megakaryocytic polyploidization. Nocodazole and 12-O-tetradecanoylphorbol-13-acetate (TPA) were used to induce megakaryocytic differentiation in K562 cell line. The ploidy distribution and CD41 expression of treated K562 cells were evaluated by flow cytometry. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we analyzed the h-nuc mRNA expression on treated K562 cells. Mature megakaryocyte-like polyploid cells were detected at day 5-7 of treatment with nocodazole. TPA also had a similar effect on K562 cells, but it was much weaker than that of nocodazole. The analysis of ploidy of nocodazole-treated K562 cells showed that nocodazole preferentially induced polyploidization of K562 cell line with a pronounced increase of the cells 8N at day 7 of culture. Expression of CD41, a differentiation-related phenotype, was significantly induced by TPA after 7 days of treatment, showing that functional maturation was mainly induced by TPA. In contrast, there was no significant increase in CD41 expression in nocodazole-treated K562 cells, suggesting that polyploidization and functional maturation are separately regulated during megakaryocytopoiesis. RT-PCR analysis indicated that h-nuc mRNA increased after 72 hours in the presence of nocodazole, preceding the induction of polyploidization. Our data indicate that h-nuc might play a role in polyploidization during megakaryocytic differentiation via inhibition of septum formation.

  12. Transferred BCR/ABL DNA from K562 extracellular vesicles causes chronic myeloid leukemia in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Jin Cai

    Full Text Available Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs. The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML, could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.

  13. Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.

    Science.gov (United States)

    Pilzer, David; Saar, Moran; Koya, Keizo; Fishelson, Zvi

    2010-03-15

    Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

  14. Safranal, a Crocus sativus L constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study.

    Science.gov (United States)

    Geromichalos, George D; Papadopoulos, Theophanis; Sahpazidou, Despina; Sinakos, Zacharias

    2014-12-01

    Crocin, a main constituent of Crocus sativus L (saffron), has been found to inhibit the growth of K-562 human chronic myelogenous leukemia (CML) cells expressing Bcr-Abl protein tyrosine kinase activity. The aim of our study is to investigate the ability of the bioactive saffron's constituents, crocin (CRC) and safranal (SFR), to inhibit the Bcr-Abl protein activity employing an in silico approach, as well as the in vitro effect of these compounds on K-562 growth and gene expression of Bcr-Abl. In silico molecular docking studies revealed that mostly SFR can be attached to Bcr-Abl protein, positioned inside the protein's binding cavity at the same place with the drug used in the treatment of CML, imatinib mesylate (IM). The predicted polar interactions and hydrophobic contacts constructing a hydrophobic cavity inside the active site, explain the observed inhibitory activity. Cytotoxicity experiments showed that SFR and CRC mediate cytotoxic response to K562 cells. In vitro studies on the expression of Bcr-Abl gene revealed that SFR and in a lesser degree IM inhibited the expression of the gene, while in contrast CRC induced an increase. The ultimate goal was to evaluate the existence of a potential antitumor activity of saffron's constituents SFR and CRC.

  15. A new disposable electrode for electrochemical study of leukemia K562 cells and anticancer drug sensitivity test.

    Science.gov (United States)

    Yu, Chunmei; Zhu, Zhenkun; Wang, Li; Wang, Qiuhong; Bao, Ning; Gu, Haiying

    2014-03-15

    Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. In this work, a new disposable electrochemical cell sensor with low cost and simple fabrication was proposed to study the electrochemical behavior of leukemia K562 cells and the effect of anticancer drugs on cell viability. The analytical device was integrated by using ITO glass as the substrate of working electrodes and paper as the electrolytic cell. The cyclic voltammetry of the K562 cells at the disposable electrode exhibited an irreversible anodic peak and the peak current is proportional to the cell number. This anodic peak is attributed to the oxidation of guanine in cells involving two protons per transfer of two electrons. For the drug sensitivity tests, arsenic trioxide and cyclophosphamide were added to cell culture media. As a result, the electrochemical responses of the K562 cells decreased significantly. The cytotoxicity curves and results obtained corresponded well with the results of CCK-8 assays. In comparison to conventional methods, the proposed method is simple, rapid and inexpensive. More importantly, the developed sensor is supposed to be a single-use disposable device and electrodes were prepared "as new" for each experiment. We think that such disposable electrodes with these characteristics are suitable for experimental study with cancer cells or other types of pathogens for disease diagnosis, drug selection and on-site monitoring.

  16. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F+ HEL Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Axel Weber

    2015-03-01

    Full Text Available Signal transducers and activators of transcription (Stats play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML and Jak2(V617F in other myeloproliferative diseases (MPD. We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL

  17. Synergistic effect of magnetic nanoparticles of Fe3O4 with gambogic acid on apoptosis of K562 leukemia cells

    Directory of Open Access Journals (Sweden)

    Baoan Chen

    2009-11-01

    the transcription of bcl-2, nuclear factor-κB, and survivin degraded as did the expression of corresponding proteins in K562 cells. Our data suggests a potential clinical application of a combination of GA and MNPs-Fe3O4 in leukemia therapy.Keywords: gambogic acid, magnetic nanoparticles of Fe3O4, traditional Chinese medicine, K562 leukemia cells, apoptosis

  18. 稳定表达HLA-A*1101蛋白的K562细胞株的建立%Establishment of Stable Subline of K562 Cells Expressing Human Leucocyte Antigen A1101

    Institute of Scientific and Technical Information of China (English)

    查显丰; 周羽竝; 杨力建; 陈少华; 李萡; 闫小娟; 李扬秋

    2011-01-01

    The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A * 1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A* 1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3' terminal of the HLA-A' 1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A* 1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A' 1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A' 1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A' 1101-T2A-EGFP plasmid was successfully constructed; after G4I8 selection for 2 months, two sublines of K562 cells (HLA-A'1101 *K562, pEGFP-N3 * K562) expressing GFP were constructed. The expression of HLA-A * AJ101 gene could be determined in HLA-A* 1101 * K562 cell line by RT-PCR,while the pEGFP-N3 * K562 cells could not express HLA-A * A1101 gene. HLA-A* 1101 protein and GFP double positive HLA-A * 1101* K562 cells were upto 88. 5% , which was obviously higher than pEGFP-N3* K562 cells (0. 698% ) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A * 1101 * K562 cells has been established and a subline of K562 cell expressing HLA-A* 1101 protein on its cell membrane was successfully

  19. Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells.

    Science.gov (United States)

    Watanabe, T; Mitchell, T; Sariban, E; Sabbath, K; Griffin, J; Kufe, D

    1985-06-01

    We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.

  20. 细胞周期蛋白E2反义脱氧寡核苷酸对K562细胞增殖及凋亡的调控作用%Effect of cyclin E2 antisense oligonucleotide on human leukemic cell line K562

    Institute of Scientific and Technical Information of China (English)

    徐丽粉; 郭晓楠; 刘英芳

    2012-01-01

    Objective To investigate the effect of cyclin E2 antisense oligonucleotide ( ASON) on human leukemic cell line K562. Methods Cyclin E2 ASON was used in vitro culture K562 cell study. MTT assay was used to measure the growth inhibitory effect of transfection of ASON and lipofectamine TM2000. The Mrna expression levels of cyclin E2 were examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Apopsis was detected by flow cytometry and morphology method. Results Cyclin E2 ASON specifically inhibited K562 cell Mrna expression level as well as the K562 cell proliferation. After transfection with cyclin E2 ASON.K562 cells developed apoptosis. Conclusion Cyclin E2 ASON can specifically inhibit K562 cell Mrna expression levels as well as the K562 cell proliferation. After being transfected with cyclin E2 ASON,K562 cells developed apoptosis. Cyclin E2 gene is likely to be a new target for antisense nucleotides techonology therapy of leukemia.%目的 研究细胞周期蛋白E2 (cyclin E2)反义脱氧寡核苷酸(ASON)对人红白血病细胞K562增殖的调控作用.方法 采用反义技术合成ASON并与K562细胞共培养.用四甲基偶氮唑蓝(MTT)法检测转染ASON和脂质体lipofectamineTM 2000后的细胞活力,逆转录-聚合酶链式反应(RT-PCR)方法检测转染细胞cyclin E2 mRNA表达水平及流式细胞术和形态学观察检测细胞凋亡.结果 Cyclin E2特异的ASON能显著地抑制cyclin E2mRNA水平的表达(F=26.442,P<0.01);白血病细胞的生长明显受抑制(P<0.01),细胞凋亡明显增加.结果 表明反义脱氧寡核苷酸能有效地抑制K562细胞的增殖,抑制K562细胞cyclin E2 mRNA表达上调,并显著地诱导细胞凋亡.结论 脂质体转染cyclin E2的反义寡核苷酸能够有效地抑制K562细胞cyclin E2 mRNA的表达,同时对白血病细胞K562的生长有明显的抑制作用,并可诱导K562细胞凋亡.提示cyclin E2在细胞周期调控中起作用,cyclinE2基因有望

  1. Mechanism of ginseng polysaccharide on apoptosis and cell cycle in leukemia K562 cells%人参多糖对K562细胞凋亡与细胞周期的影响及其机制

    Institute of Scientific and Technical Information of China (English)

    刘艺; 陈地龙; 何轩; 姜蓉; 王建伟; 左国伟; 魏强; 赵亮; 李静

    2012-01-01

    Objective To investigate the mechanism of ginseng polysaccharide (GPS) on the cell cycle and apoptosis in leukemia K562 cells. Methods Leukemia K562 cells in logarithmic phase with density of 7 x 108L-1 were incubated with (GPS group) and without (control group) 400 mg/L GPS, respectively. The effects of GPS on K562 cell cycle and apoptosis were determined by flow cytometry ( FCM). The mRNA expression of ERK was detected by RT-PCR. The distributions and protein expressions of ERK, p-ERK, NF-kB and cyclin D, were detected by immunohistochemical staining and Western blotting. Results Compared with the control group, the apoptosis rate of the K562 cells in the GPS group significantly increased after treatment for 24, 48 and 72 h (P 0. 05), but the differences of p-ERK, NF-kB and cyclin D, were statistically significant between the GPS group and the control group ( P < 0. 05). Conclusion GPS can induce cell cycle arrest in G0/G1 phase and cell apoptosis of K562 cells in a time-dependent manner, probably through inhibiting ERK/NF-kB signaling pathway and thus reducing cyclin D, expression.%目的 探讨人参多糖(ginseng polysaccharide,GPS)诱导白血病细胞K562凋亡和周期阻滞的机制.方法 取对数生长期的K562细胞,调整密度为7×108/L,空白对照组予以常规培养;GPS组加入400 mg/L GPS.流式细胞仪测定细胞的凋亡率及细胞周期分布变化;RT-PCR检测细胞ERK表达的变化.免疫组化的方法检测细胞中p-ERK、NF-κB、Cyclin D1蛋白定位及表达量的变化.Western blot检测细胞中ERK、p-ERK、NF-κB、Cyclin D1蛋白的变化.结果 与对照组比较,K562细胞在400mg/L GPS的作用下,体外培养24、48、72 h后,GPS组细胞凋亡率显著增高(P<0.05),周期分布检测结果显示,与对照组相比,GPS组K562细胞G0/G1期细胞数量呈时间依赖性增多(P<0.05),G2+M、S期细胞数量则明显减少(P<0.05).RT-PCR检测结果显示,400 mg/L GPS处理48 h组ERK mRNA的表

  2. Changes of gene expression profile in homoharringtonine-induced leukemia multi-drug resistant cell line K562/HHT%高三尖杉酯碱诱导的白血病耐药细胞基因表达谱的变化

    Institute of Scientific and Technical Information of China (English)

    于晓林; 刘永; 陈春燕; 王涓冬; 孔德晓

    2009-01-01

    目的 探讨高三尖杉酯碱(HHT)诱导的人白血病细胞耐药相关分子.方法 在前期建立的HHT诱导的人白血病多药耐药细胞株K562/HHT的基础上,采用基因芯片技术比较K562/HHT细胞、其亲本K562细胞以及用耐药逆转剂米非司酮(RU486)作用后的K562/HHT(K562/HHT/RU486)细胞三者基因表达谱的差异,选择在这三种细胞中呈动态变化的骨髓细胞X染色体上酪氨酸激酶(BMX)基因用RT-PCR和Western blot法在转录和翻译水平进行验证,进而转染BMX基因到K562K562/HHT细胞,观察BMX过表达时这两种细胞内柔红霉素(DNR)含量的变化,确定BMX是否在K562/HHT细胞耐药形成中发挥作用.结果 耐药细胞K562/HHT与其亲本K562细胞相比,共有117个基因表达有显著差异,其中57个基因表达明显上调,60个基因表达明显下调,耐药细胞K562/HHT中多药耐药基因mdr1表达明显上调;K562/HHT/RU486细胞与K562/HHT细胞相比,13个基因表达明显上调,37个基因表达明显下调.这些差异表达的基因涉及耐药、细胞信号传导、细胞分化、细胞增殖、转录调节以及离子转运等.基因NM-001721(BMX)、NM-031459(SESN2)、NM-033642(FGF13)和AL 049309(SFRS12)在两组芯片中的表达均有显著差异.其中BMX基因在K562/HHT细胞中的表达与K562细胞相比,明显上调,在K562/HHT/RU486细胞则较K562/HHT细胞减低.进一步用RT-PCR和Western blot法检测得到了相同的结果.RT-PCR和Western blot证实BMX质粒转染的K562K562/HHT细胞BMX表达均上调,流式细胞术检测到K562K562/HHT细胞内DNR含量荧光强度分别为79.28±4.04和29.84±2.67,均较转染前荧光强度158.52±8.08和58.58±6.53显著减低.结论 BMX在高三尖杉酯碱诱导的人白血病多药耐药细胞株K562/HHT耐药性产生中发挥作用.%Objective To study the resistant related molecules of human leukemia drug resistant K562 cells(K562/HHT) induced by homoharringtonine (HHT). Methods Gene

  3. Altered gravity modulates 5-lipoxygenase in human erythroleukemia K562 cells.

    Science.gov (United States)

    Maccarrone, M; Putti, S; Finazzi Agro, A

    1998-07-01

    Mammalian lipoxygenases catalyse the first committed step in the so-called "arachidonate cascade", leading to the production of potent bioactive molecules, such as leukotrienes, lipoxins and hepoxilins. Leukotrienes interact with G protein-couple receptors involved in neuronal plasticity and T lymphocyte activation, lipoxins activate leukocytes, hepoxilines control the insulin release and stimulate the phospholipase C. Lipoxygenase (linoleate:oxygen oxidoreductase; E.C. 1.13.11.34; 5-LOX) are responsible for lymphocyte maturation and programmed death (apoptosis) of neuronal cells. Therefore, 5-LOX might be Space relevant, because among the most striking effects of Space enviroment are indeed those on T lymphocyte activation, neuronal cell growth and suspectedly apoptosis. In this study, the possible effects of the force of gravity on the activity and expression of 5-LOX have been investigated by subjecting human erythroleukemia K562 cells to simulated hypogravity or hypergravity.

  4. Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

    Science.gov (United States)

    Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2014-02-01

    The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.

  5. Complete genome of Phenylobacterium zucineum – a novel facultative intracellular bacterium isolated from human erythroleukemia cell line K562

    Directory of Open Access Journals (Sweden)

    Sun Jie

    2008-08-01

    Full Text Available Abstract Background Phenylobacterium zucineum is a recently identified facultative intracellular species isolated from the human leukemia cell line K562. Unlike the known intracellular pathogens, P. zucineum maintains a stable association with its host cell without affecting the growth and morphology of the latter. Results Here, we report the whole genome sequence of the type strain HLK1T. The genome consists of a circular chromosome (3,996,255 bp and a circular plasmid (382,976 bp. It encodes 3,861 putative proteins, 42 tRNAs, and a 16S-23S-5S rRNA operon. Comparative genomic analysis revealed that it is phylogenetically closest to Caulobacter crescentus, a model species for cell cycle research. Notably, P. zucineum has a gene that is strikingly similar, both structurally and functionally, to the cell cycle master regulator CtrA of C. crescentus, and most of the genes directly regulated by CtrA in the latter have orthologs in the former. Conclusion This work presents the first complete bacterial genome in the genus Phenylobacterium. Comparative genomic analysis indicated that the CtrA regulon is well conserved between C. crescentus and P. zucineum.

  6. Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

    Science.gov (United States)

    Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Park, Nam Gyu; Chang, Young-Chae; Lee, Young-Choon; Chung, Tae-Wook; Ha, Ki-Tae; Son, Jong-Keun

    2017-01-01

    Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells. PMID:28133573

  7. Characterization of cancer stem-like cells in a novel STI571-resistant chronic myeloid leukemia cell line%K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

    Institute of Scientific and Technical Information of China (English)

    Baijun Fang; Yongping Song; Yanli Zhang; Quande Lin; Xudong Wei

    2007-01-01

    Objective: To characterize a novel chronic myeloid leukemia (CML) cell line and to further elucidate the mechanisms of resistance to STI571. Methods: A novel K562 cell line (K562NP16) was achieved after exposure of the K562 cells to VP16. A small subpopulation (K562NP16 SP) that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by flow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results: The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 (MDR1) expression of the 170 kDa P-glycoprotein (P-gp) was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion: A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.

  8. Time-series analysis in imatinib-resistant chronic myeloid leukemia K562-cells under different drug treatments.

    Science.gov (United States)

    Zhao, Yan-Hong; Zhang, Xue-Fang; Zhao, Yan-Qiu; Bai, Fan; Qin, Fan; Sun, Jing; Dong, Ying

    2017-08-01

    Chronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein. Imatinib is the first-line treatment of CML; however, many patients are resistant to this drug. In this study, we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments. GSE24946 was downloaded from the GEO database, which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5). Three drug treatment groups were considered for this study: arsenic trioxide (ATO), AMN107, and ATO+AMN107. Each group had one sample at each time point (3, 12, 24, and 48 h). Time-series genes with a ratio of standard deviation/average (coefficient of variation) >0.15 were screened, and their expression patterns were revealed based on Short Time-series Expression Miner (STEM). Then, the functional enrichment analysis of time-series genes in each group was performed using DAVID, and the genes enriched in the top ten functional categories were extracted to detect their expression patterns. Different time-series genes were identified in the three groups, and most of them were enriched in the ribosome and oxidative phosphorylation pathways. Time-series genes in the three treatment groups had different expression patterns and functions. Time-series genes in the ATO group (e.g. CCNA2 and DAB2) were significantly associated with cell adhesion, those in the AMN107 group were related to cellular carbohydrate metabolic process, while those in the ATO+AMN107 group (e.g. AP2M1) were significantly related to cell proliferation and antigen processing. In imatinib-resistant CML cells, ATO could influence genes related to cell adhesion, AMN107 might affect genes involved in cellular carbohydrate metabolism, and the combination therapy might regulate genes involved in cell proliferation.

  9. Preliminary studies of reversing K562/ADR cell effects of abnormal savda munziq total flavonoids%维药异常黑胆质成熟剂总黄酮逆转K562/ADR细胞株作用机制初步研究

    Institute of Scientific and Technical Information of China (English)

    刘玉; 古丽巴哈·买买提; 严媚; 王学梅

    2016-01-01

    ObjectiveTo probe and analyses whether the abnormal savda munziq total flavonoids (ASMq) has a reverse effect on K562/ADR, the resistant strain to human leukemia doxorubicin. Methods First cultivated K562 and K562/ADR cells, and Chose the cytotoxicity-free dose of ASMq which had a cell inhibition rate less than 10%. Resistance index=IC50(K562/ADR)/IC50(K562)=3.075/ 0.069=44.565 22. Then determined two doses for measuring the reverse multiple. At last, calculated the IC50 of K562, K562/ADR cells and K562/ADR+ASMq cells influenced by ADR.Results IC50(K562)= 0.06, IC50(K562/ADR)=3.075, IC50(K562/ADR+X1)=3.146, IC50(K562/ADR+X2)=3.274. Result fold reversal=IC50(K562/ADR)/IC50(K562/ADR+ASMq). X1 fold reversal=IC50(K562/ADR)/IC50(K562/ ADR+X1)=3.075/3.146=0.977 432, X2 fold reversal=0.939 218. According to the results, the impact of ASMq wasn't evident among the groups.ConclusionsASMq in a small dose doesn’t have an evident impact on reversion of multidrug resistance to K562/ADR. The deeper research need to be done about whether a large dose can make a difference.%目的:探讨维药异常黑胆质成熟剂总黄酮(ASMq)是否对人白血病阿霉素耐药株K562/ADR有逆转作用。方法首先对K562K562/ADR细胞进行培养,选择对K562细胞抑制率<10%的药物浓度为 ASMq 的非细胞毒性剂量,耐药倍数=IC50(K562/ADR)/IC50(K562)=3.075/0.069=44.56522,再确定两个剂量用于逆转倍数的测定(X1,X2)。计算ADR对K562K562/ADR细胞和K562/ADR+ASMq细胞的IC50。结果 IC50(K562)=0.069,IC50(K562/ADR)=3.075, IC50(K562/ADR+X1)=3.146,IC50(K562/ADR+X2)=3.274。逆转倍数=IC50(K562/ADR)/IC50(K562/ADR+ASMq)。X1逆转倍数=IC50(K562/ADR)/IC50(K562/ADR+X1)=3.075/3.146=0.977432,X2逆转倍数=0.939218。由K562K562/ADR、K562/ADR+X1、K562/ADR+X2的IC50和计算出的逆转倍数得出,ASMq在各组之间的作用不明显。结论低剂量的ASMq对K562/ADR

  10. Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells

    Institute of Scientific and Technical Information of China (English)

    SHEN Shao-hua; GU Long-jun; LIU Pei-qing; YE Xin; CHANG Wei-shan; LI Ben-shang

    2008-01-01

    Background Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia.The resistance process is multifactorial.To realize the totaI factors involved in multidrug resistance,we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins(CRKL)using siRNA to determine its role in K562/ADM cells.Methods Altered protein expressions between K562/S(K562 ADM-sensitive cell line)and K562/ADM(K562 multidrug resistant cell line induced by adriamycin)were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile,we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis.Furthermore,we used RNA interference to silence the CRKL gene expression.Results Among the 9 differentially expressed proteins,3 were up-regulated in K562/ADM cells,while 6 were down-regulated in the K562/ADM cells compared with its parent cell line.The expression of CRKL was up-regulated significantly in K562/ADM cells,and it can be decreased by recombinant lentivirus.Moreover,the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.Conclusions The data provided the differentially expressed proteins jn K562 and jts resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer.We found CRKL may be a new protein involved in the multidrug resistanse of leukaemia cells.

  11. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  12. Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

    Institute of Scientific and Technical Information of China (English)

    Ruidong MIAO; Xiaohui XIA; Dongling YANG; Minghua LU; Qin WANG

    2008-01-01

    We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.

  13. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2013-03-01

    Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS, which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

  14. Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells

    Directory of Open Access Journals (Sweden)

    Pondel Marc D

    2003-04-01

    Full Text Available Abstract Background The peptide hormone calcitonin (CT can significantly effect the proliferation rate of CT receptor (CTR positive human cancer cells. We wish to identify additional human cancers expressing CTRs and assay the effects of CT on their growth rates and signal transduction pathways. Results The expression of the human calcitonin receptor (hCTR gene in the chronic myelogenous leukemia cell line K562 was examined. RT-PCR on total RNA extracted from K562 cells detected the presence of hCTR mRNA. Further analysis demonstrated that multiple hCTR isoforms were present. Incubation of K562 cells with salmon calcitonin (sCT, but not amylin, caused an increase in intracellular levels of cAMP similar to that induced by forskolin treatment. We further demonstrated that butyrate induced erythroid differentiation of K562 cells caused a significant decrease in hCTR mRNA levels. However, phorbol myristate acetate (PMA induced megakaryocytic differentiation of these cells had no significant effect on hCTR mRNA levels. We demonstrated that exposure to various concentrations of sCT had no effect on the cellular proliferation of K562 cells in vitro. Conclusion Chronic myelogenous k562 cells express multiple CTR isoforms. However, CT does not effect K562 proliferation rates. It is likely that the small increase in intracellular levels of cAMP following CT treatment is not sufficient to interfere with cellular growth.

  15. Effects of a new retinoic acid derivative ATPA on the proliferation and differentiation of leukemia cell line K562%新型维甲酸衍生物ATPA对白血病细胞株K562增殖及分化的影响

    Institute of Scientific and Technical Information of China (English)

    洪凡青; 陈飞虎; 王璐; 陈慧慧; 吴菲; 吴繁荣; 汪渊

    2011-01-01

    目的:本研究探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基依曲替酸酯(4-amino-2-trifluoromethyl-phenyl acitretinate,ATPA)对白血病K562细胞体外增殖和分化的作用.方法:用不同浓度的ATPA作用白血病K562细胞后,在体外通过MTT法检测和绘制细胞生长曲线来分析细胞增殖;瑞特染色法观察细胞形态学改变;氯化硝基四氮唑蓝(nitroblue tetrazolium chloride,NBT)还原实验分析细胞的分化指标;采用FCM法检测细胞表面分化抗原及细胞周期的变化.结果:ATPA呈浓度依赖性抑制K562细胞增殖,明显的抑制作用从药物作用48 h后开始出现,在72 h后作用更明显.倒置显微镜下观察发现ATPA作用后K562细胞形态趋向成熟,NBT阳性细胞率增加;G0/G1期细胞表达量增加,S期细胞表达量减少,呈G0/G1期阻滞;细胞表面分化抗原CD71表达减少.结论:ATPA对白血病K562细胞具有抑制增殖和一定的诱导分化作用.%Objective: To explore the effects of 4-amino-2-trifluoromethyl-phenyl acitretinate (ATPA)on the proliferation and differentiation of K562 leukemia cells in vitro.Methods: After K562 leukemia cells were treated with ATPA at different concentrations, cell proliferation was assessed by MTT and cell growth curve.Morphologic changes of cells were observed under an inverted microscope (oilimmersion lens) after Wright's staining.Nitroblue tetrazolium (NBT) reduction test was used to analyze cell differentiation indices.The cell cycle and the expression of the specific cell surface maturation marker CD71 were analyzed by flow cytometry (FCM).Results: The growth of K562 cells treated with ATPA was inhibited in a dose-dependent manner.The distinct inhibitory effect appeared after treatment with ATPA for 48 h, but the effect was more significant after 72 h.Morphology of K562 cells treated with ATPA was observed to be mature under an inverted microscope.NBT reduction test indicated that ATPA could increase the percentage of NBT

  16. [Silence potentiates chemosensitivity of K562 cells to SAHA].

    Science.gov (United States)

    Wang, Hou-Cai; Chen, Jing; An, Na; Yu, Teng-Teng; Li, Shou-Yun; Liu, Shuang; Wei, Hui; Rao, Qing; Wang, Min; Wang, Jian-Xiang

    2014-08-01

    Ribosomal protein S27a (RPS27a) can perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The RPS27a gene has been reported to be over-expressed in breast fibroadenomas, colorectal and renal cancers, advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. This study was purposed to explore the function of RPS27a in CML-erythroleukemia cell line K562 cells. RPS27a was silenced by short hairpin RNA (shRNA) in K562 cells. Furthermore, the proliferation changes of K562 cells was detected by MTT method after silencing the RPS27a with suberoylanilide hydroxamic acid (SAHA), then the IC50 of K562-sh1/sh2 and K562-scr cells to SAHA was measured. The results indicated that compared with K562-scr cells, the IC50 of K562-sh1/sh2 to SAHA at 24 h and 48 h decreased (P silence significantly increased the percentage of apoptotic K562-sh1/sh2 cells after incubation with 1 µmol/L, 2 µmol/L and 5 µmol/L SAHA for 24 h and 48 h as compared with that of K562-scr cells (P silence can potentiate sensitivity of K562 cells to SAHA.

  17. Role of yessotoxin in calcium and cAMP-crosstalks in primary and K-562 human lymphocytes: the effect is mediated by anchor kinase A mitochondrial proteins.

    Science.gov (United States)

    Tobío, Araceli; Fernández-Araujo, Andrea; Alfonso, Amparo; Botana, Luis M

    2012-12-01

    Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3',5'-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca(2+) ), whereas the contrary effect has been observed in a Ca(2+) -free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca(2+) increase in fresh human lymphocytes but no effect was observed on Ca(2+) pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca(2+) levels in a medium with Ca(2+) and induces an increase on Ca(2+) pools depletion followed by a Ca(2+) influx. As far as Ca(2+) modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca(2+) reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca(2+) pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca(2+) , YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca(2+) influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell

  18. [Ginsenoside Rh₂ induces apoptosis and autophagy of K562 cells by activating p38].

    Science.gov (United States)

    Liu, Xiao-Xia; Xia, Jing; Tang, Jia-Feng; Zhou, Ming-Hua; Chen, Di-Long; Liu, Ze-Hong

    2017-01-01

    To study the effect of ginseng saponin Rh₂ in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh₂; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh₂ on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh₂ at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh₂ induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh₂; Acridine and MDC staining showed that Rh₂ enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh₂ increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh₂ on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh₂ inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells. Copyright© by the Chinese Pharmaceutical Association.

  19. Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity.

    Science.gov (United States)

    Long, Mark D; van den Berg, Patrick R; Russell, James L; Singh, Prashant K; Battaglia, Sebastiano; Campbell, Moray J

    2015-09-01

    To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.

  20. Potential Role of Nucleophosmin(NPM1)Gene Mutations in K562 Leukemia Cell Invasion Phenotype%NPM1突变基因调控K562白血病细胞侵袭表型的相关机制

    Institute of Scientific and Technical Information of China (English)

    陈先春; 覃凤娴; 谭诗; 张慧娟; 邵会媛; 张伶

    2011-01-01

    Objective To investigate the role of CXCR4 and Angs in the invasion phenotype in vitro of leukemic cells with NPM1 mutations, and further study the effect of NPM1 mutations on leukemia infiltration. Methods The pEGFPC1-NPM1-mA plasmid vector was transfected into K562 cells. The cells stably expressing NPM1-mA protein were established, named as K562-mA. The expressions of CXCR4 and Ang-1/2 mRNA were assayed by quantitative Real-Time PCR ( qRT-PCR ). The expression of CXCR4 protein was assayed by Western blot and flow cytometry. Results Compared with control groups, CXCR4 mRNA and protein expression levels were significantly increased in K562-mA group. The expression of Ang-1 mRNA was markedly decreased, whereas Ang-2 mRNA expression was increased in K562-mA group. Conclusion CXCR4 and Ang-1/2 may play important roles in the invasion phenotype of leukemic cells with NPM1 mutations in vitro.%目的 探讨CXCR4、Angs在NPM1突变参与调控的白血病细胞浸润转移中的作用,以期进一步明确NPM1突变在白血病浸润转移中的调控机制.方法 通过基因转染构建稳定表达NPM1突变蛋白的K562白血病细胞株(K562-mA).qRT-PCR检测各组细胞CXCR4、Ang-1/2的mRNA表达水平;Western免疫印迹和流式细胞仪分别检测细胞CXCR4总蛋白和膜蛋白的表达.结果 建立了稳定表达NPM突变基因的K562-mA细胞株.与未处理组和空载体转染组相比,K562-mA细胞CXCR4的mRNA和蛋白表达水平显著增高;Ang-1 mRNA表达水平明显降低、Ang-2 mRNA表达水平明显增高.结论 CXCR4、Ang-1/2可能在NPM1突变调控白血病细胞的浸润转移中发挥重要作用.

  1. Myc induced miR-144/451 contributes to the acquired imatinib resistance in chronic myelogenous leukemia cell K562

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Li, E-mail: liuli029@yahoo.cn [Department of Hematology, Tangdu Hospital, The Fourth Military Medical University, Xi' an 710038 (China); Wang, Sitao; Chen, Renan; Wu, Yanlan; Zhang, Bei; Huang, Siyong; Zhang, Jingyi; Xiao, Fang; Wang, Meng; Liang, Yingmin [Department of Hematology, Tangdu Hospital, The Fourth Military Medical University, Xi' an 710038 (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Increased c-myc expression in imatinib resistant CML cells. Black-Right-Pointing-Pointer c-myc contributes the imatinib resistance in CML cells. Black-Right-Pointing-Pointer c-myc transcriptionally reduces the expression of miR-144/451 in K562R cells. Black-Right-Pointing-Pointer Restoration of miR-144/451 reverses the resistance of K562R cells to imatinib. -- Abstract: Imatinib resistance remains the big hurdle for CML therapy. Previous study reveals that c-myc is important for bcr-abl CML cell proliferation, while its role in imatinib resistance is largely unknown. In this study, we first found that c-myc expression is upregulated in imatinib resistant K562R cells, which in turn enhances the expression of miR-144/451. Knockdown of c-myc or restoration of miR-144/451 in the K562R cells sensitizes K562R cells to imatinib therapy. Our study here reveals an regulatory pathway between myc and miR-144/451 and highlights that targeting either myc or miR-144/451 might be valuable for eliminating the imatinib resistant CML cells.

  2. Homoharringtonine induces apoptosis of endothelium and down-regulates VEGF expression of K562 cells

    Institute of Scientific and Technical Information of China (English)

    叶琇锦; 林茂芳

    2004-01-01

    Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

  3. Synthesis and Evaluation of Haloacetyl, α-Bromoacryloyl and Nitrooxyacetyl Benzo[b]furan and Benzo[b]thiophene Derivatives as Potent Antiproliferative Agents Against Leukemia L1210 and K562 Cells

    Science.gov (United States)

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Carrion, Maria Dora; Cara, Carlota Lopez; Casolari, Alberto; Hamel, Ernest; Fabbri, Enrica; Gambari, Roberto

    2010-01-01

    Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. In search of new compounds with strong antiproliferative activity and simple molecular structure, we have synthesized three different series of compounds in which different substituents were linked to the 3-amino position of the 2-(3′, 4′, 5′-trimethoxybenzoyl)-benzo[b]furan or benzo[b]thiophene ring system. These substituents, corresponding to acetyl/haloacetyl, α-bromoacryloyl and nitrooxyacetyl moieties had different electrophilic properties. The benzoheterocycle parent structures were selected because of their reported bioactivities. Compounds bearing a methoxy group at the 6-position of the benzo[b]furan skeleton, were identified as potent antiproliferative agents against the human chronic myelogenous K562 and murine L1210 leukemia cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 6- to the 5- or 7-position yielded inactive compounds. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. The analysis of structure-activity relationships observed in the series of compounds described here may represent a platform for the design of more active molecules. PMID:20676361

  4. 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.

  5. 夜香树花甾体皂苷抑制K562细胞增殖及对PI3K/AKt信号通路的调控%Inhibitory Effect of the Steroid Saponins from the Flowers of Cestrum Nocturnum,Linn(SSFCN) on Proliferation of Leukemia K562 Cells and PI3K/AKt Pathway Regulation

    Institute of Scientific and Technical Information of China (English)

    罗发军; 赵世元; 钟振国

    2013-01-01

    目的 探讨夜香树花甾体皂苷(SSFCN)抑制K562细胞增殖及作用机制.方法 夜香树花用有机溶剂提取、分离并鉴定SSFCN,体外培养K562细胞,采用MTT比色法测定抑制率;流式细胞仪分析细胞周期、琼脂糖凝胶电泳检测DNA图谱的变化,Western blot 法检测用SSFCN处理的K562细胞AKt磷酸化水平的变化.结果 SSFCN能显著抑制人白血病细胞K562的生长,作用后细胞生长有明显凋亡特征性改变,凋亡率呈时间和剂量依赖性.60 mg/L 的SSFCN作用K562细胞24 h后,流式细胞仪检测出现亚G1峰及DNA电泳呈梯形条带.Western blot 结果表明,SSFCN处理K562细胞72 h后,K562细胞p-AKt磷酸化水平明显下调.结论 SSFCN具有抑制K562细胞增殖和促进凋亡的作用,SSFCN通过抑制K562细胞PI3K/AKt信号通路,从而抑制K562细胞增殖.

  6. Study on Taxol in Inhibiting Human Leukemia Cell Proliferation and Inducing Apoptosis

    Institute of Scientific and Technical Information of China (English)

    赵小英; 张晓红; 徐磊; 张行

    2004-01-01

    Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations for 12-72 hrs. Cell proliferation was evaluated by MTT assay and morphological changes of apoptosis were examined by microscopy. Cell apoptosis was determined by flow cytometry (FCM) and DNA gel electrophoresis. Results: Growth of K562 cells was inhibited by Taxol with an IC50 value of 0.84 μg/mi.Typical nuclear condensation and apoptosis bodies were observed as early as 24 hrs after a 0.5 μg/ml Taxol treatment; Apoptotic rate of the Taxol-treated K562 cells increased from 3.7% to 24.0% in 24 hrs. No DNA ladder was observed by DNA gel electrophoresis. Conclusion: Taxol could inhibit K562 cell growth and induce apoptosis in vitro.

  7. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: wanghq_doctor@hotmail.com [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)

    2010-01-01

    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.

  8. Signal transduction factors on the modulation of radiosusceptibility in K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Kwang Mo; Jeong, Soo Jin [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Youn, Seon Min [College of Medicine, Eulji Univ., Daejeon (Korea, Republic of)

    2003-09-01

    The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes the induction of apoptosis via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A [HMA] coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosis of p210{sup bcr}/{sup abl} protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the induction of a number of transcription factors and the differential gene expression in this model were investigated. K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 MeV Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with 0.25{mu}M of HMA and 25{mu}M of genistein, and the expressions and the activities of ablkinase, MAPK family, NF-KB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either. In association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF-KB activity and the TK 1 expression and activity. The effects of HMA and genistein on the radiosensitivity of the K562 cells were not related to the bcr-abl kinase activity. In this study, another signaling pathway, besides the MAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the

  9. Cytotoxicity of calotropin is through caspase activation and downregulation of anti-apoptotic proteins in K562 cells.

    Science.gov (United States)

    Wang, Shih-Chung; Lu, Mei-Chin; Chen, Hsiu-Lin; Tseng, Hsing-I; Ke, Yu-Yuan; Wu, Yang-Chang; Yang, Pei-Yu

    2009-12-01

    Calotropin is one of cardenolides isolated from milkweed used for medicinal purposes in many Asian countries. Whereas calotropin possesses cytotoxicity against several cancer cells, the mechanisms of action remain unclear. We set out to evaluate the cytotoxic mechanism of calotropin on human chronic myeloid leukemia K562 cells. Calotropin inhibited the growth of K562 cells in a time- and dose-dependent manner by G(2)/M phase arrest. It upregulated the expression of p27 leading to this arrest by downregulating the G2/M regulatory proteins, cyclins A and B, and by upregulating the cdk inhibitor, p27. Furthermore, it downregulated anti-apoptotic signaling (XIAP and survivin) and survival pathways (p-Akt and NFkappaB), leading to caspase-3 activation which resulted in the induction of apoptosis. In all, calotropin exerted its anticancer activity on K562 cells by modulating the pro-survival signaling that leads to induction of apoptosis.

  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. A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'-flanking sequence of human β-globin gene

    Institute of Scientific and Technical Information of China (English)

    HUYULONG; YADICHEN; TONGSUN; RUOLANQIAN

    1993-01-01

    It has been known that there are at least three regulatory regions (NCR1. NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1 bp) of human β-glohin geneand that the function of PCR is unique to the human erythroleukemia (Ksfi2) ceils. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 ceils. which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroidspecific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific transcriptional factor (NFE-1) ,nsing competition assay. The presence of the NFEa further supported that the funciton of the cis-acting element PCR was specitic for K562 cells. and helps us to understand the mechauism of the regulation of the expression of lmman β-globin gene in the human K562 cells.

  12. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun

    2015-09-27

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  13. Effect of ATRA on the expression of HOXA5 gene in K562 cells and its relationship with cell cycle and apoptosis.

    Science.gov (United States)

    Liu, Wen-Jun; Zhang, Teng; Guo, Qu-Lian; Liu, Chun-Yan; Bai, Yong-Qi

    2016-05-01

    Leukemia is the most common malignant disease in children with high incidence and mortality rates, and a poor treatment effect. The aim of the present study was to examine the changes in the expression of homeobox (Hox) A5 gene and its relationship with cell cycle and apoptosis through the intervention of human K562 myeloid leukemia cell line by all-trans retinoic acid (ATRA), to analyze the role of HOXA5 in the pathogenesis and development process of myeloid leukemia. The optimal concentration of ATRA to be used with K562 cells was determined using a cell counting kit‑8 (CCK‑8). After 24, 72 and 48 h following treatment of K562 cells with 10 µmol/l ATRA, cell cycle events and apoptosis were measured using flow cytometry. HOXA5 mRNA and protein expression in K562 cells was assessed by RT‑PCR and western blot analysis, and the relationship between HOXA5 expression and cell cycle and apoptosis was analyzed. The HOXA5 mRNA and protein expression levels were increased following treatment with ATRA in K562 cells. Apoptosis was increased significantly. The cell cycle was inhibited in G0/G1 phase. Cell proliferation was also inhibited. HOXA5 mRNA and protein expression rates positively correlated with cell apoptosis and the increased percentage and cell cycle of the G0/G1 phase. However, HOXA5 negatively correlated with the reduced percentage of S stage. In conclusion, the expression of HOXA5 in cells was increased following treatment with ATRA in K562 cells, in a time-dependent manner. Additionally, ATRA may inhibit the proliferation of K562 cells and promote apoptosis by upregulating the HOXA5 mRNA and protein expression.

  14. 人红白血病细胞株K562上清液对人脐静脉内皮细胞的促增殖作用%Enhancement Effect of K562 Cells Supernatant on Proliferation of Human Umbilical Vein Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    潘小兵; 曹诗运

    2009-01-01

    目的 研究人红白血病细胞株(K562)上清液对人脐静脉内皮细胞(hUVEC)增殖能力的影响.方法 以hUVEC与K562细胞共培养为K562细胞组,以hUVEC加K562细胞上清液为K562细胞上清液组,以单独培养hUVEC为对照组.采用RT-PCR技术检测各组中hUVEC周期蛋白E(cyclinE)mRNA及周期蛋白依赖性激酶2(CDK2)mRNA的表达情况.结果 K562细胞组、K562细胞上清液组中hUVEC cyclinE mRNA及CDK2 mRNA表达明显增加,与对照组相比差异均有显著性(P<0.01). 结论人白血病肿瘤细胞K562上清液能够促进hUVEC的分裂增殖.

  15. Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells.

    Science.gov (United States)

    Tuo, Yanfeng; Jiang, Shujuan; Qian, Fang; Mu, Guangqing; Liu, Peng; Guo, Yuanji; Ma, Changlu

    2015-01-01

    Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

  16. Studies on the Anti-tumor Action of active components of Menispermum dauricum to leukemia K562 cells%蝙蝠葛活性成分对人白血病K562细胞株抗肿瘤作用的研究

    Institute of Scientific and Technical Information of China (English)

    杨万山; 孙抒; 汪俊颖; 全宗学

    2014-01-01

    目的 研究蝙蝠葛活性成分体外对人白血病K562细胞株的抑制增殖和诱导凋亡作用.方法 应用MTT法检测蝙蝠葛活性成分对体外培养的人白血病K562细胞株的抑制增殖作用的影响及细胞毒活性;通过倒置显微镜对细胞进行形态学观察;通过免疫细胞化学S-P法测定凋亡关键效应酶Caspase-9、Caspase-8和Caspase-3的表达情况.结果 (1)MTT比色法结果表明,蝙蝠葛活性成分对人白血病K562细胞株的增殖抑制作用呈剂量依赖性(P<0.01);(2)倒置显微镜观察:蝙蝠葛活性成分20 μg/ml作用后早期细胞出现凋亡形态学变化:细胞膜鼓泡、凋亡小体形成等;作用晚期细胞发生膜破裂坏死;(3)免疫细胞化学结果表明:蝙蝠葛活性成分作用后加药组Caspase-9、Caspase-8和Caspase-3表达增加,与对照组相比较均有显著差异(P<0.01).结论 蝙蝠葛活性成分对人白血病K562细胞株具有抑制增殖和诱导凋亡作用.

  17. 喷他脒增强K562细胞对肿瘤坏死因子相关凋亡诱导配体诱导凋亡敏感性%Pentamidine sensitizes leukemia K562 cells to TRAIL-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    刘小珊; 蒋纪恺

    2007-01-01

    背景与目的:肿瘤坏死因子相关凋亡诱导配体(TRAIL)是一种理想的抗肿瘤药物,但许多肿瘤细胞常对TRAIL诱导凋亡耐受.本研究探讨了喷他脒增强白血病K562细胞对TRAIL诱导凋亡敏感性.方法:利用光镜形态学和Annexin V FITC/PI 双标记凋亡细胞的流式细胞仪(FACS)测定两种方法观察喷他脒预处理K562细胞并继用TRAIL后凋亡的发生.应用蛋白印迹方法观察此过程中半胱氨酸-天冬氨酸蛋白酶(Caspase)-3,-8和聚ADP核糖聚合酶(PARP)等3种蛋白的蛋白剪切与X连锁凋亡抑制蛋白(XIAP)蛋白表达的改变.结果:在10 μg/ml喷他脒作用K562细胞20 h,K562细胞未发生凋亡,继用200 ng/ml TRAIL 作用4 h后,光镜和Annexin V FITC/PI 双标记流式细胞仪方法均观察到细胞发生明显凋亡,并出现了Caspase-3,-8和PARP蛋白剪切,两者单独作用则无明显细胞凋亡发生.另外,喷他脒明显降低了XIAP表达.结论:喷他脒联合TRAIL可能成为肿瘤治疗的一种新策略.

  18. In vitro evaluation of the anti-proliferative activities of the wood essential oils of three Cedrus species against K562 human chronic myelogenous leukaemia cells.

    Science.gov (United States)

    Saab, Antoine M; Lampronti, Ilaria; Borgatti, Monica; Finotti, Alessia; Harb, Faouzi; Safi, Samir; Gambari, Roberto

    2012-01-01

    There are four kinds of Cedar: Cedrus libani naturally occurring in Lebanon, Syria and Turkey, Cedrus atlantica in Morocco and Algeria, Cedrus brevefolia in Cyprus Island and Cedrus deodara which is distributed in Himalayan Mountains. Wood essential oils obtained from C. libani, C. atlantica and C. deodara were tested for the inhibition of K562 cell proliferation and for the induction of erythroid differentiation. The wood essential oils of C. libani, C. atlantica and C. deodara inhibited the proliferation of the K562 cell line exhibiting IC(50) values 23.38 ± 1.7, 59.37 ± 2.6 and 37.09 ± 1.4 µg mL(-1), respectively. Meanwhile, C. libani wood oils induced a percentage of erythroid differentiation of 15 ± 2% at concentration 5 µg mL(-1). Cedrus deodara wood oil indicated a percentage of erythroid differentiation of 20 ± 2% at concentration 25 µg mL(-1) and C. atlantica wood oils showed a percentage of erythroid differentiation of 12 ± 1.8% at concentration 10 µg mL(-1).

  19. Cytotoxic effect of Spirulina platensis extracts on human acute leukemia Kasumi-1 and chronic myelogenous leukemia K-562 cell lines

    Directory of Open Access Journals (Sweden)

    Flor Yohana Flores Hernandez

    2017-01-01

    Conclusions: The cytotoxicity exhibited by Spirulina extract to cancer cell lines might be due to the presence of phytopigments (carotenoids, chlorophyll, phycocyanin as well as polysaccharides that were reported previously as constituents of the extract. So crude extracts of Spirulina can be used as a source to develop anticancer drugs.

  20. The effect of PG to the expression of p170 on the K562/ADR cells%裴氏升血颗粒含药血清对K562/ADR细胞p170表达的影响

    Institute of Scientific and Technical Information of China (English)

    吴玉强; 薛文翰

    2015-01-01

    目的:观察裴氏升血颗粒(PG)含药血清对人白血病细胞株K562/ADR细胞p170蛋白表达水平的影响。方法:采用MTT法测定不同浓度含药血清对K562细胞系的细胞毒作用,用流式细胞仪检测非细胞毒性浓度含药血清处理后K562/ADR细胞膜表面p170表达变化。结果:不同浓度含药血清对K562/ADR细胞无明显细胞毒作用,非细胞毒性含药血清能明显下调K562/ADR细胞p170蛋白的表达。结论:裴氏升血颗粒能够提高化疗疗效,其机制可能与下调p170蛋白表达有关。%Objective:To investigate the effect of PG herbal serum to the expression level of p170 protein on the human leukemia K562/ADR cells. Methods: Testing the cytotoxic effect of different herbal serum to K562/ADR cells line by MTT method, using Flow cytometry to determine the expressive and functional changes of p170 on K562/ADR cells membrane dealed with the herbal serum at non-cytotoxic concentration. Results:Different herbal serum had no obvious cytotoxic effect to K562/A02 cells, non-cytotoxic concentration of herbal serum can significantly lowered the expression of p170 protein on the K562/ADR cells. Conclusion: PG could improve the curative effect of chemotherapy, and its mechanism may be related to descend the expression of p170.

  1. 盐酸千金藤碱逆转K562/ADR细胞多药耐药性及其机制%Correlation between reversing effect of cepharanthine hydrochloride on multidrug resistance and P-glycoprotein expression and function of K562/ADR cells

    Institute of Scientific and Technical Information of China (English)

    彭有梅; 王宁; 王亚峰; 韩立; 张艳; 江金花; 周玉冰; 王庆端

    2012-01-01

    研究盐酸千金藤碱(cepharanthine hydrochloride,CH)逆转K562/ADR细胞多药耐药性及其机制.采用MTT法检测多柔比星(adriamycin,ADR)单用及分别与CH、维拉帕米(verapamil,VER)合用的细胞毒作用;采用流式细胞仪,测定CH对细胞内ADR蓄积、罗丹明123 (Rho123)蓄积和泵出及P糖蛋白(P-gp)表达的影响.结果表明,CH(4 μmol·L-1)使K562/ADR细胞对ADR的敏感性增加7.43倍,逆转活性是VER的3.19倍,但对K562敏感株基本无影响.同时CH浓度依赖性地增加K562/ADR细胞内ADR和Rho123的蓄积,减少Rho123的泵出,抑制P糖蛋白的表达,但对K562细胞均无明显影响.CH在体外逆转肿瘤细胞多药耐药性的作用可能与其抑制P糖蛋白的功能和表达有关.%In this study, cepharanthine hydrochloride (CH) was tested for its potential ability to modulate the expression and function of P-glycoprotein (P-gp) in the multidmg-resistant human chronic myelogenous leukemia cell line K562/ADR. Cytotoxicity of adriamycin (ADR) alone or in combination with CH or verapamil (VER) in K562 and K562/ADR cells was determined by MTT assay. Based on flow cytometric technology, the effect of CH or VER on the uptake and efflux of rhodaminel23 (Rhol23) and the accumulation of ADR in these cells was detected by measuring Rhol23 or ADR-associated mean fluorescence intensity (MFI). The effects of CH and VER on P-glycoprotein (P-gp) expression in K562 and K562/ADR cells were also measured using a flow cytometry with PE-conjugated P-glycoprotein antibody. The results show that CH significantly enhanced the sensitivity of K562/ADR cells to ADR, 4 μmol·L"1 of CH enhanced the sensitivity of K562/ADR cells to ADR by 7.43 folds, the reversal activity was 3.19 times higher than that of verapamil. However, CH had no effect on drug-sensitive K562 cells (P < 0.05). CH increased Rhol23 and ADR accumulation in a concentration-dependent manner (2-8 umol·L-1) and inhibited the efflux of Rhol23 from these cells, but

  2. Study on the Flavonoid of Huanglian Jiedu Decoction in Reversing MDR of K562/ADM%黄连解毒汤中黄酮成分逆转K562/ADM多药耐药的实验观察

    Institute of Scientific and Technical Information of China (English)

    盛国良; 林潇; 孙付军; 李贵海

    2012-01-01

    目的:观察黄连解毒汤中黄酮成分逆转肿瘤多药耐药的作用,探讨本方逆转肿瘤多药耐药的物质基础.方法:以人慢性粒细胞白血病红白血病细胞株K562的耐阿霉素(adriamycin,ADM)细胞株(K562/ADM)为细胞系,通过MTT实验观察黄芩苷、京尼平苷对K562/ADM细胞ADM的敏感性的影响,计算细胞增殖抑制率、半数抑制浓度(IC50)及耐药逆转倍数,并对细胞内ADM浓度变化进行测定.结果:黄芩苷、京尼平苷均能部分逆转K562/ADM细胞.黄芩苷、京尼平苷的IC50值分别为5.06,6.74 mg·L-1.黄芩苷、京尼平苷的耐药逆转倍数分别为1.95,1.46倍.与相应的对照组相比,K562/ADM细胞经黄芩苷(50 mg·L-1)、京尼平苷(100 mg·L-1)作用后,细胞内ADM的荧光强度高于对照组,其中黄芩苷组提高到3.6%,京尼平苷组提高到1.7%.结论:黄连解毒汤逆转肿瘤多药耐药的物质基础可能与其含有的黄芩苷、京尼平苷有关.%Objective; To observe the effect on the flavoniod of Huanglian Jiedu decoction, and discuss its material base in reversing multiple-drug resistance (MDR) of cancer so as to provide the theory basis for Huanglian Jiedu decoction in clinical practices. Method: MTT assay was adopted to test the sensitivity of baicalin and geniposide to adriamycin ( ADM ) of K562/ADM, the inhibitory rate was calculated, the value of 50% inhibition concentration (IC50) and the multiple of the reversal of drug resistance as well as the change of the concentration of the intracellular ADM were assayed by using ADM K562/ADM of human chronic myelogenous leukemia (CML) erythroleukemia K562 as cell line. Result; Both baicalin and geniposide could partly reverse K562/ADM cell. The value of IC50of baicalin and geniposide was 5. 06 mg - L~ and 6. 74 mg - L-1respectively. The multiple of the reversal of drug resistance of baicalin and geniposide was 1.95 times and 1.46 times. The fluorescence intensity of the intracellular K562/ADM was

  3. Identification of a NF-kB site in the negative regulatory element (εNRAII) of human ε-globin gene and its binding protein NF-κB p50 in the nuclei of K562 cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The developmental control of the human ε-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-κB consensus sequence resides in the negative regulatory element (-3028bp ~ -2902bp, termed ε-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp ~-2986bp) containing the NF-κB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-κB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-κB motif) could not, suggesting that the binding protein is a member of NF-κB/Rel family. Western blot assay demonstrated that the molecular weight of NF-κB protein in the nuclei of K562 cells is 50ku. We suggested that NF-κB p50 may play an important role in the regulation of human ε-globin gene expression.

  4. 蝎毒多肽提取物对白血病细胞株K562/A02荷瘤鼠耐药性的影响%Reversion Impact of Peptide Extract from Scorpion Venom on Multidrug-resistance of Leukemia Stem Cell Line K562/A02 in vivo

    Institute of Scientific and Technical Information of China (English)

    杨向东; 杨文华; 刘宝山; 伊学军; 高宏; 姚芳; 王兴丽; 闫理想

    2016-01-01

    目的 探讨蝎毒多肽(peptide extract from scorpion venom,PESV)逆转白血病干细胞(leukemiastem cells,LSC)在体内多药耐药(multidrug resistance,MDR)的分子机制.方法 以多药耐药的K562/A02细胞株成模白血病BALB/c裸鼠为例,成模鼠随机分为6组:模型对照组、阿霉素(ADM)组、PESV组、ADM+PESV(H)组、ADM+PESV(M)组、ADM+PESV(L)组.模型对照组给予等体积0.9%氯化钠溶液腹腔注射,其余各组予相应剂量ADM和(或)PESV腹腔注射,连续给药14天.第21天观察各组裸鼠移植瘤生长情况,分别检测瘤块中LSC:细胞膜上P-gp的表达,细胞质中ALDH、PI3K的变化及细胞核中MDR1、NF-κB的活性.结果 K562/A02细胞经免疫磁珠分选前后的CD34+CD38-细胞比率和IC50值分别为31.5%、(60.33±10.68)μg/ml和92.8%、(58.33±9.72)μg/ml,分选后细胞干性显著提高,而耐药性无差异性损失;各组造模裸鼠成瘤率100%.瘤体中LSC:流式细胞仪检测细胞膜上P-gp表达结果:检测对照组89.8%、ADM组91.9%、PESV组88.4%、ADM+PESV(H)组53.9%、ADM+PESV(M)组78.0%、ADM+PESV(L)组78.7%;半定量RT-PCR检测MDRl mRNA的表达:PESV组>ADM+PESV(L)组> ADM+PESV(M)组>ADM+PESV(H)组>ADM组;免疫组织化学检测ALDH,显示灰度值ADM组>PESV组>ADM+PESV(H)组>ADM+PESV(M)组>ADM+PESV(L)组;Western blot检测PI3K分子与Elisa检测NF-κB因子结果一致,在ADM组、PESV组表达上调,在ADM+PESV组中表达下调,下调强度与PESV剂量呈正相关.结论 PESV具有下调白血病干细胞膜上P-gp,细胞质内ALDH、PI3K及细胞核中MDR1、NF-κB的表达水平,增强了白血病K562/A02干细胞在体内对ADM的敏感度,逆转其多药耐药特性.

  5. In vitro radiosensitivity of human leukemia cell lines

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    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 (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  6. Antiproliferation Effects of Curcumin on the STAT5 Signaling Pathway in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Hongli Liu; Weihong Chen

    2005-01-01

    OBJECTIVE Curcumin is the major component of the spice turmeric and the yellow pigment in curry powder. Many studies have shown that curcumin (diferuloylmethane) has significant antiproliferative and apoptotic effects in cancer cells by several mechanisms. Signal transducers and activators of transcription (STAT) proteins are critical in mediating a response in hematopoietic cells. This study was designed to investigate whether curcumin is associated with proteins involved in signal transduction and activation of transcription (STAT) and to investigate the expression of signal transducers and activators of transcription and the significance of the STAT5 signalingpathway of by treating k562 cells and cells from CML patients with curcumin.METHODS The study was divided into the following groups: normal control cells (human bone marrow cells), untreated K562 cells, curcumin treated K562 cells, IFN-γ treated K562 cells, curcumin plus IFN-γ treated K562cells, and CML patient cells with and without curcumin treatment. Cell proliferation was measured by the MTT assay. The expression of STAT5 mRNA was determined by RT-PCR. The expression of the STAT5 protein was assayed by Western-blotting and the expression of STAT5 in K562 cells was examined under confocal laser-scanning microscopy. The expression of STAT5 mRNA of K562 cells was determined with in situ hybridization. EMSA was used to assess the change in binding of STAT5 with DNA in CML patient cells.RESULTS The proliferation of the K562 cells and CML primary cells was decreased in the curcumin-treated group and/or IFN-γ group. The expression of STAT5 mRNA and protein were decreased the curcumin-treated group as compared with the K562 untreated group (P<0.01). STAT5 mRNA and protein expression was decreased in the IFN-γ group compared to the untreated K562 group (P<0.01). Combined use of curcumin with IFN-γ inhibited the proliferation of K562 cells and decreased the expression of STAT5mRNA and protein of the K562

  7. A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    Science.gov (United States)

    Martinez-Castillo, Macario; Bonilla-Moreno, Raul; Aleman-Lazarini, Leticia; Meraz-Rios, Marco Antonio; Orozco, Lorena; Cedillo-Barron, Leticia; Cordova, Emilio J.

    2016-01-01

    Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562), were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB) occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562), which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP. PMID:27832139

  8. Berberine-induced apoptosis via decreasing the survivin protein in K562 cell line.

    Science.gov (United States)

    Pazhang, Yaghub; Ahmadian, Shahin; Mahmoudian, Massoud; Shafiezadeh, Mahshid

    2011-12-01

    Berberine is an isoquinoline alkaloid with multiple pharmacological activities, including anti-inflammatory and anti-diarrhea effect, the induction of apoptosis and anti-cancer effect. It has been reported that berberine exerts its anti-inflammatory effect via suppressing nuclear factor-kappa B (NF-κB) expression. Survivin and inducible nitric oxide synthase (iNOS) proteins may contribute to the causal relationship between anti-inflammatory and anti-apoptotic function. To investigate the mechanism of berberine-induced apoptotic activities, the human erythro-myeloblastoid leukemia cell line (K562 cell line) was treated with different concentrations of berberine (25-100 μM). The most significant cellular growth arrest and apoptotic effects were observed in the cells treated with 75 μM of berberine for 72 h. The results indicate that survivin and iNOS protein levels were decreased in berberine-treated cells. However, decrease in the iNOS activity did not affect the cell growth and apoptosis. Moreover, the addition of NO donor, sodium nitroprusside, to culture medium decreased the cell growth in the present cell line, but it seemed that its concentration was too low to induce apoptosis. So despite its production by iNOS in untreated cells, NO does not play a significant role in carcinogenesis in this cell line. These results indicate that the apoptotic activity of berberine may be mediated through the reduction of survivin in K562 cells, but iNOS level and its activity does not play a significant role in berberine-induced apoptosis.

  9. The modulation of radiation-induced cell death by genistein in K562 cells:Activation of thymidine kinase 1

    Institute of Scientific and Technical Information of China (English)

    Min Ho JEONG; Young Hee JIN; Eun Young KANG; Wol Soon JO; Hwan Tae PARK; Jae Dong LEE; Yeo Jin YOO; Soo Jin JEONG

    2004-01-01

    Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML)cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization(SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK 1enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition,the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.

  10. An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

    Science.gov (United States)

    Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

    2015-12-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

  11. PENGARUH EKSTRAK JAMU TERHADAP AKTIVITAS SEL NATURAL KILLER DALAM MELISIS ALUR SEL LEUKIMIA (K-562 SECARA IN VITRO [The Effects of Commercial “Jamu” Extracts on Natural Killer Cell Activity in Lysing Leukemic Cell Line (K-562 in vitro

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    Elisa Veronica D.C. 2

    2002-04-01

    Full Text Available Natural killer (NK cell consitutes white blood cells which specifically functions in lysing tumor and virus invected cells. In this research, a commercial “Jamu” was tested to observe its effect on NK cells activity against leukemic cell lines (K562 in vitro. Jamu was extracted with hot water, diluted and added into cell cultures consisted of a mixture of human peripheric limphocyte cells, as the source of the effector NK cells, and K562 cell line i.e., the target cells which were cell line derived from human leukemia and had been labelled with H3-thymidine. The mixture of the cells were made by culturing the two cells at the ratio of 50:1 and 100 : 1, respectively. The results showed that lysing activity of NK cells in the presence of “Jamu” water extract measured as lysing percentage and lysing index increased only slightly, which were not statiscally significant. It should be considered that the test used in this research represents only a part of the lysing mechanism by NK cells against the target cells. An in vivo test for a period of time will be recessary to elucidate ffurther this NK cell activity.

  12. Binding and endocytosis of monoterbium transferrin by K562 cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using isotopic labeling of human serum apotransferrin, the binding and the endocytosis of monoterbium transferrin (TbC-apotransferrin, TbC-apotransferrin- FeN) by K562 cells, a human leukemic cell line, have been investigated. There are about (8.58±2.41)×105 binding sites per cell surface at 0℃. The association constant for TbC-apo- transferrin binding is 4.1×107 mol-1@L, for TbC-apo- transferrin-FeN 2.7×107 mol-1@L at 0℃. At pH 7.4, upon warming cells to 37℃, endocytosis starts. The rate constants for the endocytosis are about 0.97 min-1 and 0.31 min-1 and the endocytosis ratio reaches 56% and 80% for TbC-apo- transferrin and TbC-apotransferrin-FeN, respectively.

  13. Clonal Expansion and Cytotoxicity of TCRVβ Subfamily T Cells Induced by CML and K562 Cells

    Institute of Scientific and Technical Information of China (English)

    YupingZHang; YangqiuLi; ShaohuaChen; LijianYang; GengxinLuo; XueliZhang

    2004-01-01

    OBJECTIVE To investigate the anti-leukemia effect, the distribution and clonal expansion of TCRVβ subfamily T cells in T cells from cord blood and adult peripheral blood induced by CML cells and K562 cells in vitro. METHODS Peripheral blood T cells from one adult donor and 3 cases of cord blood were stimulated with CML cells and K562 cells and further amplified by a suspended T cell-bulk culture,in order to induce CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the specific cytotoxicity in CML by LDH assay, the phenotype identification by indirect immunofiuorescence technique and the distribution and clonal expansion of TCRVβ subfamily by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan analysis, respectively. RESULTS Oligoclonal and oligoclonal tendency T cells with higher specific cytotoxicity from cord blood and adult peripheral blood could be induced by stimulation with CML cells and K562 cells. CONCLUSIONS Specific cytotoxic T cells for an anti-CML effect could be induced by CML cells and K562 cells .The induced T cells which have the characteristic of specific cytotoxicity against CML cells may come from the clonal expansion of TCRVβ subfamily T cells.

  14. Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

    Directory of Open Access Journals (Sweden)

    Li-Li Song

    Full Text Available Despite considerable efficacy of arsenic trioxide (As2O3 in acute promyelocytic leukemia (APL treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML, are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

  15. Musashi2 modulates K562 leukemic cell proliferation and apoptosis involving the MAPK pathway

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    Zhang, Huijuan; Tan, Shi; Wang, Juan; Chen, Shana; Quan, Jing; Xian, Jingrong; Zhang, Shuai shuai; He, Jingang; Zhang, Ling, E-mail: lingzhang@cqmu.edu.cn

    2014-01-01

    The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment. - Highlights: • Knockdown of Msi2 inhibited K562 cell growth and arrested cell cycle progression. • Knockdown of Msi2 induced K562 cell apoptosis via the regulation of Bax and Bcl-2. • The MAPK pathway was involved in the process of Msi2-mediated leukemogenesis. • Our data indicate that Msi2 is a potential new target for leukemia treatment.

  16. STAT3 contributes to NK cell recognition by modulating expression of NKG2D ligands in adriamycin-resistant K562/AO2 cells.

    Science.gov (United States)

    Cai, Xiaohui; Lu, Xuzhang; Jia, Zhuxia; Zhang, Xiuwen; Han, Wenmin; Rong, Xiao; Ma, Lingdi; Zhou, Min; Chen, Baoan

    2015-11-01

    Leukemic cells can survive after chemotherapy by acquisition of multidrug resistance genes, but other phenotypes related to escape from immune recognition remain elusive. Adriamycin-resistant K562/AO2 cells are less susceptible to elimination by NK cells compared with wild type K562 cells due to lower expression of NKG2D ligands. Treatment of K562/AO2 cells with STAT3 inhibitor VII resulted in reduced expression of multidrug resistance gene P-glycoprotein, and up-regulation of NKG2D ligands on K562/AO2 cells. Meanwhile, K562/AO2 cells treated with STAT3 inhibitor proliferated less and were more susceptible to killing by NK cells than untreated K562/AO2 cells. The enhanced cytotoxicity of NK cells against K562/AO2 cells was partly blocked by treatment of NK cells with anti-NKG2D antibodies. These data suggest that STAT3 contributes to NK cell recognition by modulating NKG2D ligands in K562/AO2 cells, which may a mechanism by which cells survive and cause relapse of leukemia.

  17. The Effects of Royal Jelly on In-Vitro Cytotoxicity of K562 Cells and Peripheral Blood Mononuclear Cells

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    SE Hosseini

    2014-02-01

    Full Text Available Abstract Background & aim: Royal jelly, secreted by worker bees, has different biological activities on cells and tissues. The aim of this study was to evaluate the effects of royal jelly on peripheral blood mononuclear cells and on the tumor category of K562 cell line. Methods: In the present experimental study, three subjects were selected separately with three repetitions. K562 (104 cells and PBMC (105 cells with different concentrations of royal jelly (5, 10, 25, 50 and 100 mg/ml were cultured under standard conditions for 48 and 72 h separately. The fatality rate on PBMC cells and K562 cancer cells was evaluated by using MTT (Tetrazolium Dye-Reduction Assay. The number of viable cells in PBMC that were exposed for 48 hours with Royal Jelly was evaluated by trypan blue staining. Data were analyzed by ANOVA. Results: The royal jelly had no cytotoxicity effect on PBMC cells but at concentration of 50 and 100 mg/mL the cytotoxicity effect were observed on k562 cells whereas, at 10 and 25 mg/ml the number of PBMC viable cells increased. Conclusion: Due to the lack of lethality of royal jelly on PBMC cells and PBMC cell viability and an increase in the fatality rate of cancer cells in the future, royal jelly can be used as a potential candidate for treatment of leukemia. Keywords: Royal jelly, K562, peripheral blood mononuclear cell

  18. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    Science.gov (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  19. Isolation of flavonoids from onion skins and their effects on K562 cell viability

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    Guo-Qing Shi

    2016-04-01

    Full Text Available To investigate the anti-proliferative activity of flavonoids from onion skins, extraction by 50% ethanol (v/v, soxhlet polar fractionation, pH gradient separation, thin-layer chromatography, and recrystallization methods were used to isolate and purify flavonoids from dry onion skins. Anti-proliferative activities of some flavonoids obtained on leukemia K562 cell line were deter-mined by MTT assay. Results showed that flavonoids of onion skins were mainly in form of quercetin, kaempferol, isorhamnetin, apigenin-7-O-β-D-glucopyranoside, quercetin-3-O-β-D-glucopyranoside, kaempferol-7-O-β-D-glucopyranoside and rutin. Quercetin and kaempferol decreased K562 cell viability, and quercetin had stronger effect. However, isorhamnetin and rutin exhibited certain proliferation-promoting effects. It suggests that ortho hydroxyl groups on B ring of onion flavonoids might be the key structural elements of their cytotoxic effects on K562 cells, and hydroxyl groups in position 3 or carbonyl groups in position 4 might be one of the structural effect elements.

  20. [Inhibitory effect of all-trans retinoic acid combined with SBA-Na on K562 and Kasumi-1 cell lines in vitro].

    Science.gov (United States)

    Chang, Cheng; Guo, Bo; Zhang, Lin; Zhu, Hong-Li; Lu, Xue-Chun; Fan, Hui; Li, Su-Xia; Yang, Bo; Liu, Yang; Zhai, Bing; Yang, Yang; Ran, Hai-Hong; Lin, Jie

    2013-08-01

    This study was aimed to investigate the effect of all-trans retinoic acid (ATRA) combined with SBA-Na on the biologic activities of human leukemia K562 and Kasumi-1 cell lines and their mechanism. The ATRA solution of 10(-6) mol/L (W1), 10(-4) mol/L (W2) and the SBA-Na solution of 100 µg/ ml (Z1) and 200 µg/ml (Z2) were prepared respectively. The K562 and Kasumi-1 cells were treated with W1, W2, Z1, Z2, W1 + Z1 and W2 + Z2 respectively, at same time, the blank control was set up. The cell morphology and growth in different treated groups were observed under light microscope. The CCK-8 method was used to detect the proliferation ability of cells, the cell growth curves were drawn, the inhibitory rate of cells was calculated. The flow cytometry with PI single staining and PI/Annexin V double stainings was used to detect the change of cell cycle and apoptosis of 2 cell lines treated with different drugs. The RQ-PCR was used to detect the change of Cyclin A mRNA expression in K562 cells. The results showed both ATRA and SBA-Na displayed inhibitory effect on cell proliferation, and the combination of these two drugs had stronger effect. As compared with the control group, the cell cycle distribution were changed obviously, and the apoptosis increased more significantly in treated groups, especially in group of ATRA combined with SBA-Na. The Cyclin A mRNA expression was up-regulated in Z1 group, while Cyclin A mRNA expression was down-regulated in other groups. It is concluded that both ATRA and SBA-Na can inhibit the proliferation of K562 and Kasumi-1 cell lines and promote their apoptosis. This effect may be stronger when both drugs combined. For K562 cells, the inhibitory effect may be accomplished through down-regulation of Cyclin A mRNA.

  1. [Ginsenoside Rh₂-induced inhibition of histone deacetylase 6 promotes K562 cells autophagy and apoptosis in vivo].

    Science.gov (United States)

    Liu, Ze-Hong; Chen, Di-Long; Jiang, Rong; Chen, Yi; Xiong, Wei; Wang, Fen; Shi, Xue-Ping; Li, Hai-Xing; Li, Jing

    2016-02-01

    To study the in vivo inhibition effect of ginsenoside Rh₂ on humanleukemia cells, and explore its mechanism from autophagy and apoptosis aspects, human leukemia K562 cells allograft tumor models were applied, and after administration of ginsenosides Rh₂ by gavage, the tumor diameter, volume and inhibitory rate were measured, and the anti-tumor activity of ginsenosides Rh₂ was observed. The levels of HAT and HDAC in tumor tissues were detected by chemical colorimetry assay, and expressions of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC6 were detected by Western blotting assay. The expression levels of vital genes closely associated with autophagy and mRNA expressions of HDAC6 and Hsp90 were detected by Real time-PCR. HE staining was used to observe apoptosis, and immunohistochemistry was used to detect the protein expressions of HDAC6, Hsp90 and activated caspases 3. The results showed that ginsenoside Rh₂ could inhibit the growth of k562 cells allograft tumor, with a tumor inhibition rate up to 53.10%. Ginsenoside Rh₂ could significantly decrease HDAC activity and decrease the expressions of HDAC1, HDAC2 and HDAC6, and inhibit the expressions of HDAC6 and HSP90, increase the expressions of vital autophagy genes (beclin-1, LC3A and LC3B). Histopathological results showed that ginsenosides Rh₂ could significantly increase the tumor apoptosis. Therefore, ginsenoside Rh₂ had good anti-tumor effect in vivo, and the mechanism maybe associated with regulating autophagy and apoptosis through HDAC6 and Hsp90 pathways and inhibiting the in vivo proliferation of tumor cells. Copyright© by the Chinese Pharmaceutical Association.

  2. SIRT1 is a critical regulator of K562 cell growth, survival, and differentiation.

    Science.gov (United States)

    Duncan, Mark T; DeLuca, Teresa A; Kuo, Hsin-Yu; Yi, Minchang; Mrksich, Milan; Miller, William M

    2016-05-15

    Inhibition of histone deacetylases (HDACi) has emerged as a promising approach in the treatment of many types of cancer, including leukemias. Among the HDACs, Class III HDACs, also known as sirtuins (SIRTs), are unique in that their function is directly related to the cell's metabolic state through their dependency on the co-factor NAD(+). In this study, we examined the relation between SIRTs and the growth, survival, and differentiation of K562 erythroleukemia cells. Using a mass spectrometry approach we previously developed, we show that SIRT expression and deacetylase activity in these cells changes greatly with differentiation state (undifferentiated vs. megakaryocytic differentiation vs. erythroid differentiation). Moreover, SIRT1 is crucially involved in regulating the differentiation state. Overexpression of wildtype (but not deacetylase mutant) SIRT1 resulted in upregulation of glycophorin A, ~2-fold increase in the mRNA levels of α, γ, ε, and ζ-globins, and spontaneous hemoglobinization. Hemin-induced differentiation was also enhanced by (and depended on) higher SIRT1 levels. Since K562 cells are bipotent, we also investigated whether SIRT1 modulation affected their ability to undergo megakaryocytic (MK) differentiation. SIRT1 was required for commitment to the MK lineage and subsequent maturation, but was not directly involved in polyploidization of either K562 cells or an already-MK-committed cell line, CHRF-288-11. The observed blockage in commitment to the MK lineage was associated with a dramatic decrease in the formation of autophagic vacuoles, which was previously shown to be required for K562 cell MK commitment. Autophagy-associated conversion of the protein LC3-I to LC3-II was greatly enhanced by overexpression of wildtype SIRT1, further suggesting a functional connection between SIRT1, autophagy, and MK differentiation. Based on its clear effects on autophagy, we also examined the effect of SIRT1 modulation on stress responses. Consistent

  3. Piceatannol bolsteres fetal haemoglobin formation in K562 cells via p38 map kinase activation and ERK inactivation

    Directory of Open Access Journals (Sweden)

    AAYUSH KUKREJA

    2015-08-01

    Full Text Available Elevation of the level of fetal haemoglobin (HbF by pharmacological agents is a safe and a promising approach for treating beta thalassemia. In this study, the effect of piceatannol was studied in human erythroleukemic K562 cells for their role in gamma-globin mRNA and HbF induction. The role of p38 mitogen activated protein kinase (MAPK and extracellular regulated protein kinase (ERK signaling pathways were also examined. It was found that piceatannol significantly increased gamma-globin mRNA and HbF levels in dose and time dependent manner in K562 cells. This was determined by enzyme linked immunosorbent assay (ELISA and western blot analysis. Pretreatment with p38 MAPK inhibitor (SB203580 obstructed the stimulatory effect of piceatannol in total and HbF activation. In contrast, no change in HbF level was observed in K562 cells when treated with ERK inhibitor (PD98059. Moreover, piceatannol activated p38 MAPK and inhibited ERK signaling pathways in K562 cells as shown by western blot analysis. Besides, the inhibitor SB203580 inhibited p38 MAPK activation when cells were pre-treated with piceatannol. In summary, piceatannol was found to be a strong inducer of HbF production in K562 cells. The results mark the role of p38 MAPK and ERK signaling as molecular targets for stimulation of HbF synthesis upon treatment with piceatannol.

  4. 地西他滨对NB4及K562细胞增殖和凋亡的影响%Effects of Decitabine on Proliferation and Apoptosis of NB4 and K562 Cells

    Institute of Scientific and Technical Information of China (English)

    韩新爱; 曾慧兰; 韩艳萍; 孙尔维

    2013-01-01

    本研究旨在观察去甲基化药物地西他滨(DAC)对NB4及K562细胞增殖和凋亡的影响.用台盼蓝染色法检测DAC对NB4及K562细胞的增殖抑制作用;流式细胞术检测不同浓度DAC作用后细胞周期的变化和CD11b的表达水平;瑞氏染色法观察药物作用后细胞形态变化;DNA梯形片段电泳分析细胞凋亡.结果表明,DAC显著抑制NB4及K562细胞的增殖(P<0.05),且呈浓度和时间依赖性,DAC作用NB4及K562细胞72 h的半数抑制浓度(IC50)分别为0.113 μmol/L和0.138 μmol/L;不同浓度DAC作用72 h后,0.15 μmol/L DAC处理组两种细胞株G0/G1期细胞比例均显著增高,而S期细胞比例降低(P<0.05);两种细胞株的髓系分化抗原CD11b表达水平均升高(P<0.05);两种细胞株经不同浓度DAC处理48 h后,0.15μmoVL DAC处理组均出现典型的凋亡梯形DNA条带.结论:DAC抑制NB4及K562细胞增殖,诱导细胞分化,促进细胞凋亡;DAC对NB4细胞作用更明显.%This study was aimed to investgate the effects of decitabine (DAC) on proliferation and apoptosis of leukemia NB4 and K562 cells. The proliferation inhibition of DAC on NB4 and K562 cells was detected by Trypan blue staining. After treatment of DAC at different concentrations, the changes of cell cycle and CD11b expression was determined by flow cytometry. The cell morphological changes were observed by Wright's staining. The DNA ladder was used to detect cell apoptosis. The results indicated that DAC significantly inhibited the proliferation of NB4 and K562 cells in dose-and time-dependent manner. The median inhibitory concentration (IC50) of DAC-treated NB4 and K562 cells for 72 h was 0.113 μmol/L and 0.138 μmol/L, respectively. After treating these two cell lines with DAC at different concentration for 72 h, the cell ratio in G0/G1 phase significantly increased, while the cell ratio in S phase obviously decreased in 0. 15 μmol/L DAC group (P < 0. 05 ). The expression levels of myeloid

  5. 茅莓及其含药血清对K562白血病细胞抑制作用的实验研究

    Institute of Scientific and Technical Information of China (English)

    许晓峰; 张学进; 冯健; 姜铁军; 付天红; 高瑞兰

    2011-01-01

    目的:观察茅莓及其含药血清体内及体外对K562细胞的抑制增殖作用.方法:通过建立荷白血病瘤裸鼠模型,观察不同浓度的茅莓水煎液对荷白血病瘤裸鼠瘤体体积、重量、组织病理学改变的影响;通过半固体琼脂培养观察不同浓度的茅莓含药血清对K562细胞集落生长的抑制作用;通过MIT法观察不同浓度的含药血清对K562白血病细胞的抑制增殖作用.结果:茅莓水煎液高、中、低剂量组瘤体体积、瘤重均较模型对照组明显减少;茅莓水煎液组及阿糖胞苷(Am-c)组光镜下可见多个坏死灶,瘤细胞肿胀、破裂,细胞核溶解,消失等现象.终浓度为10%和20%的含药血清做琼脂半固体集落培养,显示随着浓度增加,K562细胞集落形成明显减少,抑制作用呈剂量依赖性,MTT法显示终浓度10%和20%含药血清能明显抑制K562细胞增殖.结论:茅莓在荷白血病瘤裸鼠体内具有显著抑制瘤体生长的作用,在一定浓度下,其含药血清对K562细胞也有明显的抑制增殖作用.%Objective: To observe the inhibition of Japanese raspberry herb and its serum containing herb on proliferation of K562 leukemia cells in vivo and vitro.Methods: Models of K562 - bearing mice were built by subcutaneous vaccination K562 leukemia cells, mice were divided into model, positive and raspberry low, middle, high groups, each group was given corresponding medicine after vaccination 5 days, observing the changes of tumor volume, weight and histopathology in each group mice after treated 14days. Observing the inhibition of different concentration serum containing raspberry on proliferation of K562 leukemia cells by MTT in vitro.Results: The volume and weight of tumor in raspberry groups were obviously reduced, there were significant difference between each treated group and model group; the histopathology results showed that necroses of tumor cells in each dose raspberry group and Ara C group were more than

  6. [Enhancement of reversing drug resistance of K562/A02 cells to adriamycin by ultrasound-induced cavitation].

    Science.gov (United States)

    Chen, Bao-An; Meng, Qing-Qi; Wu, Wei; Gao, Feng; Shao, Ze-Ye; Ding, Jia-Hua; Gao, Chong; Sun, Xin-Chen; Cheng, Hong-Yan; Sun, Yun-Yu; Wang, Jun; Cheng, Jian; Zhao, Gang; Song, Hui-Hui; Bao, Wen; Ma, Yan; Wang, Xue-Mei

    2008-12-01

    This study was aimed to investigate the effects of low frequency and power ultrasound combined with adriamycin on apoptosis of drug-resistant leukemia cell line K562/A02 in vitro, to find out the parameters of optimal exposure, and to explore the possible mechanism reversing drug-resistance of K562/A02 cells. The K562/A02 cells in logarithmic growth phase were used in experiments. The experiments were divided into 4 groups: group control, group adriamycin (A02) alone, group ultrasound (US) alone and group A02+US. The trypan blue dye exclusion test and MTT assay were used to determine the cell viability; Wright's staining was used to detect the apoptosis; the flow cytometry was used to analyze the drug concentration, and the scanning electron microscopy was used to observe the changes of cell surface. The results showed that the significant differences in cell viability, intracellular adriamycin concentration and changes of cell membrane were found between ultrasound-treated and untreated cells in the presence of various concentration of adriamycin. The exposure to ultrasound at 20 kHZ, 0.25 W/cm2 for 60 seconds could obviously decrease LC50 of adriamycin to K562/A02 cells, while the exposure to ultrasound at 20 kHZ, 0.05 W/cm2 for 60 seconds could kill K562/A02 cells at once. After being treated by low frequency ultrasound, the small holes with diameter about 1-2 microm in the cell surface appeared. The ultrasound increased the adriamycin concentration in the cells, accelerated the formation of apoptotic bodies, and promoted apoptosis of adriamycin-resistant cells. It is concluded that the ultrasound at optimal parameters enhances inhibitory effect of adriamycin on drug-resistant cell line, thereby reverses drug-resistance of drug-resistant cell line through sound-hole effect in tumor cells resulting from ultrasound induced cavitation.

  7. Quantitative Proteomic Analysis of Bromotetrandrine and Tetrandrine in K562 Cell Line Using 18O-labeling Method

    Institute of Scientific and Technical Information of China (English)

    TAN Ying; GE Zhi-qiang; LIU Chang-xiao

    2012-01-01

    Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class Ⅰ new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method.Methods To illustrate its mechanism,a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study.Compared to normal chronic leukemia cell line K562 and K562 induced by Tet,the proteomic changes of K562 induced by W198 were investigated.In order to validate the quantitation by the 18O-labeling,the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample.Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments.Comparing the 105 identified soluble proteins' expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells,16 proteins were found with significantly altered expression levels after W198 treatment.Eight proteins were up-expressed including HMGB2,peroxiredoxin-2,and eIF4A-I,etc.Eight proteins were down-expressed including TCP-1,GRP94,GST-π,and SFGHs,etc.Compared to K562 induced by Tet,eight proteins of K562 were found with significantly altered expression levels after W198 treatment.Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a,etc.Three proteins were down-expressed including phosphoglycerate kinase 1,isoform 5 of interleukin enhancer-binding factor 3,etc.Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool,but it is not suitable for validation using a large number of samples.Other more effective methods,such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples.In total,105 soluble proteins were discovered

  8. Monocytic differentiation of K562 cells induced by proanthocyanidins from grape seeds.

    Science.gov (United States)

    Wang, Min; Wang, Li; Pan, Xiao-Jing; Zhang, Hong

    2012-01-01

    Grape seeds procyanidins can inhibit the proliferation of some cancer cell lines and have strong antioxidant activity. The purpose of this study was to investigate whether grape seeds procyanidins affect the proliferation and redifferentiation in K562 cells. The sulforhodamine B colorimetric assay and trypan blue staining were used to measure cell proliferation and survival. Morphological changes, NBT reductive activity, and surface antigens were used to detect redifferentiation of K562 cells. Intracellular reactive oxygen species (iROS) were detected by a fluorescent probe. Grape seeds procyanidins inhibited cell proliferation but the treatment did not appreciably increase lethality. After treatment with grape seeds procyanidins, a typical differentiated morphology was observed. The positive rate of CD11b and CD14 cells and NBT reductive activities increased significantly. As antioxidants, grape seeds procyanidins can induce arrest in the phase G1 and decrease iROS formation. All results indicate that the antioxidant grape seeds procyanidins are likely to induce monocytic differentiation in leukemia cells, mostly through decreasing iROS formation and inducing phase G1 arrest.

  9. File list: NoD.Bld.20.AllAg.K-562 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.20.AllAg.K-562 hg19 No description Blood K-562 ERX593133,ERX378356,SRX31877...2,DRX041639,DRX041641,ERX593131,DRX041640,DRX041642,SRX318771 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.20.AllAg.K-562.bed ...

  10. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells.

    Science.gov (United States)

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N K

    2015-09-25

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

  11. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells*

    Science.gov (United States)

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N. K.

    2015-01-01

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation. PMID:26269597

  12. Involvement of CD147 on multidrug resistance through the regulation of P-glycoprotein expression in K562/ADR leukemic cell line

    Directory of Open Access Journals (Sweden)

    Aoranit Somno

    2016-01-01

    Full Text Available The relationship between P-gp and CD147 in the regulation of MDR in leukemic cells has not been reported. This study aimed to investigate the correlation between CD147 and P-gp in the regulation of drug resistance in the K562/ADR leukemic cell line. The results showed that drug-resistant K562/ADR cells expressed significantly higher P-gp and CD147 levels than drug-free K562/ADR cells. To determine the regulatory effect of CD147 on P-gp expression, anti-CD147 antibody MEM-M6/6 significantly decreased P-gp and CD147 mRNA and protein levels. This is the first report to show that CD147 mediates MDR in leukemia through the regulation of P-gp expression.

  13. 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.

  14. Modification of antioxidative and antiapoptotic genes expression in irradiated K562 cells upon fullerenol C60(OH)24 nanoparticle treatment.

    Science.gov (United States)

    Stankov, Karmen; Borisev, Ivana; Kojic, Vesna; Rutonjski, Lazar; Bogdanovic, Gordana; Djordjevic, Aleksandar

    2013-01-01

    Recent data established the prospective applications for fullerenol (C60(OH)24) nanoparticle (FNP) in many fields, such as antioxidants, neuroprotective agents, and potential anti-radiation drugs. Leukemia cell sensitization to apoptosis induced by ionizing radiation is achieved by upregulation of ROS production and/or downregulation of antioxidative enzymes. Therefore, our aim was to analyze the potential role of fullerenol nanoparticle in modulation of the leukemic cellular response to irradiation. We used the qRT-PCR to analyze the expression level of mRNA for 11 genes in irradiated and FNP pre-treated irradiated K562 cells, and compared the gene expression level with the overall cell survival. Our results of the improved cell survival in FNP-treated irradiated cells and significant overexpression of anti-apoptotic Bcl-2 and Bcl-xL and cytoprotective genes such as GSTA4, MnSOD, NOS, CAT and HO-1 genes, may indicate that FNP exerts cytoprotective function in K562 leukemic cells, rendering K562 cells more tolerant to radiotherapy.

  15. Evaluation of Morpholino Antisense Oligos’ Role on BCR-ABL Gene Silencing in the K562 Cell Line

    Directory of Open Access Journals (Sweden)

    Bahman Delalat

    2010-01-01

    Full Text Available Objective: Chronic myeloid leukemia (CML develops when a hematopoietic stem cellacquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cellsto have a greater than normal proliferation rate. Scientists attempt to improve the CMLtreatment process by silencing the BCR/ABL oncogene. In this work, we used morpholinoantisense oligos to silence the BCR/ABL oncogene.Materials and Methods: In this study, the K562 was used as a BCR/ABL fusion-genepositive cell line and the Jurkat cell line as a control. We explored the inhibiting capacityof morpholino antisense oligos in the the expression of the BCR/ABL oncogene andstudied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation ofapoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos.Results: Prolonged exposure of the K562 cell line to the morpholino antisense oligostargeted against the BCR-ABL gene showed proliferation inhibition as its main feature.After western blotting, we found that complete silencing of BCR/ABL was achieved, butflow cytometric analysis showed no broad apoptosis.Conclusion: The results indicate that the Morpholino antisense oligo is able to inhibitp210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR.

  16. Effects of highly purified anthraquinoid compounds from Aloe vera on sensitive and multidrug resistant leukemia cells.

    Science.gov (United States)

    Grimaudo, S; Tolomeo, M; Gancitano, R; Dalessandro, N; Aiello, E

    1997-01-01

    Folk medicine has attributed antitumor properties to preparations from Aloe vera. We have studied the effects of five purified compounds from the plant on human K562 leukemia and on its multidrug resistant (MDR) variant, K562/R. The glycosides aloin A and B, aloesin and aloeresin were devoid of antitumor activity up to 200 mu M concentrations. Only the aglycone aloe emodin produced reproducible antitumor effects, which, interestingly, were more pronounced in the MDR, P-glycoprotein overexpressing, cell line. Its IC50 was in fact 29 mu M in K562 and 10.5 mu M in K562/R. Aloe emodine caused mainly cytostasis and accumulation of the cells in the S and G(2)-M phases of the cell cycle during the first 48 h of treatment. Thereafter, massive cell death ensued. Research on the antitumor activity of compounds extracted from Aloe vera probably deserves continuation.

  17. Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Xing-Xiang Peng; Amit K. Tiwari; Hsiang-Chun Wu; Zhe-Sheng Chen

    2012-01-01

    Imatinib,a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI),has revolutionized the treatment of chronic myelogenous leukemia (CML).However,development of multidrug resistance(MDR) limits the use of imatinib.In the present study,we aimed to investigate the mechanisms of cellular resistance to imatinib in CML.Therefore,we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process.While characterizing the phenotype of these cells,we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells.In addition,these cells were cross-resistant to second- and third-generation BCR-ABL TKIs.Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells.In addition,accumulation of [14C]6-mercaptopurine (6-MP) was decreased,whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells.These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells.

  18. Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

    2016-09-02

    Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

  19. Human Wharton's jelly mesenchymal stem cell secretome display antiproliferative effect on leukemia cell line and produce additive cytotoxic effect in combination with doxorubicin.

    Science.gov (United States)

    Hendijani, Fatemeh; Javanmard, Shaghayegh Haghjooy; Sadeghi-aliabadi, Hojjat

    2015-06-01

    Mesenchymal stem cell (MSC) therapy moves toward clinic progressively. Recent evidences establish anticancer effect of mesenchymal stem cells. However multiple factors including type of cancer, MSC source, study design, and animal model play role in final outcome. Wharton's jelly - a newly approved source of MSCs - possesses superiorities to bone marrow as the conventional source; therefore investigation of its medical effects can produce beneficial results. In this survey we examined cytotoxic and proapoptotic effect of human Wharton's jelly MSC secretome on K562 human leukemia cells. MSCs were isolated from human Wharton's jelly of umbilical cord by explant culture method, then characterized according to ISCT criteria (morphology and plastic adherence, surface antigenicity and differentiation potential). MSC secretome was collected and its cytotoxic and proapoptotic effects on K562 cells in combination with doxorubicin were evaluated using BrdU cell proliferation assay and Annexin V-PI staining. Our results showed antiproliferative effect of mesenchymal stem cell secretome on K562 cancer cells, the effect was also added to cytotoxic effect of doxorubicin without induction of drug resistance. Human Wharton's jelly derived mesenchymal stem cells exerted cytotoxic effect on leukemia cells. Addition of that effect to anticancer effect of chemotherapeutic agents can leads to cytotoxic drug dose reduction and diminished side effects. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Effects of ginsenoside Rg3 on proliferation and induced differentiation of erythroleukemia cell line K562 in vitro%人参皂甙Rg3在体外对白血病K562细胞增殖和诱导分化的影响

    Institute of Scientific and Technical Information of China (English)

    肖凤; 刘彬; 张建平; 王伴青; 方木水; 胡玮; 孙续禄; 朱清仙

    2012-01-01

    背景:文献报道人参皂甙Rg3具有抗肿瘤作用,但对白血病作用研究很少.目的:观察人参皂甙Rg3对白血病K562细胞的作用,并探讨其相关机制.方法:以白血病K562细胞为靶细胞,实验分为对照组和人参皂甙Rg3组,人参皂甙Rg3组分别添加10,20,40,80,100 mg/L Rg3.结果与结论:MTT检测结果显示,不同浓度人参皂甙Rg3组K562细胞生长抑制率均显著高于对照组(P < 0.05~0.01).NBT结果显示,人参皂甙Rg3组培养1,2,3 d K562细胞还原率均高于对照组(P < 0.01);人参皂甙Rg3组部分K562细胞体积变小,核仁消失,同时阳性细胞增多,细胞向成熟分化;流式细胞仪检测显示人参皂甙Rg3组培养2 d细胞周期G2期增高了3.84倍.提示人参皂甙Rg3在体外可抑制K562细胞增殖活性,其机制可能与人参皂甙Rg3将K562细胞周期阻滞在G2期,使细胞不能进行正常的有丝分裂,从而抑制细胞增殖有关.%BACKGROUND: Evidence exists that ginsenoside Rg3 can inhibit the growth of carcinoma cells. However, there are fewer studies describing ginsenoside Rg3 effects on leukemia.OBJECTIVE: To investigate the effects of ginsenoside Rg3 on proliferation of erythroleukemia cell line K562 and the underlying mechanism.METHODS: Erythroleukemia cell line K562 was used as target cell. The cells were divided into a control group and an Rg3 group. 10, 20, 40, 80, 100 mg/L Rg3 was added in the Rg3 group.RESULTS AND CONCLUSION: The MTT colorimetric assay indicated that K562 growth inhibition rate was significantly higher in the Rg3 groups than in the control group (P < 0.05-0.01). The nitroblue tetrazolium assay showed that after culture for 1,2, 3 days, the reducing power of K562 cells in the Rg3 group was significantly higher than in the control group (P < 0.01). In the Rg3 group, some K563 cell somas became small, nucleoli disappeared, nitroblue tetrazolium positive cells increased, and cells developed toward maturation. Flow cytometry detection showed

  1. Radioimmunoassay of haemoglobin F in K 562 cells following induction with renin substrate and erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Rosenloef, K.; Fyhrquist, F.; Hortling, L.; Groenhagen-Riska, C. (Helsinki Univ. (Finland). Minerva Inst. for Medical Research; Helsinki Univ. (Finland). 4th Dept. of Medicine)

    1985-06-01

    To test the hypothesis of renin substrate (RS: angiotensinogen) being a precursor of erythropoietin (EP), the capacity of RS and EP to induce Hb synthesis was compared in cultured human erythroid leukaemia cells of the K 562 line after prestimulation with haemin. For this purpose a radioimmunoassay for haemoglobin F (HbF) was developed. This assay was shown to be specific for HbF, reproducible, and sensitive for 0.1 ng of HbF. The cells were induced by RS and EP to increased HbF production. Cells stimulated with RS or EP showed increased benzidine staining. These data support the hypothesis that renin substrate is a likely precursor of erythropoietin.

  2. 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.

  3. ZM-66, a New Podophyllotoxin Derivative Inhibits Proliferation and Induces Apoptosis in K562/ADM Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Li; Hong-jie Li; Jian-sheng zhi; Hong Chen; Wen-li Xie

    2014-01-01

    Objective To investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM. Methods The K562/ADM cells were treated with varying concentrations (0, 1, 2, 4×10-3 mmol/L) of ZM-66 or etoposide for 24 hours. The proliferation was detected by Sulforhodamine B Sodium Salt (SRB) assay and apoptosis was detected by flow cytometry analysis and fluorescent staining. In addition, the expression levels of p53 and bax genes in K562/ADM cells were detected by RT-PCR analysis. The level of P-glycoprotein (P-gp), P53 and Bax protein in K562/ADM cells were detected by Western blot assay. Results SRB assay demonstrated that etoposide had little inhibitory effect on K562/ADM cells, whereas ZM-66 (1, 2, 4×10-3 mmol/L) had significantly inhibitory effect on K562/ADM cells (all P Conclusion ZM-66 is able to induce cell death by apoptosis in vitro, as a result of the reverse of the apoptosis resistance in drug-resistant K562/ADM cells by modulating expression of key factors associated with apoptosis induction.

  4. 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.

  5. 逆转录病毒介导的HO-1基因在尼洛替尼诱导K562/A02耐药细胞凋亡中的作用%The effect of retrovirus-mediated HO-1 gent on chronic myeloid leukemia resistance cell KS62/A02 apoptosis induced lay nilotinib

    Institute of Scientific and Technical Information of China (English)

    陈埕; 王季石; 秦东; 杨远; 于艳艳; 方琴

    2012-01-01

    目的 研究逆转录病毒介导的血红素加氧酶-1(HO-1)基因在尼洛替尼(Nilotinib,AMN107)诱导慢性髓性白血病(CML)耐药细胞凋亡中的作用.方法 制备高病毒滴度的逆转录病毒载体pQCXIP-EGFP-HO-1,建立稳定转染HO-1基因的K562/A02细胞.采用RT-PCR鉴定K562/A02细胞中HO-1基因的表达.RT-PCR和Western blot法检测AMNl07作用24 h后K562/A02细胞中HO-1 mRNA和蛋白的表达,采用MTT法观察细胞增殖变化,通过实时荧光定量PCR(RQ-PCR)法检测bcr-abl融合基凶的表达.通过流式细胞术检测细胞凋亡和细胞周期分布.结果 成功构建重组逆转录病毒载体,并建立稳定转染HO-1基因的K562/A02细胞系;证实转基冈组细胞HO-1 mRNA显著表达.AMN107作用3组细胞后,转基因组细胞HO-1 mRNA和蛋白的表达明显高于转空载体组和未转染组,差异有统计学意义(P<0.05);MTT法检测显示AMN107处理后细胞增殖受抑.而转基因组细胞存活率明显高于转空载体组和未转染组,差异有统计学意义(P<0.05);RQ-PCR法检测结果显示,10 ILLmol/L AMNl07抑制bcr-abl基因的表达,但转基因组的bcr-abl基因表达水平(Ct值18.15±0.18)高于转空载体组(20.32±0.20)和未转染组(20.51±0.21),差异有统计学意义(P<0.05);流式细胞术检测结果显示10 μmol/L AMNl07作用24 h后,转基因组、转空载体组、未转染组细胞凋亡率分别为(17.26±0.23)%、(39.47±0.17)%、(41.84±0.09)%.未加药转基因组、未加药未转染组细胞凋亡率分别为(3.74±0.03)%、(5.91±0.08)%;细胞周期分析结果显示经AMNl07处理后,Go/G1期和S期细胞明显减少,细胞阻滞在G2/M期,而转基因细胞组细胞周期改变不如转空载体组、未转染组明显.结论 AMN107可抑制CML耐药细胞增殖且诱导细胞凋亡,HO-1基因在CML耐药细胞凋亡过程中起保护作用,与促进CML耐药细胞的生长有关.

  6. Antileukemic Effect of Tualang Honey on Acute and Chronic Leukemia Cell Lines

    Science.gov (United States)

    Nik Man, Nik Muhd Khuzaimi; Hassan, Rosline; Ang, Cheng Yong; Abdullah, Abu Dzarr; Mohd Radzi, Muhammad Amiro Rasheeq; Sulaiman, Siti Amrah

    2015-01-01

    Complementary medicine using natural product as antitumor is on the rise. Much research has been performed on Tualang Honey and it was shown to have therapeutic potential in wound healing, and antimicrobial activity and be antiproliferative against several cancer models such as human osteosarcoma (HOS), human breast (MCF-7 and MDA-MB-231), and cervical (HeLa) cancer cell lines. To date, there was limited study on antileukemic properties of Tualang (Koompassia excelsa) Honey. The aim of this study was to evaluate the antileukemic effect of Tualang Honey on acute and chronic leukemia cell lines. Leukemia cell lines (K562 and MV4-11) and human mononuclear cell isolated from peripheral blood were grown in RPM1 1640 culture medium. The cells were incubated with increasing concentrations of Tualang Honey. After incubation, the evaluation of viability and apoptosis was performed. The morphological changes of leukemia cells were the presence of cytoplasmic blebs followed by apoptotic bodies and round shape of cells. IC50 against K562 and MV4-11 was determined. Tualang Honey gave 53.9% and 50.6% apoptosis activity on K562 and MV4-11, respectively, while on human mononuclear cell it was 37.4%. Tualang Honey has the apoptosis-inducing ability for acute and chronic myeloid leukemia (K562 and MV4-11) cell lines. PMID:26613081

  7. Antileukemic Effect of Tualang Honey on Acute and Chronic Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Nik Muhd Khuzaimi Nik Man

    2015-01-01

    Full Text Available Complementary medicine using natural product as antitumor is on the rise. Much research has been performed on Tualang Honey and it was shown to have therapeutic potential in wound healing, and antimicrobial activity and be antiproliferative against several cancer models such as human osteosarcoma (HOS, human breast (MCF-7 and MDA-MB-231, and cervical (HeLa cancer cell lines. To date, there was limited study on antileukemic properties of Tualang (Koompassia excelsa Honey. The aim of this study was to evaluate the antileukemic effect of Tualang Honey on acute and chronic leukemia cell lines. Leukemia cell lines (K562 and MV4-11 and human mononuclear cell isolated from peripheral blood were grown in RPM1 1640 culture medium. The cells were incubated with increasing concentrations of Tualang Honey. After incubation, the evaluation of viability and apoptosis was performed. The morphological changes of leukemia cells were the presence of cytoplasmic blebs followed by apoptotic bodies and round shape of cells. IC50 against K562 and MV4-11 was determined. Tualang Honey gave 53.9% and 50.6% apoptosis activity on K562 and MV4-11, respectively, while on human mononuclear cell it was 37.4%. Tualang Honey has the apoptosis-inducing ability for acute and chronic myeloid leukemia (K562 and MV4-11 cell lines.

  8. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  9. Effects of arsenic trioxide on the methylation of TMS1 gene in K562 cells

    Institute of Scientific and Technical Information of China (English)

    李洪丽

    2014-01-01

    Objective To detect the methylation status of TMS1gene and its demethylation by arsenic trioxide(As2O2)in K562 cells.Methods K562 cells were treated with different concentrations of As2O2for 48 hours.Methylationspecific PCR(MSP)was used to determine the methylation status of TMS1.RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein.

  10. Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

    Institute of Scientific and Technical Information of China (English)

    伍晓菲; 陈智超; 刘仲萍; 周浩; 游泳; 黎纬明; 邹萍

    2004-01-01

    To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry.Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2 O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.

  11. The anti-cancer effect of Propranolol in K562 cell line: an in vitro study

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    S Bastani

    2016-03-01

    Full Text Available Introduction: Β-AR receptors are one of the proteins involved in cancer and stress. The therapeutic activity of β-blockers such as propranolol is attributed to the blockade of β1-adrenergic receptors (ARs. In this study, the effect of propranolol on the viability of K562 cell line was examined. Material and methods: In order to assessment of anti-tumoral effects of propranolol, different concentrations of propranolol were prepared. K562 cells were treated with different concentrations of propranolol, then the percentage of inhibitory effect of propranolol on K562 cell viability at different times (24, 48 and 72 hours was estimated by MTT assay. Gel electrophoresis of DNA and DAPI staining were used for apoptosis investigation. Statistical comparisons were performed using two-sample t-test, Nominal significance level of each univariate test was 0.05. Results: Propranolol decreased viability of K562 cell line. The inhibitory effect of propranolol is time- and concentration-dependent, thus in higher concentrations and 72 hours after treatment, the maximum inhibitory effect was observed. (P<0.05. As the results showed, Propranolol induces apoptosis in K562 cell line. Conclusions: With respect to the inhibitory effect of propranolol on cell viability and its apoptotic effect on K562 cell line, this drug may be used for cancer therapy.

  12. 半夏醇提取液逆转多药耐药细胞系K562/A02的耐药性%Reversal of Multirug Resistance in K562/A02 Cell by Alcoholic Extract of Pinellia ternate

    Institute of Scientific and Technical Information of China (English)

    彭向前; 冯玮; 张文会

    2012-01-01

    Objective; To investigate the effect and mechanism of alcoholic extract of Pinellia ternate at non-cytotoxic concentration (inhibit rate≤ 5% ) reversing the multidrug resistant (MDR) leukemia cell lines K562/A02. Method; Cells were seeded into 72-well plates at 2 x 108/L with varying concentrations (2.5, 5, 10, 20, 40 μg o L-1 ) of P. ternate for 72 h, MTT colorimetry was used to determine the cytotoxic effect of alcoholic extract of P. ternate and the sensibility to adriamycin of K562/A02 cell line. Flow cytometry was used to determine the expressive and functional changes of glycoprotein-170 (pl70) on the cell membrane dealed with alcoholic extract of P. ternate at non-cytotoxic concentration. And daunorubicin concentration in the cells was detected by flow cytometry. The statistical treatment with SPSS 16. 0 was applied to deal with all the experimental data. Result: Alcoholic extract of P. ternate had definite cytotoxic effect on K562/A02, the non-cytotoxic dose of oxymatrine was 6. 5 μg ·L-1 . Non-cytotoxic dose of alcoholic extract of P. ternate can significantly decrease theq medium inhibition concentration ( IC50 ) value of K562/ADM cell line to adriamycin ( ADM ) . The IC50 was obviously reduced from (30. 9 ±0. 11) μg·L-1 to (10.1 ±0.21) μg·L-1 , alcoholic extract of P. ternate at non-cytotoxic concentration could partly reverse MDR of K562/A02 (3.06times) , and could descend the expression of pl70 from 91.21% to 45.12% (P < 0. 01 ) . The exosmosis test of daunorubicin showed that intracellular concentration of chemotherapeutics concentration increased evidently. Conclusion; Alcoholic extract of P. ternate is able to partly reverse MDR of K562/A02 cell line could descend the expression of P1 70, and can restrain the function of pumping chemotherapeutics out of the cell and enhance the efficient intracellular concentration of chemotherapeutics which can kill leukemia cells of multidrug resistance, and partly reversed MDR of K562/A02.%目的:观

  13. Amsacrine suppresses matrix metalloproteinase-2 (MMP-2)/MMP-9 expression in human leukemia cells.

    Science.gov (United States)

    Liu, Wen-Hsin; Chen, Ying-Jung; Chien, Jen-Hung; Chang, Long-Sen

    2014-05-01

    This study explores the suppression mechanism of amsacrine (4-(9-Acridinylamino)-N-(methanesulfonyl)-m-anisidine hydrochloride) on matrix metalloproteinase-2 (MMP-2) and MMP-9 expression in human leukemia cells. Amsacrine attenuated cell invasion with decreased MMP-2/MMP-9 protein expression and mRNA levels in U937, Jurkat, HL-60, K562, KU812, and MEG-01 cells. Moreover, amsacrine reduced both MMP-2/MMP-9 promoter luciferase activity and MMP-2/MMP-9 mRNA stability in leukemia cells. Studies on amsacrine-treated U937 cells revealed that amsacrine-elicited ROS generation induced JNK and p38 MAPK activation but reduced the phospho-ERK level. Amsacrine-induced ERK inactivation and p38 MAPK/JNK activation were demonstrated to suppress MMP-2/MMP-9 promoter luciferase activity and promote MMP-2/MMP-9 mRNA decay, respectively. p38 MAPK/JNK activation led to up-regulation of protein phosphatase 2A catalytic subunit α (PP2Acα) in amsacrine-treated U937 cells. Okadaic acid (PP2A inhibitor) treatment increased MMP-2/MMP-9 mRNA stability in amsacrine-treated cells, whereas PP2Acα over-expression increased MMP-2/MMP-9 mRNA decay. Amsacrine-induced MMP-2/MMP-9 down-regulation was also related to PP2Acα up-regulation on Jurkat, HL-60, K562, KU812, and MEG-01 cells. Collectively, our data indicate that amsacrine induces MMP-2/MMP-9 down-regulation via simultaneous suppression of genetic transcription and mRNA stability in human leukemia cells.

  14. CRISPR/Cas9-Directed Reassignment of the GATA1 Initiation Codon in K562 Cells to Recapitulate AML in Down Syndrome

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    Kevin M. Bloh

    2017-06-01

    Full Text Available Using a CRISPR/Cas9 system, we have reengineered a translational start site in the GATA1 gene in K562 cells. This mutation accounts largely for the onset of myeloid leukemia in Down syndrome (ML-DS. For this reengineering, we utilized CRISPR/Cas9 to generate mammalian cell lines that express truncated versions of the Gata1s protein similar to that seen in ML-DS, as determined by analyzing specific genetic alterations resulting from CRISPR/Cas9 cleavage. During this work, 73 cell lines were clonally expanded, with allelic variance analyzed. Using Tracking of Indels by DEcomposition (TIDE and Sanger sequencing, we defined the DNA sequence and variations within each allele. We found significant heterogeneity between alleles in the same clonally expanded cell, as well as among alleles from other clonal expansions. Our data demonstrate and highlight the importance of the randomness of resection promoted by non-homologous end joining after CRISPR/Cas9 cleavage in cells undergoing genetic reengineering. Such heterogeneity must be fully characterized to predict altered functionality inside target tissues and to accurately interpret the associated phenotype. Our data suggest that in cases where the objective is to rearrange specific nucleotides to redirect gene expression in human cells, it is imperative to analyze genetic composition at the individual allelic level.

  15. Apoptosis Induction in Human Leukemia Cell Lines by Gold Nanoparticles Synthesized Using the Green Biosynthetic Approach

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    Farideh Namvar

    2015-01-01

    Full Text Available Gold nanoparticles were grown on Sargassum muticum water extract (S-GNPs using the green biosynthetic approach. The nanoparticles were characterized using UV-visible spectroscopy, zeta potential, and transmission electron microscopy (TEM. The resulting S-GNPs were spherical and crystalline with a size of <10 nm. The in vitro anticancer activity was demonstrated in human leukemia cell lines. The cancer cells were treated with different concentrations of S-GNPs, and calorimetric (MTT assay used for the cytotoxicity test, which resulted in an IC50 value of 4.22 ± 1.12, 5.71 ± 1.4, 6.55 ± 0.9, and 7.29 ± 1.7 μg/mL for each of the K562, HL-60, Jurkat, and CEM-ss cells, respectively. Thus, the K562 was selected for the next experiments. Furthermore, apoptosis induction was confirmed by Hoechst 33342, annexin V staining, and caspase-3/-9 activity tests. The cell cycle analysis exhibited a significant increase in the accumulation of S-GNPs treated cells at the sub-G1 phase, demonstrating the induction of apoptosis by S-GNPs. The nature of the inhibition of cancer cell growth by S-GNPs could open the way for further research in the design of green synthesis therapeutic agents, particularly in nanomedicine, for the treatment of cancer.

  16. SIRT1 enhances drug resistance of CML K562 cells%SIRT1增强人慢性粒系白血病K562细胞耐药性

    Institute of Scientific and Technical Information of China (English)

    毛蓓蓓; 宋崴; 吕湘; 陈厚早; 刘德培

    2011-01-01

    Objective To explore the function of SIRT1 in drug resistance of chronic myelogenous leukemia (CML) K562 cells. Methods K562 cells stably expressing dominate negative SIRT1-H363Y or SIRT1 shRNA were treated with H2O2 and etoposide. Cleaved caspase3, BAX and -γ-H2A. X signals were detected by Western blot; MCF-7 and 293A cells overexpressing wild type SIRT1 were treated with H2O2 and etoposide. Γ-H2A. X was detected by Western blot. Results SIRT1-H363Y and SIRT1 shRNA expression in K562 cells could enhance the cleavage of caspase3 and the expression of BAX, but inhibit the formation ofy-H2A. X induced by H2O2(control vs SIRT1-H363 Y: 0. 54 ± 0. 03 vs 0. 23 ± 0. 01) ( P < 0. 01) and etoposide (control vs SIRT1-H363 Y: 1. 36 ± 0. 20 vs0.68±0. 14) (P<0. 05); (control vs S1RT1 RNAi: 0. 68 ±0. 06 vs 0.44 ±0. 04) (P <0. 01). On the contrary, overexpression of wild type SIRT1 in MCF-7 and 293 A cells promotes the formation of 7-H2A. X (MCF-7: 0.26±0.04 vs0.46±0.02)(P<0.01);(293A: 0 vs 0. 42 ±0. 09) (P <0. 05). Conclusions SIRT1 may enhance the drug resistance and signal for DNA repair in K562 cells.%目的 研究Ⅲ类去乙酰化酶SIRT1对人慢性粒系白血病K562细胞耐药性的影响及其机制.方法 分别将酶活性缺失突变型SIRT1-H363Y和SIRT1 shRNA表达质粒转染K562细胞,G418筛选稳定克隆后用H2O2和依托泊甙(etoposide)处理细胞,Western blot检测caspase3剪切,BAX表达及DNA损伤标志物H2A.X磷酸化;在MCF-7和293A中过表达野生型SIRT1,检测H2O2和etoposide处理后的H2A.X磷酸化.结果 K562细胞中SIRT1-H363Y表达和SIRT1干扰均能促进H2O2和etoposide诱导的caspase3剪切及BAX表达,并显著抑制H2O2诱导的H2A.X磷酸化(对照vs SIRT1-H363Y:0.54 +0.03vs0.23±0.01)(P<0.01)和etoposide(对照vs SIRT1 H363Y:1.36±0.20vs0.68±0.14)(P <0.05);(对照vs SIRT1 RNAi:0.68±0.06vs0.44±0.04)(P<0.01);在MCF-7和293A细胞中,野生型SIRT1过表达能明显增加etoposide诱导的H2A

  17. Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells In Vitro and In Vivo.

    Science.gov (United States)

    Atashrazm, Farzaneh; Lowenthal, Ray M; Woods, Gregory M; Holloway, Adele F; Karpiniec, Samuel S; Dickinson, Joanne L

    2016-03-01

    Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti-tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti-tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60, and K562 were treated with fucoidan and cell cycle, cell proliferation, and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase-dependent as it was significantly attenuated by pre-treatment with a pan-caspase inhibitor. P21/WAF1/CIP1 was significantly up-regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down-regulated the activation of AKT through hypo-phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia.

  18. ESTABLISHMENT OF K562/ADM/VER CELL SUBLINE RESISTANT TO VERAPAMIL AND ITS RESISTANT MECHANISM

    Institute of Scientific and Technical Information of China (English)

    谢佐福; 周冬梅; 林贤东; 林声; 吴允昆

    2001-01-01

    Objective: To understand whether verapamil (VER) resistance development in the multidrug-resistant cell line and its mechanism. Methods: K562/ADM/VER cell subline resistant to verapamil was established through a gradual increase of VER concentration in the media. MTT method was used to assay resistance to VER, cross resistance to dipyriamole (DPM), cyclosporin A (CsA) in the cells, and HPLC and spectrofluorometer to detect intracellular accumulation of VER or ADM respectively, as well as S-P immunocytochemical technique for detection of genes expression. Results: It were observed that 7.9-fold increase in VER resistance, significantly reduced intracellular accumulation of VER or ADM and also develop across resistance to DPM and CsA in K562/ADM/VER cells, compared with its parent cell, K562/ADM. High-level of p-glycoprotein(pgp), middle-level of p53, p16, was present in two cell lines without expression of GSTPI, C-myc, C-myc, C-fos and C-erbB-2. Bc1-2 protein expression was found only in K562/ADM cells. Conclusion: K562/ADM cells were capable of being induced to develop resistance to VER.

  19. Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

    Science.gov (United States)

    Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

    2015-01-01

    Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

  20. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2012-11-15

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  1. Construction and characterization of hGM-CSF-expressing K-562 cell line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3.1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3.1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell li...

  2. Gene expression profiles of human promyelocytic leukemia cell lines exposed to volatile organic compounds.

    Science.gov (United States)

    Sarma, Sailendra Nath; Kim, Youn-Jung; Ryu, Jae-Chun

    2010-05-27

    Benzene, toluene, o-xylene, ethylbenzene, trichloroethylene and dichloromethane are the most widely used volatile organic compounds (VOCs), and their toxic mechanisms are still undefined. This study analyzed the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells exposed to VOCs using a 35-K whole human genome oligonucleotide microarray to ascertain potential biomarkers. Genes with a significantly increased expression levels (over 1.5-fold and p-values p53 signaling pathway, apoptosis, and natural killer cell-mediated cytotoxicity pathway. Functionally important immune response- and apoptosis-related genes were further validated by real-time RT-PCR. The results showed that IFIT1, IFIT2, IFIT3, USP18, INFGR2, PMAIP1, GADD45A, NFKBIA, TNFAIP3, and BIRC3 genes altered their expression profiles in a dose-dependent manner. Similar expressions profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, both gene expression profiles and gene ontology analysis have elucidated potential gene-based biomarkers and provided insights into the mechanism underlying the response of human leukemia cell lines to VOC exposure.

  3. K562 cells display different vulnerability to H₂O₂ induced oxidative stress in differing cell cycle phases.

    Science.gov (United States)

    Akcakaya, Handan; Dal, Fulya; Tok, Sabiha; Cinar, Suzan-Adin; Nurten, Rustem

    2015-02-01

    Oxidative stress can be defined as the increase of oxidizing agents like reactive oxygen and nitrogen species, or the imbalance between the antioxidative defense mechanism and oxidants. Cell cycle checkpoint response can be defined as the arrest of the cell cycle functioning after damaging chemical exposure. This temporary arrest may be a period of time given to the cells to repair the DNA damage before entering the cycle again and completing mitosis. In order to determine the effects of oxidative stress on several cell cycle phases, human erytroleukemia cell line (K562) was synchronized with mimosine and genistein, and cell cycle analysis carried out. Synchronized cells were exposed to oxidative stress with hydrogen peroxide (H2O2) at several concentrations and different times. Changes on mitochondria membrane potential (ΔΨm) of K562 cells were analyzed in G1, S, and G2 /M using Rhodamine 123 (Rho 123). To determine apoptosis and necrosis, stressed cells were stained with Annexin V (AnnV) and propidium iodide (PI) for flow cytometry. Changes were observed in the ΔΨm of synchronized and asynchronized cells that were exposed to oxidative stress. Synchronized cells in S phase proved resistant to the effects of oxidative stress and synchronized cells at G2 /M phase were sensitive to the effects of H2O2 -induced oxidative stress at 500 μM and above.

  4. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Mizutani, Naoki [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); College of Life and Health Sciences, Chubu University, Kasugai (Japan); Omori, Yukari [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Suzuki, Motoshi [Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kyogashima, Mamoru [Department of Microbiology and Molecular Biology, Nihon Pharmaceutical University, Saitama (Japan); Nakamura, Mitsuhiro [Department of Drug Information, Gifu Pharmaceutical University, Gifu (Japan); Tamiya-Koizumi, Keiko [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Nozawa, Yoshinori [Tokai Gakuin University, Kakamigahara (Japan); Murate, Takashi, E-mail: murate@isc.chubu.ac.jp [College of Life and Health Sciences, Chubu University, Kasugai (Japan)

    2016-02-19

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.

  5. Electrochemical K-562 cells sensor based on origami paper device for point-of-care testing.

    Science.gov (United States)

    Ge, Shenguang; Zhang, Lina; Zhang, Yan; Liu, Haiyun; Huang, Jiadong; Yan, Mei; Yu, Jinghua

    2015-12-01

    A low-cost, simple, portable and sensitive paper-based electrochemical sensor was established for the detection of K-562 cell in point-of-care testing. The hybrid material of 3D Au nanoparticles/graphene (3D Au NPs/GN) with high specific surface area and ionic liquid (IL) with widened electrochemical windows improved the good biocompatibility and high conductivity was modified on paper working electrode (PWE) by the classic assembly method and then employed as the sensing surface. IL could not only enhance the electron transfer ability but also provide sensing recognition interface for the conjugation of Con A with cells, with the cell capture efficiency and the sensitivity of biosensor strengthened simultaneously. Concanavalin A (Con A) immobilization matrix was used to capture cells. As proof-of-concept, the paper-based electrochemical sensor for the detection of K-562 cells was developed. With such sandwich-type assay format, K-562 cells as model cells were captured on the surface of Con A/IL/3D AuNPs@GN/PWE. Con A-labeled dendritic PdAg NPs were captured on the surface of K-562 cells. Such dendritic PdAg NPs worked as catalysts promoting the oxidation of thionine (TH) by H2O2 which was released from K-562 cells via the stimulation of phorbol 12-myristate-13-acetate (PMA). Therefore, the current signal response was dependent on the amount of PdAg NPs and the concentration of H2O2, the latter of which corresponded with the releasing amount from cells. So, the detection method of K-562 cell was also developed. Under optimized experimental conditions, 1.5×10(-14) mol of H2O2 releasing from each cell was calculated. The linear range and the detection limit for K-562 cells were determined to be 1.0×10(3)-5.0×10(6) cells/mL and 200 cells/mL, respectively. Such as-prepared sensor showed excellent analytical performance with good fabrication reproducibility, acceptable precision and satisfied accuracy, providing a novel protocol in point-of-care testing of cells.

  6. H-ferritin-regulated microRNAs modulate gene expression in K562 cells.

    Directory of Open Access Journals (Sweden)

    Flavia Biamonte

    Full Text Available In a previous study, we showed that the silencing of the heavy subunit (FHC offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC comparing it with K562 transduced with scrambled RNA (K562shRNA. Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, "Cell Death and Survival, Hematological System Development and Function, Hematopoiesis", is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.

  7. Dopamine inhibits proliferation, induces differentiation and apoptosis of K562 leukaemia cells

    Institute of Scientific and Technical Information of China (English)

    HE Qun; YUAN Lin-bo

    2007-01-01

    Background Dopamine exerts its effects mainly in nervous system through D1, D2 or D3 receptors. There are few reports dealing with the effects of dopamine on leukaemia cells. However, some dopamine agonists or antagonists do show biological effects on some types of leukaemia cells. Here, we report the effects of dopamine on the proliferation,differentiation and apoptosis of K562 leukaemia cells.Methods Proliferation was determined by MTT assay and cell counting both in liquid and semisolid cultures.Differentiation was verified by morphology, benzidine staining and flow cytometry. Apoptosis was checked by Hoechst 33258 staining and flow cytometry. The two groups were untreated group and treated group (dopamine 10-9 mol/L-10-4mol/L).Results In liquid culture, MTT assay and colony assay, dopamine inhibited proliferation of K562 cells. Inhibition rate was 29.28% at 10-6 mol/L and 36.10% at 10-5 mol/L after culture for 5 days in MTT assay. In benzidine staining and CD71 expression, dopamine induced K562 cells toward erythroid differentiation by increased 155% at 10-6 mol/L and by 171% at 10-5 mol/L after culture for 5 days in benzidine staining. In Hoechst 33258 staining and flow cytometry,dopamine induced K562 cells toward apoptosis. The sub G1 peak stained by PI was 14.23% at 10-4 mol/L dopamine after culture for 3 days compared with the control (0.81%) in flow cytometry.Conclusion Dopamine inhibites proliferation and induces both differentiation and apoptosis of K562 leukaemia cells.

  8. H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

    Science.gov (United States)

    Biamonte, Flavia; Zolea, Fabiana; Bisognin, Andrea; Di Sanzo, Maddalena; Saccoman, Claudia; Scumaci, Domenica; Aversa, Ilenia; Panebianco, Mariafranca; Faniello, Maria Concetta; Bortoluzzi, Stefania; Cuda, Giovanni; Costanzo, Francesco

    2015-01-01

    In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs. PMID:25815883

  9. Induction of cellular senescence by doxorubicin is associated with upregulated miR-375 and induction of autophagy in K562 cells.

    Directory of Open Access Journals (Sweden)

    Ming-Yu Yang

    Full Text Available BACKGROUND: Cellular senescence is a specialized form of growth arrest that is generally irreversible. Upregulated p16, p53, and p21 expression and silencing of E2F target genes have been characterized to promote the establishment of senescence. It can be further aided by the transcriptional repression of proliferation-associated genes by the action of HP1γ, HMGA, and DNMT proteins to produce a repressive chromatin environment. Therefore, senescence has been suggested to functions as a natural brake for tumor development and plays a critical role in tumor suppression and aging. METHODOLOGY/PRINCIPAL FINDINGS: An in vitro senescence model has been established by using K562 cells treated with 50 nM doxorubicin (DOX. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells ought to be independent of p53 and p16-pRb pathways. Indeed, no change in the expression of the typical senescence-associated premalignant cell markers in the DOX-induced senescent K562 cells was found. MicroRNA profiling revealed upregulated miR-375 in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor was able to reverse the proliferation ability suppressed by DOX (p<0.05 and overexpression of miR-375 suppressed the normal proliferation of K562 cells. Upregulated miR-375 expression was associated with downregulated expression of 14-3-3zeta and SP1 genes. Autophagy was also investigated since DOX treatment was able to induce cells entering senescence and eventually lead to cell death. Among the 24 human autophagy-related genes examined, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. CONCLUSIONS/SIGNIFICANCE: This study has demonstrated that in the absence of p53 and p16, the induction of senescence by DOX was associated with upregulation of miR-375 and autophagy initiation. The anti-proliferative function of miR-375 is possibly exerted, at least in part

  10. Triptolide Inhibits Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression in Human Colon Cancer and Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    Xiangmin TONG; Shui ZHENG; Jie JIN; Lifen ZHU; Yinjun LOU; Hangping YAO

    2007-01-01

    Triptolide (TP), a traditional Chinese medicine, has been reported to be effective in the treatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines. This study investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia (K562), and elucidates the possible molecular mechanism involved. SW114 and K562 cells were treated with different doses of TP (0, 5, 10, 20, or 50 ng/ml). The cell viability was assessed by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation of both tumor cell lines in a dose-dependent manner. To further investigate its mechanisms, the products prostaglandin E2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that TP strongly inhibited the production of NO and PGE2. Consistent with these results, the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulated both at the mRNA level and the protein expression level, as shown by real-time RT-PCR and Western blotting. These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activity could be involved in the antitumor mechanisms of TP.

  11. The differentiation effect of low-dose cytosine arabinoside is disturbed in PU.1-knockdown K562 cells.

    Science.gov (United States)

    Nakano, Hiroko; Yanagita, Akane; Takahashi, Shinichiro

    2014-07-01

    We recently demonstrated by using PU.1-knockdown K562 (K562 PU.1KD) cells stably expressing PU.1 short inhibitory RNAs and PU.1-overexpressing K562 (K562 PU.1OE) cells, that therapeutic concentrations of 5-aza-2'-deoxycytidine (5-azadC) induce erythroid differentiation of these cells and that the PU.1 expression level is closely associated with the differentiating and apoptotic effects of 5-azadC on K562 cells. In this study, we investigated whether the effects of low-dose cytosine arabinoside (Ara-C), which is another erythroid differentiation inducer in K562 cells, is associated with the expression level of PU.1 in these cells. As a result, we demonstrated that the effect of Ara-C on cell viability and differentiation, as determined by the WST-8 assay and β-globin mRNA expression analysis, respectively, was suppressed in K562 PU.1KD cells compared to their controls. Collectively, these findings suggest that sufficient expression of PU.1 is indispensable for the erythroid differentiation of K562 cells.

  12. Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

    Directory of Open Access Journals (Sweden)

    VIVIAN M. RUMJANEK

    2001-03-01

    Full Text Available Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado

  13. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

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    Juan Liu

    2016-04-01

    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  14. Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

    Science.gov (United States)

    Kucherenko, Yuliya V; Lang, Florian

    2014-07-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

  15. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

    Directory of Open Access Journals (Sweden)

    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  16. Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv%盐霉素抑制耐格列卫的人慢性粒细胞白血病细胞株K562/Glv增殖并诱导其凋亡

    Institute of Scientific and Technical Information of China (English)

    徐霜清; 祝爱珍; 刘成成; 许婷婷; 陈小宇; 刘革修

    2012-01-01

    AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec - resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK - 8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential ( △Ψm ), reactive oxygen species ( ROS ) and the concentration of intracellular Ca 2+ ( [ Ca 2+ ) in K562/Glv cells. The activity of caspase - 3 , - 8 and - 9 was measured by the method of colorimetry. The levels of cyto-chrome C, Bcl - 2, Bax, (3 - catenin and phosphorylated low - density lipoprotein receptor - related protein 6 ( p - LRP6 ) were determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose — dependent manner. Salinomycin at concentration of 0. 2 μmol/L inhibited the growth of the cells with the inhibitory rate of ( 36.70 ± 2. 31 )% . The cell apoptotic rate was ( 19. 66 ± 2. 23 )% . Salinomycin at concentration of 0. 2 μmol/L decreased the level of △Ψm, and increased the levels of ROS, cytochrome C and [ Ca 2+]i in the cells. Salinomycin also increased the activity of caspase — 3, — 8 and — 9 in the cells, reduced the ratio of Bel — 2/Bax, and attenuated the levels of (3 — catenin and p — LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec - resistant myeloid leukemia cell line K562/Glv via Bcl - 2/Bax and mitochondria - dependent pathways, and inhibits the cell growth through Wnt signal pathway.%目的:探讨盐霉素对耐格列卫的人慢性粒细胞白血病细胞株K562/Glv抑制增殖和诱导凋亡的作用及其机制.方法:采用CCK-8的方法检测盐霉素对K562/Glv细胞生长的抑制作用;流式细胞术检测细胞凋亡、活性氧、细胞内Ca2+浓度([Ca2+]i)和线粒体膜电位(ΔΨm);比色法检测caspase-3、-8和-9活性;Western blotting 分析细胞色素C、Bcl-2、Bax、β-catenin和磷酸化

  17. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

  18. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

    Science.gov (United States)

    Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation.

  19. 蛇毒组分对K562阿霉素敏感株和耐药株的抑制作用%Snake venom components inhibits growth of K562 adriamycin-sensitive and-resistant strains

    Institute of Scientific and Technical Information of China (English)

    杨昌山; 何慧华; 董伟华

    2013-01-01

    目的:通过对中华眼镜蛇毒进行分离和各组分的初筛,寻找逆转K562对阿霉素耐药的活性成分KD-Ⅲ-1,为今后研究肿瘤耐药性奠定工作基础。方法:通过凝胶分离得到的蛇毒组分分别作用于K562对阿霉素耐药株K562/A和敏感株K562/S,筛选有效活性组分;通过荧光探针Rh123测定P-gp蛋白活性和PI染色进一步确定该组分的逆转K562/A的耐药活性。结果:分别给予2μg/mL阿霉素( Adr)和各浓度蛇毒组分处理24 h后,可以发现蛇毒组分对阿霉素敏感株K562/S和耐药株K562/A都有明显的抑制作用,并呈现出剂量-效应关系。1μg/mL蛇毒组分处理组与2μg/mL蛇毒处理组抑制作用明显;在药物持续作用48 h后对K562/A的活性仍有抑制作用在0.5、1、2μg/mL的蛇毒组分组表现的更加明显。通过Rho外排实验发现蛇毒粗毒组平均荧光强度(MFI)与阴性对照组没有明显差异,而2.5μg/mL蛇毒组分组的MFI明显降低,与对照组相比具有统计学意义(P<0.05)。2.5μg/mL蛇毒粗毒和分离组分分别对K562/S敏感株和K562/A耐药株作用3 h后,K562/S的PI染色阳性率明显升高,而K562/A的PI染色阳性率并没有明显升高。结论:蛇毒组分KD-Ⅲ-1对K562/A和K562/S细胞均有明显抑制的作用,抑制作用可能与诱导凋亡有关。%Objective:To explore the active ingredient, KD-Ⅲ-1, for reversal of K562 resistance to adriamycin ( Adr) via separation and primary screening of individual components of cobra venom, thus offering the sound basis for future study on tumor drug resistance. Methods: The K562 Adr-resistant strains, K562/A, and -sensitive strains, K562/S, were incubated with isolated snake venom components derived from gel separation, for exploration of the effective component. The P-gp protein activity was measured by the fluorescent probe Rh123, and the PI staining was applied to determine the resistance reversal capacity of the components

  20. 60 Coγ射线辐射致LyGDI断裂对K562细胞的延迟性凋亡作用%Delayed K562 cell apoptosis promoted by cleaved LyGDI after 60Co γ-rays irradiation

    Institute of Scientific and Technical Information of China (English)

    孙华丽; 段卫明; 邵彦彦; 肖海楠; 周新文

    2010-01-01

    目的 采用不同剂量的60 Coγ射线照射人红白血病细胞系K562,探究鸟苷酸解离抑制因子-2(LyGDI)在细胞延迟性死亡中的作用以及其调控机制.方法 细胞流式仪PI单染检测细胞周期,藻红B染色观察细胞的凋亡,Western blot分析LyGDI和Rac1蛋白的表达,免疫荧光检测Rac1蛋白在细胞内的分布.结果 K562细胞被阻滞在G2/M期的百分率为71.3%,K562细胞早期凋亡率较低,8 Gy60Co γ射线照射后24 h,凋亡率为14%,呈现出延迟性的细胞凋亡;K562细胞在4 Gy的γ射线照射24 h后,可见LyGDI发生断裂,总的Rac1蛋白表达未发生明显变化,但其在细胞内的分布发生改变,Rac1转移至细胞膜上与少量核内分布,Rac1脱离LyGDI而激活.结论 LyGDI具有增加细胞延迟性凋亡的作用,其断裂使Rac1转移至细胞膜上,诱导Rac1的激活,从而促进了细胞的凋亡.%Objective To elucidate the function and regulatory mechanism of LyGDI involved delayed cell death in the human K562 cells and HL-60 cells induced by 60Co γ-rays. Methods Erythrosine B cells staining was used to count the apoptosis rate. PI staining and flow cytometry were applied to check the cell cycle. The expression of LYGDI and Rac1 was resolved by Western blot by using monoclonal antibody of LyGDI and Racl. The distribution of Racl protein in cells was observed with immunofluorescence by using the confocal microscope. Results The K562 cells showed G2/M phase arrest and the percent age was 71.3%. The apoptosis rate was very low at early post-irradiation stage in the K562 cells. The apoptosis rate was 14% in the K562 cells at 24 h post-irradiation with 8 Gy of γ-rays, and delayed cell apoptosis was present. LyGDI was cleaved in the K562 cells irradiated by 4 Gy 60 Co γ-rays after 24 hours post-irradiation. The expression of Racl protein was not altered at all, but the distribution was changed in the irradiated cells while the Racl protein moved to cell membrane and a little in cell

  1. siRNA沉默Ikaros基因对K562细胞中γ珠蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    马艳; 李冉; 吴新忠; 胡珺; 曾玉

    2012-01-01

    @@ 地中海贫血,即β珠蛋白合成障碍性贫血,简称地贫,是一种单基因缺陷的遗传性疾病,它是由于β珠蛋白多肽链合成减少或缺失,α链与非α链(β、γ、δ)之间不平衡所导致的以红细胞无效生成为特征的血红蛋白病.重型β-地贫常常有严重的临床症状,多采取规则的输血及祛铁治疗,但大多数病人最终还是死于铁过载相关的心脏疾病,平均寿命不满20岁[1].%To interfere the Ikaros gene with siRNA, and observe the expression of y-globin in K562 human erythroleukemia cells. Method, Three siRNA-Ikaros(siRNAl,siRNA2 and siRNA3) were designed and transacted to K562 cells by Lipolectamine 2000. The optimal siRNA se-quence of Ikaros gene silencing was screened by semiquantitative RT-PCR analysis. Real-time quantitative PCR and Western blotting mehtods were used to detect the expression of Ikaros and y-globin mRNA and protein levels alter Ikaros gene silencing. Hemoglobin content of the K562 cells were detected by using benzidine staining method. Results, The transaction rate of K562 cells by the Lipolectamine 2000 could reach up to 58. 7%. SiRNA3 could effectively interfere the expression of Ikaros gene, and the inhibited efficiency could reach up to 86. 7%. SiRNA3 could enhance the expression of y-globin and the hemoglobin content of K562 cells. Conclusion; SiRNA3 can interfere the expression of Ikaros and en-

  2. Reversal in multidrug resistance by magnetic nanoparticle of Fe3O4 loaded with adriamycin and tetrandrine in K562/A02 leukemic cells

    Directory of Open Access Journals (Sweden)

    Baoan Chen

    2008-06-01

    loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02. We propose that Fe3O4-MNPs loaded with ADM and Tet probably have synergetic effect on reversal in multidrug resistance.Keywords: magnetic nanoparticles, tetrandrine, adriamycin, multidrug resistance reversal, leukemia K562/A02

  3. DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

    Science.gov (United States)

    Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

    2015-07-31

    We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Simvastatin induces NFκB/p65 down-regulation and JNK1/c-Jun/ATF-2 activation, leading to matrix metalloproteinase-9 (MMP-9) but not MMP-2 down-regulation in human leukemia cells.

    Science.gov (United States)

    Chen, Ying-Jung; Chang, Long-Sen

    2014-12-15

    The aim of the present study was to explore the signaling pathways associated with the effect of simvastatin on matrix metalloproteinase-2 (MMP-2)/MMP-9 expression in human leukemia K562 cells. In sharp contrast to its insignificant effect on MMP-2, simvastatin down-regulated MMP-9 protein expression and mRNA levels in K562 cells. Simvastatin-induced Pin1 down-regulation evoked NFκB/p65 degradation. Meanwhile, simvastatin induced JNK-mediated c-Jun and ATF-2 activation. Over-expression of Pin1 suppressed simvastatin-induced MMP-9 down-regulation. Treatment with SP600125 (a JNK inhibitor) or knock-down of JNK1 reduced MMP-2 expression in simvastatin-treated cells. Simvastatin enhanced the binding of c-Jun/ATF-2 with the MMP-2 promoter. Down-regulation of c-Jun or ATF-2 by siRNA revealed that c-Jun/ATF-2 activation was crucial for MMP-2 expression. Suppression of p65 activation or knock-down of Pin1 by shRNA reduced MMP-2 and MMP-9 expression in K562 cells. Over-expression of constitutively active JNK1 rescued MMP-2 expression in Pin1 shRNA-transfected cells. Simvastatin treatment also suppressed MMP-9 but not MMP-2 expression in human leukemia U937 and KU812 cells. Taken together, our data indicate that simvastatin-induced p65 instability leads to MMP-9 down-regulation in leukemia cells, while simvastatin-induced JNK1/c-Jun/ATF-2 activation maintains the MMP-2 expression underlying p65 down-regulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

    Science.gov (United States)

    Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

    2017-06-01

    Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

  6. 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.

  7. Animal experimental study of inhibitory effect of suicide genes on K562 cell neoplasia%自杀基因抑制K562细胞成瘤的动物实验研究

    Institute of Scientific and Technical Information of China (English)

    万德胜; 姜义荣; 陈万宁

    2008-01-01

    目的 探讨自杀基因胞嘧啶脱氨酶(CD)和胸苷激酶(TK)在小鼠体内对K562细胞的抑制作用.方法 将在慢病毒介导下已转染CDgly TK的K562细胞种植于裸鼠皮下,观察其成瘤情况和对前体药物[环氧鸟苷(GCV)、5-氟胞嘧啶(5-FC)]的敏感性.结果 种植K562细胞和K562/CDg1y TK细胞的裸鼠分别在(7.0±1.2)和(7.1±0.9)d后长出肉眼可见的瘤块.两组裸鼠生存期分别是(52.2±5.3)和(54.2±3.7)d,差异无统计学意义(P>0.05);接种K562/CDgly TK细胞的小鼠经GCV和5-FC处理后,其裸鼠分别在(26.9±1.7)、(25.7±1.9)d后肉眼可见肿瘤生长,对照组接种K562细胞后(6.9±1.7)d生长出肉眼可见肿瘤,两者差异有统计学意义(P<0.01).结论 自杀基因在体内对K562细胞有明显的抑制作用,能增强前体药物GCV和5-FC对肿瘤细胞的药物作用.

  8. Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yin TONG; Yan-yan LIU; Liang-shun YOU; Wen-bin QIAN

    2012-01-01

    Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML)cells and its mechanisms of action are unknown.In this study,the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML)cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60,and the CML cell line K562 were used.Cell viability was quantitated using MTT assay.Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining,which were followed by flow cytometry and fluorescence microscopy analysis,respectively.Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot.Autophagy was studied using electron microscopy,the acridine orange staining method,and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines,the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line)were resistant to perifosine (2.5-20 μmol/L)in respect to inhibiting cell growth and inducing apoptosis.Perifosine (2.5,5,and 10 μmol/L)inhibited Akt and its phosphorylation in AML cells,but not in CML cells.Treatment with perifosine (20 μmol/L)resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II.Treatment of CML cells with perifosine (5,10,and 20 μmol/L)dose-dependently upregulated AGT5,but not Beclin 1 at the protein level.Furthermore,inhibition of autophagyby chloroquine (40 nmol/L)significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro,which is due to perifosine-induced protective autophagy and upregulation of ATG5.

  9. The Ethanol Extract of Fructus trichosanthis Promotes Fetal Hemoglobin Production via p38 MAPK Activation and ERK Inactivation in K562 Cells

    Directory of Open Access Journals (Sweden)

    Hui Li

    2011-01-01

    Full Text Available Pharmacological stimulation of fetal hemoglobin (HbF expression may be a promising approach for the treatment of beta-thalassemia. In this study, the effects of Fructus trichosanthis (FT were investigated in human erythroleukemic K562 cells for their gamma-globin mRNA and HbF-induction activities. The role of signaling pathways, including extracellular regulated protein kinase (ERK and p38 mitogen-activated protein kinase (MAPK, was also investigated. It was found that the ethanol extract of FT significantly increased gamma-globin mRNA and HbF levels, determined by real-time reverse transcription polymerase chain reaction and enzyme linked immunosorbent assay, respectively, in dose- and time-dependent manner. Total Hb (THb levels were also elevated in the concentrations without cytotoxicity (<80 μg mL−1. Pre-treatment with p38 MAPK inhibitor SB203580 blocked the stimulatory effects of FT extract in total and HbF induction. In contrast, no change in HbF was observed when treated with ERK inhibitor PD98059. Furthermore, FT ethanol extract activated p38 MAPK and inhibited ERK signaling pathways in K562 cells, as revealed in western blotting analysis. In addition, SB203580 significantly abolished p38 MAPK activation when the cells were treated with FT. In summary, the ethanol extract of FT was found to be a potent inducer of HbF synthesis in K562 cells. The present data delineated the role of ERK and p38 MAPK signaling as molecular targets for pharmacologic stimulation of HbF production upon FT treatment.

  10. EFFECT OF BcL-2 ANTISENSE DRUG WITH DIFFERENT STRUCTURE ON THE BIOLOGICAL FUNCTION OF K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    雷小勇; 张洹; 何冬梅

    2004-01-01

    Objective: To study the differences and similarities of the antisense drugs with different structures on the biological functions of K562 cells. Methods: Cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis and agarose gel electrophoresis of DNA fragmentation were also performed. The expression level of protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Results: PNA targeting the coding region of the Bcl-2 messenger RNA could effectively inhibit K562 cell viability, down-regulate the synthesis of the Bcl-2 protein and increase cell apoptosis. By 72 h after the Bcl-2 antisense PNA treatment, K562 cells showed more reduction in the level of Bcl-2 protein compared with cells treated with the antisense ODN. After treatment with 10μmol/L of Bcl-2 antisense PNA or antisense ODN for 72 h, apoptotic rates of K562 cells were 13.15±1.13 and 11.72±1.12, respectively. Furthermore, there was significant difference in the percentage of apoptotic cells between antisense PNA group and antisense ODN group. Conclusion: The results suggest that antisense PNA targeting the coding region of Bcl-2 mRNA has better antisense effects than the antisense oligonucleotides on inducing apoptosis of K562 cells.

  11. Phenylalanine sensitive K562-D cells for the analysis of the biochemical impact of excess amino acid.

    Science.gov (United States)

    Sanayama, Yoshitami; Matsumoto, Akio; Shimojo, Naoki; Kohno, Yoichi; Nakaya, Haruaki

    2014-11-06

    Although it is recognized that the abnormal accumulation of amino acid is a cause of the symptoms in metabolic disease such as phenylketonuria (PKU), the relationship between disease severity and serum amino acid levels is not well understood due to the lack of experimental model. Here, we present a novel in vitro cellular model using K562-D cells that proliferate slowly in the presence of excessive amount of phenylalanine within the clinically observed range, but not phenylpyruvate. The increased expression of the L-type amino acid transporter (LAT2) and its adapter protein 4F2 heavy chain appeared to be responsible for the higher sensitivity to phenylalanine in K562-D cells. Supplementation with valine over phenylalanine effectively restored cell proliferation, although other amino acids did not improve K562-D cell proliferation over phenylalanine. Biochemical analysis revealed mammalian target of rapamycin complex (mTORC) as a terminal target of phenylalanine in K562-D cell proliferation, and supplementation of valine restored mTORC1 activity. Our results show that K562-D cell can be a potent tool for the investigation of PKU at the molecular level and to explore new therapeutic approaches to the disease.

  12. Inhibitory Effects of Phellinus Linteus Intracellular Polysaccharide on the Proliferation of Leukemic Cells K562%桑黄胞内多糖对白血病细胞K562的增殖抑制效应研究

    Institute of Scientific and Technical Information of China (English)

    史新强; 沈业寿; 卫自; 马金宝; 合肥

    2007-01-01

    背景与目的:观察桑黄胞内多糖(PLIP)对体外培养的人白血病细胞K562的增殖抑制作用,并初步探讨其作用机制.材料和方法:采用MTT法及台盼蓝拒染法测定细胞增殖抑制率和生长曲线,Hoechst-PI双荧光染色,流式细胞术和DNA琼脂糖凝胶电泳检测细胞凋亡.结果:PLIP在25 μg/ml、50 μg/ml、100 μg/ml、200 μg/ml、400 μg/ml、800 μg/ml剂量时均能显著抑制K562细胞增殖(P<0.05),其中400 μg/ml剂量时抑制率最高,达52.55%,半数抑制浓度(IC50)为262.36 μg/ml,流式细胞仪检测PLIP在100 μg/ml、200 μg/ml、400 μg/ml剂量时K562细胞凋亡率分别为5.72%、13.57%、19.39%,并能诱导K562细胞出现凋亡所具有的形态学及生化特征.结论:PLIP对体外培养的人白血病细胞K562生长增殖具有明显的抑制作用,其作用机制可能与诱导K562细胞凋亡和影响细胞周期有关.

  13. Anti - K562 cells effect mediated by dendritic cells derived from cord blood pulsed with exosomes%Exosomes致敏的脐血树突细胞介导的抗K562细胞作用

    Institute of Scientific and Technical Information of China (English)

    卜宁; 李奇灵; 孙秉中; 张涛; 冯琦; 乔庆大

    2006-01-01

    目的分离K562细胞释放的exosomes,致敏脐血树突细胞(dendritic cell,DCs),观察其对细胞毒性T淋巴细胞(cytotoxic Tlymphocytes,CTLs)的激活效应.方法离心超滤和蔗糖密度梯度离心法分离K562细胞释放的exosomes,固相免疫电镜法(SPIEM)制备exosomes的HSP70、ICAM-1及ABL免疫电镜标本.常规方法从脐血单个核细胞诱导DCs并分离T细胞,将K562细胞来源的exosomes冲击或未冲击的DCs与T细胞共培养.MTT比色法检测体外细胞毒活性.结果K562细胞分泌的exosomes为直径50~100nm的膜性微囊.Exosomes致敏的脐血DCs激活CTLs的能力显著高于肿瘤冻融抗原致敏的DCs组,在效靶比为50:1时,两组CTLs对K562细胞的杀伤率为(68.4%vs35.3%,P<0.05).结论K562细胞分泌的exosomes负载脐血DCs后活化CTLs,有抗肿瘤活性.

  14. Molecular cross talk between Notch1, Shh and Akt pathways during erythroid differentiation of K562 and HEL cell lines.

    Science.gov (United States)

    Roy, Anita; Haldar, Srijan; Basak, Nandini Pal; Banerjee, Subrata

    2014-01-01

    Erythropoiesis is a tightly regulated process dependent on extrinsic signals conveyed by the bone marrow niche. The signalling pathways thus activated or repressed do not act in isolation; rather an intricate cross talk among these pathways ensues homoeostasis within the erythroid compartment. In this study, we describe the effects of two such signalling pathways namely the Notch1 and the Shh pathway on erythropoiesis in immortalised K562 and HEL cell lines as well as the cross talk that ensues between them. We show that while activation of the Notch1 pathway inhibits differentiation of erythroid lineage cell lines as well as in in-vitro primary erythroid cultures from the human CD34(+) cells; Shh pathway favours erythroid differentiation. Further, the Notch1 pathway activates the Akt pathway and constitutively active Akt partially mimics the effect of Notch1 activation on erythropoiesis. Moreover, the Notch1, Akt and Shh pathways were found to cross talk with each other. In this process, activation of Notch1 was found to down regulate the Shh pathway independent of Akt activation. Significantly, Notch1 not only down regulated the Shh pathway, but also inhibited recombinant Shh mediated erythropoiesis. Our study thus reveals an intricate crosstalk among the Notch1, Shh and Akt pathways wherein Notch1 emerges as a key regulator of erythropoiesis.

  15. The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

    Institute of Scientific and Technical Information of China (English)

    BEN Yali; LIU Deli; ZHU Dali; ZHU Derui; LUO Qin

    2006-01-01

    The difference of sensitivity to indole- 3-acetic acid ( IAA ) combined with horseradish peroxidase (HRP) in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTT assay. The cell cycle and apoptosis of K562 and BXPC- 3 cells were examined by a fluorescence flow cytometer (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC- 3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

  16. The recognition of N-glycans by the lectin ArtinM mediates cell death of a human myeloid leukemia cell line.

    Directory of Open Access Journals (Sweden)

    Fernanda Caroline Carvalho

    Full Text Available ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit, interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50 = 10 µg/mL, as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.

  17. Apoptosis Induced by Microbubble-Assisted Acoustic Cavitation in K562 Cells: The Predominant Role of the Cyclosporin A-Dependent Mitochondrial Permeability Transition Pore.

    Science.gov (United States)

    Zhao, Lu; Feng, Yi; Shi, Aiwei; Zong, Yujin; Wan, Mingxi

    2015-10-01

    Acoustic cavitation of microbubbles has been described as inducing tumor cell apoptosis that is partly associated with mitochondrial dysfunction; however, the exact mechanisms have not been fully characterized. Here, low-intensity pulsed ultrasound (1 MHz, 0.3-MPa peak negative pressure, 10% duty cycle and 1-kHz pulse repetition frequency) was applied to K562 chronic myelogenous leukemia cells for 1 min with 10% (v/v) SonoVue microbubbles. After ultrasound exposure, the apoptotic index was determined by flow cytometry with annexin V-fluorescein isothiocyanate/propidium iodide. In addition, mitochondrial membrane potential (ΔΨm) was determined with the JC-1 assay. Translocation of apoptosis-associated protein cytochrome c was evaluated by Western blotting. We found that microbubble-assisted acoustic cavitation can increase the cellular apoptotic index, mitochondrial depolarization and cytochrome c release in K562 cells, compared with ultrasound treatment alone. Furthermore, mitochondrial dysfunction and apoptosis were significantly inhibited by cyclosporin A, a classic inhibitor of the mitochondrial permeability transition pore; however, the inhibitor of Bax protein, Bax-inhibiting peptide, could not suppress these effects. Our results suggest that mitochondrial permeability transition pore opening is involved in mitochondrial dysfunction after exposure to microbubble-assisted acoustic cavitation. Moreover, the release of cytochrome c from the mitochondria is dependent on cyclosporin A-sensitive mitochondrial permeability transition pore opening, but not formation of the Bax-voltage dependent anion channel complex or Bax oligomeric pores. These data provide more insight into the mechanisms underlying mitochondrial dysfunction induced by acoustic cavitation and can be used as a basis for therapy. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  18. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibit proliferation of leukemia cells.

    Science.gov (United States)

    Ji, Yuan; Ma, Yongbin; Chen, Xiang; Ji, Xianyan; Gao, Jianyi; Zhang, Lei; Ye, Kai; Qiao, Fuhao; Dai, Yao; Wang, Hui; Wen, Xiangmei; Lin, Jiang; Hu, Jiabo

    2017-08-01

    Human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSCs) are able to inhibit proliferation of leukemia cells. Microvesicles released from human embryonic stem cell derived-mesenchymal stem cells (hESC‑MSC‑MVs) might play an important part in antitumor activity. Microvesicles were isolated by ultracentrifugation and identified under a scanning electron microscopy and transmission electron microscope separately. After 48-h cocultured with hESC‑MSCs and hESC‑MSC‑MVs, the number of K562 and HL60 was counted and tumor cell viability was measured by CCK8 assay. The expression of proteins Bcl-2 and Bax were estimated by western blotting. Transmission electron microscope and western blot analysis were adopted to evaluate the autophagy level. Results showed that both hESC‑MSCs and hESC‑MSC‑MVs inhibited proliferation of leukemia cells in a concentration-dependent manner. hESC‑MSC‑MVs reduced the ratio of Bcl/Bax, enhanced the protein level of Beclin-1 and LC3-II conversion, thus upregulating autophagy and apoptosis. In conclusion, microvesicles released from human embryonic stem cell derived-mesenchymal stem cells inhibited tumor growth and stimulated autophagy and excessive autophagy might induce apoptosis.

  19. Cordycepin regulates GSK-3β/β-catenin signaling in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Bor-Sheng Ko

    Full Text Available BACKGROUND: Leukemia stem cells (LSCs are a limitless cell source for the initiation and maintenance of leukemia. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs. Therefore, targeting β-catenin is considered a therapeutic strategy for the treatment of leukemia. The goal of this study was to explore whether cordycepin, an active component of the traditional medicine Cordyceps sinensis, regulates β-catenin expression in leukemia cells. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found that cordycepin significantly suppressed cell proliferation in all malignant cancer cells, including U937, K562, A549, HepG2, SK-Hep1 and MCF7 in a dose-dependent manner. However, cordycepin reduced β-catenin levels in U937, K562 and THP1 leukemia cells and had no effect on other solid cancer cells. In addition, treatment with cordycepin significantly suppressed leukemia colony formation in soft agar assay. Cordycepin enhanced proteasome-dependent degradation and inhibited nuclear translocation of β-catenin in leukemia cells. Cordycepin-reduced β-catenin stability was restored by the addition of a pharmacological inhibitor of GSK-3β, indicating that cordycepin-suppressed β-catenin stability is mediated by the activation of GSK-3β. Furthermore, cordycepin abolished the effect of Wnt3a-induced β-catenin in leukemia cells. In addition, cordycepin-impaired β-catenin is regulated by Akt activation but is not significantly influenced by AMPK or mTOR signal pathways. SIGNIFICANCE: Our findings show for the first time that codycepin selectively reduces β-catenin stability in leukemia but not in other solid tumor cells. This suppressive effect is mediated by regulating GSK-3β. A synergistic combination of cordycepin with other treatments should be used as a novel strategy to eradicate leukemia via elimination of LSCs.

  20. Growth-inhibiting Effect of Patrinia Scabra Bunge Lignanoid on K562 Cells%糙叶败酱总木脂素对K562细胞体外生长的影响

    Institute of Scientific and Technical Information of China (English)

    陈茹; 赵健雄; 王学习; 李世刚; 王惠娟

    2007-01-01

    目的:探讨糙叶败酱提取物总木脂素对K562细胞体外生长的影响及作用机制.方法:采用四甲基偶氮唑盐比色法(monotetrazolium test,MTT)测定糙叶败酱总木脂素对K562细胞生长的影响;采用电镜技术对其进行形态学观察;采用流式细胞仪对其进行DNA含量分析,检测经药物作用后的细胞凋亡率.结果:糙叶败酱总木脂素对K562细胞生长有明显的抑制作用,抑制作用随药物浓度增加及作用时间延长而有加强趋势,细胞经糙叶败酱总木脂素作用96h,IC50 21.16μg/mL.电镜下观察到经糙叶败酱总木脂素作用48h后,一部分K562细胞体积变小,胞浆浓缩,胞核染色质聚集或边集,核碎裂,凋亡小体形成,呈典型的凋亡形态特征.流式细胞仪可检测到经糙叶败酱总木脂素诱导K562细胞48h后产生明显的亚二倍体峰,细胞凋亡率为10.1%.结论:糙叶败酱总木脂素可抑制K562细胞的生长,作用的机制与其诱导K562细胞凋亡有关.

  1. Differentiation of Erythroleukemia K562 Cells Induced by Piperine%胡椒碱诱导人红白血病细胞株K562的分化

    Institute of Scientific and Technical Information of China (English)

    宋其芳; 瞿燕春; 郑红波; 张高华; 林鸿刚; 杨金亮

    2008-01-01

    背景与目的:白血病是一种血液系统的恶性肿瘤,诱导分化是治疗白血病非常有效的方法之一.胡椒碱(piperine)是一种从胡椒属植物中提取的生物碱,具有镇静、抗炎、抗肿瘤等多种药理活性.本研究旨在探讨胡椒碱对K562细胞的增殖抑制和诱导分化作用.方法:采用台盼蓝染色计数法绘制生长曲线和流式细胞术检测细胞周期及细胞凋亡.观察胡椒碱对K562细胞增殖的影响;通过观察细胞形态学变化、检测硝基蓝四氮唑(nitroblue tetrazolium,NBT)还原能力、流式细胞术检测细胞表面标志CD33和CD14的变化,探讨胡椒碱对K562细胞的诱导分化作用.结果:20μmol/L和40μmol/L胡椒碱可诱导K562细胞向巨噬细胞和单核系细胞分化.40 μmol/L胡椒碱作用3 d,K562细胞的NBT还原阳性率由(8.5±1.9)%上升到(76.7±5.3)%;20 μmol/L胡椒碱作用3 d,流式细胞术结果显示细胞表面分化抗原CD33的平均荧光强度(mean fluorescence intensity,MFI)下降42.05%(P<0.01),而CD14的MFI则升高了1倍(P<0.01);20 μmol/L以上浓度的胡椒碱对K562细胞的增殖具有抑制作用,其抑制作用随时间的延长或剂量的增加有增强的趋势.结论:胡椒碱可诱导K562细胞向巨噬和单核系细胞分化.

  2. Antizyme1基因转染对K562细胞增殖与凋亡的影响%The Effect of Antizymel Transfection on Cell Proliferation and Cell Apoptosis in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    姜立; 马文丽; 李晋; 彭翼飞; 徐兵; 郑文岭

    2007-01-01

    目的: 研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷( As2O3)诱导凋亡时的影响.方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体.脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系.采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化.并通过RT-PCR方法检测antizyme1转染对cyclin D1和survivin基因表达的影响.结果:获得稳定表达antizyme1的K562pAZ1m细胞株后,其增殖能力明显减慢.cyclin D1基因表达降低,细胞主要停滞于G0/G1期.在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低.结论:AZ1基因能够抑制K562细胞增殖,通过对cyclin D1的负调控使细胞周期停滞于G0/G1期.并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用.

  3. Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

    Indian Academy of Sciences (India)

    Feng Liu; Shi-Ying Liu; Ping Xu; Zhen-Hua Xie; Guo-Ping Cai; Yu-Yang Jiang

    2008-03-01

    According to the method used in our laboratory, our group synthesized (DIPP-Trp)2-Lys-OCH3. It inhibited the proliferation of K562 and HeLa cells in a dose- and time-dependent manner with an IC50 of 15.12 and 42.23 M, respectively. (DIPP-Trp)2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)2-Lys-OCH3 not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G2/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)2-Lys-OCH3 is a potential anticancer drug that intervenes in the signalling pathway.

  4. H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

    KAUST Repository

    Zolea, Fabiana

    2015-10-09

    The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

  5. Hemin-dependent induction and internalization of CD38 in K562 cells.

    Science.gov (United States)

    Yalcintepe, Leman; Ercelen, Sebnem; Adin-Cinar, Suzan; Badur, Selim; Tiryaki, Demir; Bermek, Engin

    2003-10-01

    The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP-ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time-dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD(+) glycohydrolase and ADP-ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP-ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti-CD38 monoclonal antibody. SDS-PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin-dependent appearance of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin-induced expression of CD38 was followed by its down-regulation.

  6. Effect of STAT5 decoy oligodeoxynucleotides on expression profile of apoptosis-related genes in K562 cells by cDNA microarray%cDNA芯片检测STAT5诱骗核苷酸对K562细胞凋亡相关基因的影响

    Institute of Scientific and Technical Information of China (English)

    曾建明; 冯文莉; 王小中; 史梅; 涂植光; 黄宗干

    2006-01-01

    目的:转录因子STAT5在慢性粒细胞白血病(chronic myeloid leukemia,CML)中组成性激活,并与CML细胞恶性表型密切相关,本研究用cDNA芯片检测方法探讨诱骗核苷酸(decoy ODNs)抑制STAT5对白血病K562细胞株凋亡相关基因的影响.方法:分别提取decoy ODNs处理前后的K562细胞总RNA,逆转录合成Cy3、Cy5标记的cDNA探针,与人14K基因表达谱cDNA芯片(V2.0)杂交,扫描获得数据后,用Genespring软件分析对照组和实验组的差异表达基因,半定量RT-PCR验证cDNA芯片结果.结果:2张cDNA芯片检测重复性良好(R=0.9799).在13 824个标记的基因中,检出413个上调基因,其中包括:TIAF1、GAB1、GYPA、DAPK3、TNFRSF1B、IRF1和PML等在内的18个凋亡相关基因;在检出的332个下调基因中,发现PIM1、CCND2、CCND1、MYC和BCL2L1等在内的11个凋亡相关基因;部分基因经半定量RT-PCR证实与cDNA芯片结果一致.结论:Decoy ODNs抑制STAT5信号通路后可引起K562细胞多种凋亡相关基因的变化,本实验为进一步研究STAT5信号通路所调控的凋亡相关基因提供了依据.

  7. Characterization of miRNomes in Acute and Chronic Myeloid Leukemia Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Qian Xiong; Jiangwei Yan; Songnian Hu; Xiangdong Fang; Yadong Yang; Hai Wang; Jie Li; Shaobin Wang; Yanming Li; Yaran Yang; Kan Cai; Xiuyan Ruan

    2014-01-01

    Myeloid leukemias are highly diverse diseases and have been shown to be associated with microRNA (miRNA) expression aberrations. The present study involved an in-depth miRNome analysis of two human acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and one human chronic myeloid leukemia (CML) cell line, K562, via massively parallel signature sequenc-ing. mRNA expression profiles of these cell lines that were established previously in our lab facil-itated an integrative analysis of miRNA and mRNA expression patterns. miRNA expression profiling followed by differential expression analysis and target prediction suggested numerous miRNA signatures in AML and CML cell lines. Some miRNAs may act as either tumor suppres-sors or oncomiRs in AML and CML by targeting key genes in AML and CML pathways. Expres-sion patterns of cell type-specific miRNAs could partially reflect the characteristics of K562, HL-60 and THP-1 cell lines, such as actin filament-based processes, responsiveness to stimulus and phag-ocytic activity. miRNAs may also regulate myeloid differentiation, since they usually suppress dif-ferentiation regulators. Our study provides a resource to further investigate the employment of miRNAs in human leukemia subtyping, leukemogenesis and myeloid development. In addition, the distinctive miRNA signatures may be potential candidates for the clinical diagnosis, prognosis and treatment of myeloid leukemias.

  8. 核苷类似物对K562细胞的抗增殖和诱导分化作用%Nucleoside analog inhibiting proliferation and inducing differentiation of K562 cells

    Institute of Scientific and Technical Information of China (English)

    居小萍; 王健民; 周虹; 夏放; 袁有忠

    2001-01-01

    目的:探讨核苷类似物阿昔洛韦(ACV)、更昔洛韦(GCV)对人白血病细胞的抗增殖和诱导分化作用.方法:将ACV、GCV分别加入K562细胞培养4 d,测定其活细胞数目、克隆形成率、联苯胺染色阳性率,观察光镜形态、组织化学染色,并进行流式细胞分析、端粒酶检测.结果:在ACV、GCV的作用下,K562细胞的增殖受抑,生长分数降低,并且向具有合成血红蛋白能力的细胞分化.结论:核苷类似物ACV、GCV对K562细胞具有抑制增殖和诱导分化作用,该结果为慢性粒细胞白血病的治疗提示了新的途径.

  9. 糙叶败酱总木脂素对K562细胞基因表达的影响%EFFECT OF PATRINIA SCABRA BUNGE LIGNANS ON GENE EXPRESSION PROFILING OF K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    高佩媛; 程卫东

    2008-01-01

    [目的]研究糙叶败酱总木脂素对白血病K562细胞基因表达的影响.探讨糙叶败酱治疗白血病的药理作用机制.[方法]分别提取药物治疗组表模型组K562细胞总RNA, Cy3和Cy5荧光标记,反转录分别合成cDNA探针后,与基因表达谱芯片杂交,ScanArray Lite扫描仪扫描芯片,Quantarray软件分析表达信号.[结果]总木脂素处理前后比较差异表达的基因共有147个,其中表达上调的有68个,表达下调的有79个.[结论]糙叶败酱总木脂素对K562细胞基因表达具有调控作用,从分子水平阐释了糙叶败酱治疗白血病的药理作用机制.

  10. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

    Directory of Open Access Journals (Sweden)

    Halsey Christina

    2012-08-01

    Full Text Available Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.

  11. Apoptosis induced by(DIPP-L-Leu)2-L-Lys-OCH3 in K562 cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Apoptosis as a mechanism of deleting cells from tissues plays an important role in physiological and varieties of pathological situations, especially cancer conditions. In order to search for tumor cells apoptosis inducers, the inhibition effects on K562 cells of N-phosphoryl dipeptide methyl esters were studied by MTT assays, and (DIPP-L-Leu)2- L-Lys-OCH3 was the compound which had the best activity. From the studies of the typical apoptotic morphologic changes, DNA agarose gel electrophoresis, and flow cytometry analysis, it could be concluded that (DIPP-L-Leu)2- L-Lys-OCH3 could induce apoptosis of K562 cells in a dose-dependent manner, and the IC50 was 22.66 mol/L according to MTT assays.

  12. Different photodynamic effect between continuous wave and pulsed laser irradiation modes in k562 cells in vitro

    Science.gov (United States)

    Klimenko, V. V.; Bogdanov, A. A.; Knyazev, N. A.; Rusanov, A. A.; Dubina, M. V.

    2014-10-01

    Photodynamic therapy is a cancer treatment method is used primarily continuous mode laser radiation. At high power density irradiation occurs intense consumption of molecular oxygen and this caused hypoxic tumor tissue, which leads to inefficiency PDT. In this paper, pulsed and continuous irradiation modes during PDT photosensitizer Radachlorin were compared. A mathematical model for the generation of singlet oxygen 1O2 in tumor cells during photodynamic therapy with tissue oxygenation was developed. Our study theoretically and experimentally demonstrates the increased singlet oxygen generation efficiency in a pulsed irradiation mode compared to continuous wave mode with the same power density 20mW/cm2. Experimental in vitro showed that pulsed irradiation mode mostly induces apoptosis k562 tumor cells at irradiation doses of k562 1.25 - 2.5J/cm2 while the continuous mode induced necrosis.

  13. Experimental study on K562 cells and hematopietic stemcells with Fuzheng-Quxie Tablet%扶正祛邪丹对K562细胞株及造血干细胞的影响

    Institute of Scientific and Technical Information of China (English)

    王展翔; 许勇钢; 麻柔; 杨经敏; 刘锋; 郑金福; 廖军鲜

    2001-01-01

    目的:研究扶正祛邪丹对K562细胞株和小鼠造血干细胞的影响,探讨扶正祛邪丹治疗骨髓增生异常综合征的疗效机理。方法:扶正祛邪丹含药兔血清培养K562细胞株,用流式细胞仪检测P53蛋白表达、细胞活力、细胞凋亡与细胞周期;用造血干细胞培养法观察扶正祛邪丹对BALB/C小鼠骨髓造血干细胞增殖的影响。结果:扶正祛邪丹对K562细胞株P53蛋白表达有抑制作用,对红系造血祖细胞有促进作用。不能诱导细胞凋亡,对细胞增殖周期无明显影响,对小鼠骨髓粒系造血干细胞无明显促进作用。结论:扶正祛邪丹促进红系造血干细胞增殖,降低P53蛋白表达,可能为其主要疗效机理。%Objective:To study the mechanism of Fuzheng Quxie Tablet (FZQXD) in treating myelodysplastic syndromes (MDS).Methods: The in vitro effect of FZQXD on K562 cell line and hematopietic stem cells were studied with flow cytometery and cell cultuer.Results:FZQXD inhibited P53 protein expression,markedly promoted the proliferation of colony forming unit-erythrocytes(CFU-E).no effect on K562 cell in inducing apoptosis and cell-cycle.Conclusions:That FZQXD slightly inhibited P53 protein expression and markedly promoted the proliferation of colony forming unit-erythrocytes (CFU-E) might be one of the mechanisms in treating myelodysplastic syndromes.

  14. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    Science.gov (United States)

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice.

  15. Effect of NF-κB signaling pathway on the formation of multidrug resistance in K562/A02 cells%NF-κB信号通路的激活对白血病细胞K562/A02多药耐药的影响

    Institute of Scientific and Technical Information of China (English)

    孙海英; 李德志; 徐开林; 李振宇; 曾令宇; 鹿群先

    2009-01-01

    目的 研究K562细胞株及其耐药细胞株K562/A02 NF-κ B活性的差异性表达,探讨白血病多药耐药(MDR)发病机制.方法 用MTT法检测K562/A02的耐药倍数,观察细胞生长的形态学变化,PI单染法检测K562/S、K562/A02细胞周期分布,并用RT-PCR方法榆测mRNA的表达、流式细胞仪检测P糖蛋白(P-gp)表达及其功能,Western blotting方法检测细胞核NF-κB p65表达,比较K562K562/A02白血病耐药细胞株之间的差异性.结果 与K562细胞不同,耐药细胞株K562/A02细胞生长特性发生改变,旱半贴壁生长,与K562/S细胞相比,K562/A02细胞的G0/G1期、S期比例增高,G2/M期细胞比例减低,差异有统计学意义(P<0.05),凋亡细胞比例差异无统计学意义(P>0.05),且检测到mdrl mRNA及细胞膜P-gp表达增高以及细胞核内NF-κB p65表达明显增加.结论 NF-κ B信号通路的激活即NF-κ B p65细胞核内转位可能参与了白血病MDR的形成.%Objective To explore the different expression of NF-κB in both K562 and its multidrug resistant cell line K562/A02 and discuss the mechanism of muhidrug resistance(MDR). Methods To detect the growing feature of the cells. Flow cytometry was used to analys the difference between the distribution profile of K562/S and K562/A02 cell. MTT colorimetry was used to determine the cytotoxic effect of adramycin, and expression of mdrl gene was detected by semi-quantitative reverse transcriptase poly-merase chain reaction (RT-PCR) in K562 and K562/A02 cells. FACS was used to determine the expression and function of glycoprotein (P-gp) on the cell membrane. Western blotting was used to determine the NF-κB p65protein in nueleus. Results There was a difference between K562 and K562/A02 cells growed in a halfadherent way rather than suspending ones, there were increases in the percentage number of cells at G0/G1 and S phases(P <0.05). This was mirrored by a decreasing number of cells within the G2/M phase(P<0.05). Butthere was

  16. Up-regulation of NKG2D ligand ULBP2 by matrine in K562 cells and the underlying molecular mechanisms

    Institute of Scientific and Technical Information of China (English)

    马玲娣

    2014-01-01

    Objective To probe matrine acting on natural killer cell(NK)activating receptor NKG2D ligands expression in CML cell line K562 and its underlying molecular mechanism.Methods The expression of NKG2D ligands(major histocompatibility complex class I chain-related molecule A or B(MICA/B),UL16-binding proteins(ULBP)1,2,and 3 on K562 cells were analyzed before and after treated with matrine by FCM.The cytotoxic sensitivity of K562 to NK cell was detected by FCM after

  17. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  18. Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines.

    Science.gov (United States)

    Ebrahimnezhad Darzi, Salimeh; Amirghofran, Zahra

    2013-06-01

    Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50 µg/ml on Jurkat (85.66 ± 4.9%) and K562 cells (65.04 ± 0.93%) at 24 h after treatment (p officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells.

  19. [Influence of polypeptide extract from scorpion venom on PI3K and p-Akt signaling protein expression and cell proliferation of K562 cells].

    Science.gov (United States)

    Yu, Wen-Jun; Yang, Wen-Hua; Yang, Xiang-Dong; Shi, Zhe-Xin; Wang, Xing-Li; Hao, Zheng; Zhang, Jia

    2012-08-01

    This study was aimed to investigate the effect of polypeptide extract from scorpion venom (PESV) on PI3K, p-Akt signal protein regulating K562 cell apoptosis and its mechanism. The K562 cells were cultured with PESV for different time, the cell growth curve was determined by MTT method, the levels of PI3K and p-Akt proteins were detected by Western blot. The results showed that as compared with control group, the apoptosis rate of K562 cells treated with PESV increased, the levels of PI3K and p-Akt expression decreased. It is concluded that the PESV inhibits the proliferation and promotes the apoptosis of K562 cells probably through suppressing the expression of PI3K and p-Akt signal proteins.

  20. Effects of K562 cells with over-expression of MHC class Ⅰ chain-related protein A on phagocytosis of dendritic cells%K562细胞过表达MHCⅠ类相关抗原A对树突状细胞吞噬功能的影响

    Institute of Scientific and Technical Information of China (English)

    张跃; 邵小青; 陈贝; 季明春; 龚卫娟

    2012-01-01

    Objective To observe how apoptosed K562 cells with over-expression of MHC class I chain-related protein A (MICA) affects phagocytic function of dendritic cells. Methods At first a K562 cell line with ectopic MICA expression, called K562-MICA, was generated by gene trans-fection and G418 screen. Both K562 and K562-MICA cells were stained with fluorescent CFSE, treated with mitomycin C, and then co-cultured with THP1 cells or dendritic cells derived from peripheral blood monocytes overnight. Phagocytic activities were evaluated through detection of percentage of ap-optotic cells by flow cytometry. Meanwhile, some activating receptors on THP1 cells and NKG2D expression on DC were measured by flow cytometry. Finally NKG2D neutralizing antibody was added to cell co-culture system to observe whether phagocytosis of DC would be varied correspondingly. Results K562-MICA cells stimulated THP1 cell to enhance expression of CD86 and MICA, but had no effects on HLA-DR and NKG2D expression. Compared with K562 cells, apoptotic bodies from K562-MICA cells were more susceptible to be uptake by DC. Apoptosed K562-MICA cells induced DC to increase NKG2D expression. In addition, NKG2D antibody could significantly inhibit phagocytosis of DC. Conclusion MICA over-expression on K562 cells promoted phagocytic function of DC, and the function depended on NKG2D expression on DC.%目的 观察过表达MHC Ⅰ类相关抗原A(MICA)的K562细胞,体外经诱导凋亡后,对树突状细胞(DC)吞噬功能的影响.方法 首先利用脂质体介导的基因转染技术和G418筛选过程,建立稳定表达MICA的K562细胞(K562-MICA).其次分别取K562K562-MICA细胞经CFSE标记,并用丝裂霉素C诱导凋亡,与单核细胞系THP1或外周血单核细胞来源的DC共孵育过夜,流式细胞术检测2种细胞吞噬凋亡小体的活性.同时检测THPI细胞表面相关活化性受体的表达,以及DC表面NKG2D受体的情况.最后,在细胞共培养体系中加入NKG2D抗体,观

  1. The best time of cytotoxicity for extracted cell wall from Lactobacillus casei and paracasei in K562 cell line

    Directory of Open Access Journals (Sweden)

    Riki M

    2013-02-01

    Full Text Available Background: The aim of this study was to evaluate the effect of extracted cell walls from Lactobacillus casei and Lactobacillus paracasei as probiotic bacteria (isolated from common carp intestine on K562 and the role of cell concentration on the results of MTT [3-(4,5-Dimethylthiazol-2-yl2,5- Diphenyl tetrazolium Bromide] test.Methods: For this purpose, bacteria were cultured in specific medium (MRS broth at anaerobic condition for 24-48 hour. After incubation period culture medium was centri-fuged, then the cells were washed twice with PBS buffer to remove additional medium. Finally, collected bacterial cell disrupted by Sonication and cell walls were separated from other components by centrifugation. After that, different concentrations of cell walls (500, 1000, 2000 and 4000 µg/ml were prepared in RPMI medium for each bacteria, separately. Then anticancer properties of the cell walls were determined in vitro at 12, 24, 48 and 72 h, also the effect of K562 concentration was assayed with MTT technique.Results: The results showed extracted cell wall from both probiotic statistically (P=0.098 have anti turmeric properties in K562 and their properties will arise in relation with concentration. As well as, we found that the number of cell had not any affect on the result of MTT assay.Conclusion: We conclude that the cytotoxicity property of extracted cell wall is related in the type of bacteria, but this anticancer property would warrant further study on the clinical application of extracted cell wall.

  2. PESV对K562细胞BCR/ABL融合基因及凋亡调控因子Bcl-2、Bad表达的影响%The Effects of PESV on the Expression of BCR/ABL Fusion Gene and Bcl-2, Bad of Apoptosis Regulators on the K562 Cells

    Institute of Scientific and Technical Information of China (English)

    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    Objective: To investigate the PESV of K562 cells BCR / ABL fusion gene and apoptosis regulators bcl-2 and bad expression. Methods: K562 cells were cultured in vitro, by PESV for different times, the apoptosis rate by flow cytometry, fluorescence quantitative RT-PCR detection of BCR / ABL, Bcl-2, Bad mRNA level changes. Results: Compared with the control group, PESV treated K562 cells, apoptosis increased, BCR / ABL fusion gene reduced expression, anti-apoptotic gene Bcl-2 mRNA expression decreased, pro-apoptotic gene Bad mRNA expression. Conclusion: PESV reduced in K562 cells can reduce the BCR / ABL fusion gene, may regulate the expression of Bcl-2 and Bad, inhibit proliferation of K562 cells and promote their apoptosis.%目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.

  3. Effects of phenolic alkaloids from Menispermum dauricum on inducing the multidrug resistance cell line K562/MDR1 to apoptosis and reversing their multidrug resistance%蝙蝠葛酚性碱诱导多药耐药细胞系K562/MDR1凋亡及逆转耐药性的研究

    Institute of Scientific and Technical Information of China (English)

    何志一; 刘相辉; 刚宏林

    2010-01-01

    目的:探讨蝙蝠葛酚性碱(phenolic alkaloids from Menispermum dauricum,PAMD)诱导多药耐药(multidrug resistance,MDR)细胞系K562/MDR1凋亡及逆转耐药性的作用.方法:四甲基偶氮唑蓝(MTT)比色法检测K562/S及K562/MDR1细胞对不同浓度PAMD的敏感性,并计算半数抑制浓度(IC50).膜联蛋白V (Annexin V)-异硫氰酸荧光素(FITC)+碘化丙啶(PI)双参数检测细胞凋亡百分率变化,分析在PAMD的作用下,两种细胞对伊马替尼(IM,STI571)敏感性的变化.结果:PAMD可诱导两种细胞凋亡,其低剂量72 h时对K562/S和K562/MDR1细胞的凋亡率分别为(10.92±1.03)%和(8.12±0.98)%,并可提高伊马替尼对K562/MDR1的凋亡率.PAMD可显著逆转K562/MDR1细胞对伊马替尼的耐药性,其逆转倍数为2.22.结论:PAMD对K562/S和K562/MDR1细胞具有诱导凋亡作用,同时具有逆转白血病细胞株K562/MDR1多药耐药性、回归靶位的作用.

  4. Antitumor activity of 6-(cyclohexylamino)-1, 3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione and Its Ti(IV), Zn(II), Fe(III), and Pd(II) complexes on K562 and Jurkat cell lines.

    Science.gov (United States)

    Shabani, Fahmideh; Ghammamy, Shahriar; Mehrani, Khayroallah; Teimouri, Mohammad Bagher; Soleimani, Masoud; Kaviani, Saeid

    2008-01-01

    (6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione) abbreviated as CDP was synthesized and characterized. Ti(IV), Zn(II), Fe(III), and Pd(II) metal complexes of this ligand are prepared by the reaction of salts of Ti(IV), Zn(II), Fe(III), and Pd(II) with CDP in acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR, (1)H NMR, (13)C NMR, and UV-Visible spectroscopy. All complexes were characterized by several techniques using elemental analysis (C, H, N), FT-IR, electronic spectra, and molar conductance measurements. The elemental analysis data suggest the stoichiometry to be 1:1 [M:L] ratio formation. The molar conductance measurements reveal the presence of 1:1 electrolytic nature complexes. These new complexes showed excellent antitumor activity against two kinds of cancer cells that are K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells.

  5. Phytochemicals and Cytotoxicity of Launaea procumbens on Human Cancer Cell Lines

    Science.gov (United States)

    Rawat, Preeti; Saroj, Lokesh M.; Kumar, Anil; Singh, Tryambak D.; Tewari, SK.; Pal, Mahesh

    2016-01-01

    Background: The plant Launaea procumbens belongs to the family Asteraceae and traditionally used in the treatment rheumatism, kidney, liver dysfunctions and eye diseases. In the present study Phytochemical analysis and fractions of methanolic extract of L. procumbens leaves were tested in vitro for their cytotoxicity. Objectives: Phytochemical analysis and cytotoxic activity of methanolic extract and fractions of Launaea procumbens against four cancer cell lines K562, HeLa, MIA-Pa-Ca-2 and MCF-2 by SRB assay. Materials and Methods: Powdered leaves of Launaea procumbens were extracted sequentially with hexane, ethyl acetate, butanol and water by cold extraction. Phytochemical analysis and cytotoxicity assay were carried out for these fractions using SRB assay against four human cancer cell lines, namely leukemia (K562), cervix (HeLa), pancreatic (MIA-Pa-Ca-2) and breast (MCF-7). Results: Ethyl acetate extract exerts potent cytotoxicity against human leukemia (K562), cervix (HeLa) and breast (MCF-7) cell lines IC50 value of 25.30±0.50, 19.80±0.10 and 36.90±4.90 μg/ml respectively. Moderately cytotoxic effect found in hexane extract IC50 value of 41±8 and 48.20±0.50 μg/ml against leukemia (K562), and breast (MCF-7) cancer cell line respectively. The Chemical composition analyzed by GC-MS showed considerable differences in solvent fractions of Launaea procumbens. Conclusion: This study revealed the cytotoxic potential of ethyl acetate and hexane fractions of L. procumbens leaves on different cancer cell lines. SUMMARY Ethyl acetate and Hexane fractions of Launaea procumbens plant exhibit cytotoxicity. Among the different fractions Ethyl acetate showed relatively higher cytotoxicity.Ethyl acetate found more cytotoxic against leukemia (K 562), cervix (HeLa) and breast (MCF-7) cancer cell lines. Moderete cytotoxicity found in hexane fraction against leukemia (K 562) and breast (MCF-7) cancer cell line.GC-MS results showed L. procumbens is a rich source of 1-H

  6. Isolation and purification of antibacterial peptides with anti- K562 activity from the Tenebrio molitor Linnaeus larvae%黄粉虫幼虫抗肿瘤细胞K562抗菌肽的分离纯化

    Institute of Scientific and Technical Information of China (English)

    刘颜岗; 程璟侠; 赵瑞君; 樊宏英

    2009-01-01

    目的 从黄粉虫幼虫体内分离纯化具有抗肿瘤细胞K562的抗菌肽.方法 通过超声诱导黄粉虫幼虫大量表达抗菌肽.然后经过研磨、离心、固相萃取、反相高效液相色谱分离纯化黄粉虫幼虫抗菌肽,采用四甲基偶氮噻唑蓝(MTY)比色法和光镜观察法筛选对K562有杀伤作用的抗菌肽.结果 离心上清液上样固相萃取柱,经10%、30%、80%的乙腈水溶液洗脱,只有80%的乙腈水溶液分离组分有活性(P<0.01).该活性组分经反相高效液相色谱纯化后分离出5个具有抗肿瘤活性的峰物质,这5个峰活性都较强(P<0.01),其中峰9、峰4能初步确定为抗菌肽,其他3种有待于进一步的实验证明.结论 黄粉虫幼虫体内存在抗肿瘤细胞K562的抗菌肽和抗菌物质,而且不止一种.%Objective To isolate and purify the antimicrobial peptides with anti-tumor cell K562 activity from the Tenebrio molitor Linnaeus larvae. Methods Antimicrobial peptides of Tenebrio molitor L.larvae induced by ultrasonic waves were isolated and purified by trituration, centrifugalization, solid phase extraction (SPE) and reversed-phase high-performance liquid chromatography (RP-HPLC). Then the antimicrobial peptides with anti-K562 activity were sieved by MTT colorimetric method and light microscope observation. Results Supematant eluted with 10%, 30%, 80% acetonitrile (ACN) in aqueous solution by solid phase extraction, of which, only 80% was active (P<0.01). Five anti-tumor peaks appeared after purification by RP-HPLC, which all had strong activity (P<0.01). Only 9 and 4 peaks could initially identified as antimicrobial peptides, the others still need to be proved. Conclusion There are antimicmbial peptides and anti-hacterial substances which have anti-KS62 activity in the Tenebrio molitor L.larvae, and more than one.

  7. Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation.

    Science.gov (United States)

    Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes.

  8. siRNA Delivery Improvement by Co-formulation of Different Modified Polymers in Erythroleukemic Cell Line K562

    Directory of Open Access Journals (Sweden)

    Mazdak Ganjalikhani hakemi

    2013-09-01

    Full Text Available Objective(s: siRNA may be a very promising tool for treatment of various diseases especially in cancer therapy due to high specificity. One of the main hurdles applications of siRNAs in vivo is optimization of the delivery strategy, especially the carrier systems. The aim of this study was to optimize siRNA delivery into suspended erythroleukemic cell line K562. Materials and Methods: We applied polyethyleneimine (PEI and oligoethyleneimine (OEI derivatives alone or their co-formulation with different agents such as chloroquine (a drug known to alter lysosomal pH and thus to inhibit lysosomal degradation of macromolecules, DOPE (lipophilic agent, succinic acid (introduction of negatively charged to polymer and transferrin (the ligand of transferring receptor which is over-expressed in many types of tumors and hematopoietic cells. Results: In this study it was shown that utilizing a combination of 70% OEI-HA10 (ten hexyl acrylate residues per one OEI chain plus 30% of transferin-PEI with Luc-siRNA was highly effective for transfecting K562 cell. This co-formulation silenced luciferase activity up to 70% after short time without any significant inhibition in the luciferase activity in siCONTROL wells. Conclusion: In conclusion, the combination of modified PEI with transferrin and OEI by hexyl acrylate may increase siRNA delivery and reduce toxicity in hematopoietic suspended cells.

  9. The biocompatibility of materials used in printed circuit board technologies with respect to primary neuronal and K562 cells.

    Science.gov (United States)

    Mazzuferi, Manuela; Bovolenta, Roberta; Bocchi, Massimo; Braun, Tanja; Bauer, Joerg; Jung, Erik; Iafelice, Bruno; Guerrieri, Roberto; Destro, Federica; Borgatti, Monica; Bianchi, Nicoletta; Simonato, Michele; Gambari, Roberto

    2010-02-01

    Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PCBs are characterized by low production costs and large-scale development, both essential elements in the frame of disposable applications. LOAC platforms have been employed not only for diagnostic and/or analytical purposes, but also for identification and isolation of eukaryotic cells, including cancer and stem cells. Accordingly, the compatibility of the employed materials with the biological system under analysis is critical for the development of LOAC devices to be proposed for efficient and safe cell isolation. In this study, we analyzed the in-vitro compatibility of a large set of materials and surface treatments used for LOAC development and evaluation with quasi-standard PCB processes. Biocompatibility was analyzed on hippocampal primary cells (a model of attached cell cultures), in comparison with the reference K562 cell line (a model of cells growing in suspension). We demonstrate here that some of the materials under study alter survival, organization, morphology and adhesion capacity of hippocampal cells, and inhibit growth and differentiation of K562 cells. Nonetheless, a subset of the materials tested did not negatively affect these functions, thus demonstrating that PCB technology, with some limitations, is suitable for the realization of LOAC devices well compatible at least with these preparations. (c) 2009 Elsevier Ltd. All rights reserved.

  10. Synthesis, Crystal Structure and Inhibition of N-2-Thiophenesulfonyl-α-L-phenylalanine Ethyl Ester on K562 Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The title compound N-2-thiophenesulfonyl-α-L-phenylalanine ethyl ester has been synthesized. Complete assignments were achieved by IR, MS, 1H NMR and single-crystal X-ray diffraction analyses. Using MTT assay, the inhibitory rate of the title compound on K562 cells (chronic myeloid 1eukemic cells) was measured and the result of preliminary bioassay showed that the title compound possesses antiproliferation effects on K562 cells. In order to investigate the relationship between structure and activity of the target compound, we report its crystal structure and biological behavior in the present paper. Crystallographic data: C15H17NO4S2, Mr = 339.42, monoclinic, space group P21, flack = -0.15(12), a = 5.7916(10), b = 11.5078(19), c = 12.924(2) (A), β = 97.781(3)°, Z = 2, V = 853.4(2) (A)3, Dc = 1.321 g/cm3, F(000) = 356, -7≤h≤7, -10≤k≤14, -15≤l≤15, R = 0.0628, wR = 0.1540 and μ(MoKα) = 0.327 mm-1. The molecule comprises a benzene and a thiofuran rings, and the intramolecular N(1)-H(1A)…O(1) makes a five-membered ring of O(1)-C(6)-C(5)-N(1)-H(1A).

  11. A novel erythroid differen tiation related gene EDRF2 inhibited α-globin gene expression in K562 cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In previous studies, we found that EDRF2 was an erythroid differentiation related factor, whose expression was markedly upregulated during erythroid differentiation. It suggested that this factor played a role in erythropoiesis.By using rapid amplification of cDNA ends technology, we cloned EDRF2 gene 5 '- and 3 '-cDNA ends successfully.Transfection and Northern blot analysis demonstrated that EDRF2 inhibited mRNA expression of α-globin gene, while did not regulate γ-globin gene expression. Gel shift assay confirmed that DNA-binding activity of erythroid specific transcription factors GATA-1, NF-E2 and AP1 was not af fected by either forced overexpression or artificial down regulation of EDRF2 gene in K562 cells. However, we de tected that the negative regulator of expression of GATA-1 transcription factor was increased in EDRF2 overexpressed K562 cells. These results strongly suggested that EDRF2 was involved in α-globin gene expression and erythroid differen tiation and served as a negative regulator of PU.1 transcrip tion factor.

  12. Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death.

    Science.gov (United States)

    Grüllich, Carsten; Friske, Viktoria; Finke, Jürgen

    2008-09-01

    Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

  13. Prokaryotic Expression and Purification of sCAR-TSP-1, and Induced Apoptosis in Leukemic Cells k562%融合蛋白sCAR-TSP-1原核表达纯化及诱导白血病细胞K562凋亡的研究

    Institute of Scientific and Technical Information of China (English)

    梁天祥; 谌贺宽子; 陈磊; 武虎; 唐斌

    2012-01-01

    According to thrombospondin-l(TSP-l) amino acid sequence(RFYWMWK), to the ade-novirus receptor sCAR as template, the 8 amino acids corresponding gene nucleotide amplified by PCR from sCAR, sCAR-TSP-1, connected to the expression vector pQE30, is transformed into E. coli M15 obtained after engineering bacteria. The strain induced by IPTG, efficient expression with histidine label in the form of inclusion body of the fusion protein sCAR-TSP-1. Inclusion body after urea denaturation dissolved, PBS dilution refolding, Ni ion affinity chromatography purification, and obtains the target proteia SDS-PAGE analysis shows, there is an obvious specificity protein band. At the same time, the experimental results show that the fusion protein in sCAR-TSP-1 cells of leukemia cell K562 has obvious apoptosis.%根据thrombospondin-1( TSP-1)氨基酸序列(RFYVVMWK),以已有的腺病毒受体sCAR为模板,将8个氨基酸对应基因核苷酸通过PCR扩增于sCAR之后,得到sCAR-TSP-1,连接到表达载体pQE30上,转化大肠杆菌M15后获得工程茵.该菌株经IPTG诱导后,高效表达出带有组氨酸标签以包涵体形式存在的融合蛋白;sARTSP-1.包涵体经过尿素变性溶解、PBS稀释复性、Ni离子亲和层析柱纯化,获得目的蛋白.SDS-PAGE分析表明,有一条明显的特异性蛋白条带.同时细胞实验结果表明融合蛋白sCAR-TSP-1对白血病细胞K562有明显凋亡作用.

  14. Optimization of experimental human leukemia models (review

    Directory of Open Access Journals (Sweden)

    D. D. Pankov

    2012-01-01

    Full Text Available Actual problem of assessing immunotherapy prospects including antigenpecific cell therapy using animal models was covered in this review.Describe the various groups of currently existing animal models and methods of their creating – from different immunodeficient mice to severalvariants of tumor cells engraftment in them. The review addresses the possibility of tumor stem cells studying using mouse models for the leukemia treatment with adoptive cell therapy including WT1. Also issues of human leukemia cells migration and proliferation in a mice withdifferent immunodeficiency degree are discussed. To assess the potential immunotherapy efficacy comparison of immunodeficient mouse model with clinical situation in oncology patients after chemotherapy is proposed.

  15. Optimization of experimental human leukemia models (review

    Directory of Open Access Journals (Sweden)

    D. D. Pankov

    2014-07-01

    Full Text Available Actual problem of assessing immunotherapy prospects including antigenpecific cell therapy using animal models was covered in this review.Describe the various groups of currently existing animal models and methods of their creating – from different immunodeficient mice to severalvariants of tumor cells engraftment in them. The review addresses the possibility of tumor stem cells studying using mouse models for the leukemia treatment with adoptive cell therapy including WT1. Also issues of human leukemia cells migration and proliferation in a mice withdifferent immunodeficiency degree are discussed. To assess the potential immunotherapy efficacy comparison of immunodeficient mouse model with clinical situation in oncology patients after chemotherapy is proposed.

  16. By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, stichoposide D inhibits growth of leukemia xenografts.

    Science.gov (United States)

    Yun, Seong-Hoon; Park, Eun-Seon; Shin, Sung-Won; Ju, Mi-Ha; Han, Jin-Yeong; Jeong, Jin-Sook; Kim, Sung-Hyun; Stonik, Valentin A; Kwak, Jong-Young; Park, Joo-In

    2015-09-29

    Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-β-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia.

  17. Silencing HCCR2 expression inhibits the proliferation of leukemia cells by inducing apoptosis and promoting cell cycle arrest.

    Science.gov (United States)

    Qiao, Shu-Kai; Ren, Han-Yun; Shi, Yong-Jin; Liu, Wei

    2013-12-01

    The human cervical cancer oncogene (HCCR2) has been found to be overexpressed in a variety of human malignant tumors cells, and its function is related to cell cycle progression and survival. However, the molecular mechanisms of action of HCCR2 in leukemia remain unclear. In this study, we used the RNA interference strategy to investigate the effects of HCCR2 knockdown in the K562 leukemia cell line, and to explore the potential mechanisms involved. Following transfection with small interfering RNA (siRNA) targeting HCCR2 (HCCR2-siRNA), we examined the effects of HCCR2 knockdown on cell morphology, cell proliferation, cell cycle progression and apoptosis in K562 cells. Morphological changes were evaluated by Wright-Giemsa staining. Cell cycle progression and apoptosis were measured by flow cytometry. The expression levels of genes related to the cell cycle and apoptosis were detected by quantitative RT-PCR (qRT-PCR) and western blot analysis. HCCR2 expression at the mRNA and protein level was significantly decreased following transfection with plasmids expressing HCCR2-siRNA. Silencing HCCR2 expression significantly suppressed cell proliferation, induced G1 cell cycle arrest and promoted the apoptosis of K562 cells. Additionally, we found that the expression of Bax, p53 and p21 was significantly increased, while Bcl-2 expression was significantly decreased in the HCCR2-siRNA-transfected cells. However, the expression of p27 was not affected. These results suggest that the HCCR2 gene plays an important role in the tumorigenesis of leukemia, thus making it an attractive therapeutic target for acute leukemia.

  18. 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.

  19. YCD/5-FC自杀基因治疗系统对K562B白血病细胞杀伤作用的小鼠体内实验研究%Study on the in vivo killing activity of YCD/5-FC gene therapy system on K562B cells

    Institute of Scientific and Technical Information of China (English)

    张雨生; 王健民; 周虹; 翟勇平

    2002-01-01

    目的了解酵母菌胞嘧啶脱氨酶/5-氟胞嘧啶(YCD/5-FC)系统在体内对转基因高致瘤性K562细胞(K562B细胞)的杀伤效应.方法以高滴度逆转录病毒转染K562B细胞并筛选出阳性转染克隆YCD-K562B;12只SCID小鼠分为治疗及对照组,在小鼠左右两侧近前肢处腹部皮下注射YCD-K562B及K562B细胞,成瘤后治疗组腹腔注射500 mg/kg 5-FC共10 d,对照组腹腔注射生理盐水,观察瘤体相对体积变化及病理变化.结果瘤细胞接种第21天,瘤体相对体积分别为:YCD-K562B+5-FC组2.922±0.581,YCD-K562B+生理盐水组24.434±4.790,K562B+5-FC组22.701±2.350,K562B+生理盐水组24.460±1.670;YCD-K562B+5-FC组与YCD-K562B+生理盐水组相比差异非常显著(P=0.000*!1),K562B+5-FC组及K562B+生理盐水组相比,差异无显著性(P=0.096),表明5-FC对转YCD基因的K562B白血病细胞有明显的杀伤效应,而对未转基因细胞的生长无影响;YCD-K562B+5-FC 组瘤体于瘤细胞接种后第12~第15天(5-FC治疗的第3~第6天)有所缩小(最小的相对体积为0.681),病理检查可见5-FC治疗组瘤体有以小动脉血管为中心的坏死.结论 YCD/5-FC系统在体内对转YCD基因K562B细胞有明显的杀伤效应.

  20. Anti-leukemic activity of Wattakaka volubilis leaf extract against human myeloid leukemia cell lines.

    Science.gov (United States)

    Nandi, Debkumar; Besra, Shila Elizabeth; Vedasiromoni, Joseph Rajan; Giri, Venkatachalam Sesha; Rana, Prince; Jaisankar, Parasuraman

    2012-12-18

    Wattakaka volubilis has been traditionally used in Ayurvedic medicine in India for treatment of several ailments such as bronchial asthma, inflammations, tumors, piles, leucoderma, application to boils, rat bite etc. The present study was designed to investigate anti-leukemic activity of the crude aqueous methanolic extract and to identify active compounds from the leaves of Wattakaka volubilis. The leaves of Wattakaka volubilis were extracted with aqueous methanol. Liquid-liquid fractionation of the crude methanolic extract with different organic solvents was done and the fractions were screened for in vitro anti-leukemic activity using different leukemic cell lines. The active fractions were then subjected to chromatographic separation for isolation of bioactive compounds. Structure of isolated compound was elucidated by spectroscopic methods. The in vitro anti-leukemic activities of different extracts of the leaves and isolated compound WVP were studied in U-937, HL-60 and K-562 cell-lines by using cell count, MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] and DNA laddering assays, flow-cytometric and confocal microscopic techniques. Kaempferol-3-O-[α-l-rhamnopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→6)-O]-β-d-glucopyranoside (WVP) was isolated from crude leaves extract of Wattakaka volubilis. Both the n-butanolic extract (WVB) of Wattakaka volubilis and its isolate WVP were found to be responsible for in vitro anti-leukemic activity. The IC(50) values of WVB were found be 120, 100 and 50(μg/ml) in U937, K562, and HL-60 cell lines, respectively. Whereas, the pure isolate WVP exhibited anti-leukemic activity with IC(50) values of 13.5, 10.8, and 13.2(μg/ml) in U937, K562, and HL-60 cell lines, respectively. The flow-cytometric analysis confirms that the cell cycle arrest occurs at G1 phase in case of U937 and K562 cell lines and G2/M phase in case of HL60 cell lines. Similarly both confocal microsocopic analysis and DNA laddering assay

  1. Effects of Huangqi (Hex) on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells

    Institute of Scientific and Technical Information of China (English)

    Xaio-Dong CHENG; Chun-Hui HOU; Xue-Jun ZHANG; Heng-Yue XIE; Wei-Ying ZHOU; Lei YANG; Shu-Bing ZHANG; Ruo-Lan QIAN

    2004-01-01

    Huangqi(Astragalus membranaceus),a traditional Chinese medicine,has been used to ameliorate side effects of cancer chemotherapy in China.However,little is known about its molecular mechanisms.Here we show that induction ofK562 or HEL cells with 1.5 mg/mi of Huangqi(Hex)(Components extracted from Huangqi)for 3-5 d results in the expression of ?-globin gene in both cell lines and leads to terminal differentiation.Moreover,the apoptosis in HEL cells can be induced by increasing concentration of Huangqi(Hex)to 4.5 mg/ml for 3-5 d.Upregulation ofApaf-1,caspase-3 and acetylcholinesterase(AChE)in HEL cells may playa crucial role in the process of apoptosis.The prospect of inducing expression of adult(β)globin gene and apoptosis selectively in cancer cells is obviously attractive from a therapeutic point of view.

  2. Induction of apoptosis in human leukemia cells through the production of reactive oxygen species and activation of HMOX1 and Noxa by benzene, toluene, and o-xylene.

    Science.gov (United States)

    Sarma, Sailendra Nath; Kim, Youn-Jung; Song, Mee; Ryu, Jae-Chun

    2011-02-27

    Whereas benzene (BZ) is a well-known human carcinogen, toluene (TOL) and o-xylene (o-XY) are not; however, all three compounds are important environmental pollutants. Although BZ, TOL, and o-XY have been shown to induce apoptosis in vitro, their mechanism of toxicity remains unclear. In this study, we sought to identify the apoptotic pathway(s) activated by BZ, TOL, and o-XY in human HL-60 promyelocytic leukemia cells. Cell cycle analysis by propidium iodide (PI) staining and flow cytometric analyses of Annexin V/PI double-stained cells revealed similar patterns of apoptosis following BZ, TOL, and o-XY exposure. Though reactive oxygen species (ROS) production contributes significantly to BZ metabolite-induced apoptotic cell death, we hypothesized that BZ, TOL, and o-XY can themselves trigger ROS production, leading to the activation of apoptotic signaling. Dose-dependent increases in ROS production and significant tail moments were observed in HL-60 cells exposed to all three compounds. Real-time RT-PCR revealed increased HMOX1 and Noxa expression in BZ-, TOL-, and o-XY-treated HL-60 cells, confirming the results of previous microarray analyses. Similar expression profiles were found in human K562 erythromyeloblastoid leukemia cells and human U937 leukemic monocyte lymphoma cells. Pretreatment with the ROS scavenger N-acetyl cysteine decreased the effects of exposure to BZ, TOL, and o-XY. In summary, this study provides useful insights into the mechanism of BZ-, TOL-, and o-XY-induced apoptosis in leukemia cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  3. β-carotene treatment alters the cellular death process in oxidative stress-induced K562 cells.

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    Akçakaya, Handan; Tok, Sabiha; Dal, Fulya; Cinar, Suzan Adin; Nurten, Rustem

    2017-03-01

    Oxidizing agents (e.g., H2 O2 ) cause structural and functional disruptions of molecules by affecting lipids, proteins, and nucleic acids. As a result, cellular mechanisms related to disrupted macro molecules are affected and cell death is induced. Oxidative damage can be prevented at a certain point by antioxidants or the damage can be reversed. In this work, we studied the cellular response against oxidative stress induced by H2 O2 and antioxidant-oxidant (β-carotene-H2 O2 ) interactions in terms of time, concentration, and treatment method (pre-, co-, and post) in K562 cells. We showed that co- or post-treatment with β-carotene did not protect cells from the damage of oxidative stress furthermore co- and post-β-carotene-treated oxidative stress induced cells showed similar results with only H2 O2 treated cells. However, β-carotene pre-treatment prevented oxidative damage induced by H2 O2 at concentrations lower than 1,000 μM compared with only H2 O2 -treated and co- and post-β-carotene-treated oxidative stress-induced cells in terms of studied cellular parameters (mitochondrial membrane potential [Δψm ], cell cycle and apoptosis). Prevention effect of β-carotene pre-treatment was lost at concentrations higher than 1,000 μM H2 O2 (2-10 mM). These findings suggest that β-carotene pre-treatment alters the effects of oxidative damage induced by H2 O2 and cell death processes in K562 cells.

  4. Evaluation of Synergetic Anticancer Activity of Berberine and Curcumin on Different Models of A549, Hep-G2, MCF-7, Jurkat, and K562 Cell Lines

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    Acharya Balakrishna

    2015-01-01

    Full Text Available Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562. All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 104 cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1, Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL. The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells, and blank (only medium. The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models.

  5. Characterization of cells resistant to the potent histone deacetylase inhibitor spiruchostatin B (SP-B) and effect of overexpressed p21waf1/cip1 on the SP-B resistance or susceptibility of human leukemia cells.

    Science.gov (United States)

    Kanno, Syu-Ichi; Maeda, Naoyuki; Tomizawa, Ayako; Yomogida, Shin; Katoh, Tadashi; Ishikawa, Masaaki

    2012-09-01

    We previously showed that the B cell leukemia cell line NALM-6 had the highest susceptibility among a number of leukemia cell lines to spiruchostatin B (SP-B), a potent histone deacetylase (HDAC) inhibitor. We also showed that SP-B-induced cytotoxicity depended on induction of apoptosis that was mediated by p21waf1/cip1 expression. In the present study, we generated and characterized a stable, SP-B-resistant NALM-6 cell line (NALM-6/SP-B) by continuous exposure to SP-B, starting with a low SP-B concentration. NALM-6/SP-B cells were also more resistant to FK228, which has a similar chemical structure to SP-B, and were slightly more resistant to the P-gp substrates doxorubicin and vincristine than parental cells, but displayed similar susceptibility to other HDAC inhibitors and to paclitaxel as the parental cells. There was little change in the basal mRNA expression of HDAC1, p53, Bax, Bcl-2, Fas, caspase-3, c-Myc and MDR1 in NALM-6/SP-B compared to parental cells, but the mRNA expression of p21waf1/cip1 was decreased. The introduction of an exogenous p21waf1/cip1 expression vector restored SP-B induction of NALM-6/SP-B cell apoptosis. Moreover, overexpressed p21waf1/cip1 enhanced SP-B induction of the apoptosis of the human erythroleukemia leukemia cell line K562 which is less susceptible to SP-B than NALM-6 cells. These results suggest that downregulation of p21waf1/cip1, which is a characteristic feature of NALM-6/SP-B cells, was important for their resistance to SP-B, and that this SP-B resistance could be overcome by the introduction of exogenous p21waf1/cip1. Furthermore, introduction of p21waf1/cip1 to other leukemia cells such as K562 may enhance their susceptibility to SP-B. This is the first report of the characterization of SP-B-resistant cells and of the effect of overexpressed p21waf1/cip1 on the resistance or susceptibility of human leukemia cells to SP-B.

  6. Influence of Polypeptide Extract from Scorpion Venom on PI3K and p-Akt Signaling Protein Expression and Cell Proliferation of K562 Cells%蝎毒多肽提取物对K562细胞PI3K和p-Akt信号蛋白表达及细胞增殖的影响

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    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    This study was aimed to investigate the effect of polypeptide extract from scorpion venom (PESV) on PDK, p-Akt signal protein regulating K562 cell apoptosis and its mechanism. The K562 cells were cultured with PESV for different time, the cell growth curve was determined by MTT method, the levels of PDK and p-Akt proteins were detected by Western blot. The results showed that as compared with control group, the apoptosis rate of K562 cells treated with PESV increased, the levels of PDK and p-Akt expression decreased. It is concluded that the PESV inhibits the proliferation and promotes the apoptosis of K562 cells probably through suppressing the expression of PDK and p-Akt signal proteins.%本研究旨在探讨蝎毒多肽提取物(PESV)对人慢性髓系白血病细胞系K562细胞凋亡调控的PI3K和p-Akt信号蛋白的影响及作用机制.将体外培养的K562细胞,经PESV处理不同时间后,通过MTT法检测细胞增殖曲线,Western blot法检测PI3K及p-Akt蛋白水平变化.结果表明,与对照组相比,PESV处理后K562细胞凋亡率增加,PI3K及p-Akt蛋白表达降低.结论:PESV可能通过抑制凋亡调控的PI3K、p-Akt信号蛋白,抑制K562细胞增殖,促进细胞凋亡.

  7. Diatrizoate, Iopromide and Iotrolan Enhanced Cytotoxicity of Daunorubicin in Multidrug Resistant K562/adr Cells: Impaired the Mitochondrial and Inhibited the P-Glycoprotein Function

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    Nitaya S.N. Ayudhya

    2009-01-01

    Full Text Available Multidrug resistance was an obstacle in cancer chemotherapy because the cells decreased their intracellular drug accumulation by energy-dependent compounds efflux pumps such as P-glycoprotein (P-gp. This study observed some iodinated radiographic contrast media, diatrizoate, iopromide and iotrolan affected the cellular energetic state and the kinetics of P-gp in drug-sensitive K562 and drug resistant K562/adr cell lines using spectrophotometer and spectrofluorometer. By colorimetric MTT assay, it was found that contrast media (0-3500 µM had no effect on both K562 and K562/adr cell viabilities, but in co-treatment with daunorubicin (DNR, diatrizoate decreased cell viability in K562/adr cells by decreasing ICso of DNR from 610.7 ±74.5 nM to 360±108.9 nM. The change in cellular energetic state was studied using rhodamine B as a probe to estimate mitochondrial membrane potential (ΔΨm. The results showed that 3500 µM diatrizoate decreased ΔΨm from 162.2±0.3 mV to 86.9±9.9 mV in K562/adr cells. The kinetics of P-gp-mediated efflux of DNR could be reduced by diatrizoate from 0 (no inhibition to 0.65±0.11. This inhibition could be partially prevented in co-incubation with 20 nM concanamycin A or 10 µM cytochalasin B. Among the three molecules, diatrizoate showed the best efficiency. It could be proposed for further studies that diatrizoate could be used as MDR identification or MDR imaging and also acted as MDR sensitizing agent in cancer treatments.

  8. Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

    Science.gov (United States)

    Zhang, Lixia; Li, Yan; Li, Xiaoyan; Zhang, Qing; Qiu, Shaowei; Zhang, Qi; Wang, Min; Xing, Haiyan; Rao, Qing; Tian, Zheng; Tang, Kejing; Wang, Jianxiang; Mi, Yingchang

    2017-09-01

    The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryl iodine]-1, 3-diphenyl-2-thiobarbituric acid (UCF-101) treatment was detected with reverse transcription-quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF-101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF-101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF-101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF-101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF-101, resulting in the upregulation of the WT1 protein level. However; UCF-101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF-101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal-related kinase (ERK1/2) pathway markedly and persistently, but UCF-101

  9. Autonomous growth potential of leukemia blast cells is associated with poor prognosis in human acute leukemias

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    Jakubowski Ann A

    2009-12-01

    Full Text Available Abstract We have described a severe combined immunodeficiency (SCID mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the growth potential of leukemic blasts from 133 patients with acute leukemia, (67 acute lymphoblastic leukemia (ALL and 66 acute myeloid leukemia (AML in the animals after subcutaneous inoculation without conditioning treatment. The blasts displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 leukemias, 45 (33.8% displayed an aggressive growth pattern, 14 (10.5% displayed an indolent growth pattern and 74 (55.6% did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was nearly 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. In addition, we demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.

  10. 地西他滨对K562细胞γ-珠蛋白基因表达及细胞增殖的影响%Effects of Decitabine on γ-globin Gene Expression and Proliferation in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    谢冬梅; 赖永榕; 刘容容; 谭滨彬; 杨高晖

    2012-01-01

    目的 研究地西他滨对K562细胞γ-珠蛋白基因表达及细胞增殖的影响,探讨药物治疗β-地中海贫血的新思路.方法 分别用不同浓度(0 nmol/L、50 nmol/L、100 nmol/L、200 nmol/L、300 nmol/L)地西他滨作用于K562细胞24 h、48 h、72 h,用CCK-8检测药物对K562细胞存活率的影响,采用流式细胞术检测细胞凋亡率,采用实时荧光定量RT-PCR检测γ-珠蛋白基因表达.结果 地西他滨对K562细胞生长有抑制作用,作用后细胞凋亡率增加,γ-珠蛋白基因表达显著提高(P<0.05).结论 地西他滨可以提高K562细胞γ-珠蛋白基因表达.%Objective To investigate the effects of decitabine on γ-globin gene expression( HBG ) and proliferation in K562 cells,and provide a new way for β-thalassemia gene therapy. Methods K562 cells were treated with different concentrations of decitabine( 0 nmol/L,50 nmol/L,100 nmol/L,200 nmol/L,300 nmol/L ) for 24 h,48 h,72 h,respectively. The survival of K562 cells was examined by cell counting kit-8( CCK8 ),the Annexin V +/PI- cells were detected by flow cytometry( FCM ). Real-time quantitative reverse transcription polymerase chain reaction( RT-PCR ) was applied to detect the expression of γ-globin gene. Results K562 cells were inhibited by decitabine,and the significant increase in apoptosis and expression of γ-globin gene was found( P < 0.05 ). Conclusion Decitabine can improve γ-globin gene expression in K562cell.

  11. Effect of Thalidomide on Apoptosis of K562 Cells and its Vascular Endothelial Growth Factor Secretion%不同浓度沙利度胺对K562细胞凋亡及血管内皮生长因子分泌的影响

    Institute of Scientific and Technical Information of China (English)

    张玉高; 韩丽英; 陈枫; 赵华

    2011-01-01

    Objective To investigate the effect of thalidomide on apoptosis of k562 cells and its vascular endothelial growth factor secretion. Methods K562 cells were cultured in vitro with 0. 5, 1.0. And 2. 0 mmol/L thalidomide for 24, 48, 72, and 96 hours. Morphology of the K562 cells was observed by the Wright-Giemsa staining method. Methylthiazolyl tetrozolium (MTT) assay was used to determine the cell growth. The rate of apoptosis was analyzed by flow cytometry (FCM) with annexin V-fluorescein isothiocyanate/propidium iodide (AnnexinV-FITC/PI) double-staining method. Agarose gel electrophoresis was used to detected Deoxyribonucleic acid Ladder (DNA Ladder). The concentration of VEGF was quantified by the enzyme-linked immunosorbent assay (ELISA). Results Cultured for 24 or 48 hours, thalidomide had no effect on the proliferation of the K562 cells. But after cultured for 72 hours, thalidomide began to inhibit the growth of the K562 cells at the concentration of 1. 0 and 2. 0 mmol/L (P< 0. 001). After cultured for 96 hours, the proliferation of the K562 cells was inhibited too at the concentration of 0. 5 mmol/L thalidomide (P<0. 001). Thus, thalidomide inhibited the growth of the K562 cells with a dose-and time-dependent manner to some extent. After exposure to thalidomide for 72 hours, K562 cells underwent typical morphological changes of apoptosis such as vaculization, the budding of cytoplasm, chromatin condensation, margination, shrunken nucleus and apoptotic body. The results of flow cytometry showed that thalidomide could obviously increase the rates of the apoptosis of K562 cells (P<0. 001). After treated with thalidomide for 72 hours,DNA was extracted for Agarose gel electrophoresis and typical DNA ladder strips were observed. The secretion of VEGF was inhibited when exposure to thalidomide for 48 hours(P<0. 001), and there was negative correlation between VEGF.concentrations and apoptotic rates(r=- 0. 789). Conclusions Thalidomide could inhibite the growth of the

  12. PESV对K562细胞PI3K、p-Akt通路抑制作用的研究

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    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    目的 探讨PESV对人慢性粒细胞白血病细胞系K562细胞凋亡调控的PI3K、p - Akt信号蛋白的影响及作用机制方法 将体外培养K562细胞,经PESV处理不同时间后,通过MTT检测细胞生长曲线,Western blot检测PI3K及p- Akt蛋白水平变化.结果 与对照组相比,PESV处理后K562细胞,凋亡率增加,PI3K及p- Akt表达降低.结论 PESV可能通过抑制凋亡调控的PI3K、p- Akt信号蛋白,从而达到抑制K562细胞系增殖,促进细胞凋亡的作用.

  13. Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias

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    Fang Hongbo; Liu Jing; Guo Dan; Liu Peixiang; Zhao Yongliang

    2014-01-01

    Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.

  14. Upregulation and activation of caspase-3 or caspase-8 and elevation of intracellular free calcium mediated apoptosis of indomethacin-induced K562 cells

    Institute of Scientific and Technical Information of China (English)

    张广森; 周光飚; 戴崇文

    2004-01-01

    Background A nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell opoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis.Methods K562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L, 800 μmol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/ Am probe labeling combined with LSCM. Results Indomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400-800 μmol/L). Western blot results showed upregrulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis.Conclusions Activation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.

  15. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Majuri, R. (Minerva Foundation Institute for Medical Research, Helsinki (Finland))

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of {sup 35}S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with {sup 35}S. The same two bands were observed if the cell surface proteins were labeled with {sup 125}I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author).

  16. 人参皂苷对K562细胞增殖与分化相关基因表达的影响%The effect of ginsenoside on mRNA expression of proliferation and differentiationrelated genes in K562 cells

    Institute of Scientific and Technical Information of China (English)

    危建安; 胡永珍; 韩凌

    2011-01-01

    Objective To explore the potential mechanisms of ginsenosides on K562 cell proliferation and differentiation. Methods Relative quantification RT-PCR with SYBR Green dye was used to determine the expression of Lif, Ccndl, Mdm2, Erbb2 ,p53 and myc mRNA in K562 cells treated with ginsenoside (20 μg/mL) at 36 h. Results mRNA expression of Lif was significantly up-regulated(P < 0.05 ), and that of myc was markedly down-regulated (P < 0.05) in K562 cells when treated with traginsenoside。 mRNA levels of Mdm2,Erbb2 and Ccndl were not significantly changed after treatment of ginsenoside (P > 0. 05 ). mRNA expression of p53 was very weak in K562 cells, so that was not statistically analysed. Conclusion The effect of ginsenoside on proliferative inhibition and differential induction of K562 cells is related to up-regulation of Lif mRNA and down - regulation of myc mRNA respectively.%目的 探讨人参皂苷影响K562细胞增殖、分化的可能分子机制.方法 采用real-time PCR技术和SYBR Green染料法检测人参皂苷(20 μg/mL)作用于K562细胞36 h时间点Lif、Ccnd1、Mdm2、Erbb2、p53和myc等6个基因mRNA相对表达水平.结果 人参皂苷能显著促进K562细胞Lif mRNA的表达(P<0.05),对myc mRNA表达有显著抑制作用(P<0.05),对Mdm2、Erbb2和Ccnd1等mRNA表达无显著影响.结论 人参皂苷对K562细胞的诱导分化和增殖抑制作用可能分别与Lif mRNA表达上调和mycmRNA表达下调相关.

  17. 苦参碱对K562细胞端粒酶hTERT-mRNA表达及其酶活性影响作用的研究%Effects of Matrine on hTERT-mRNA Expression and Telomerase Activity in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    李旭芬; 张苏展; 郑树; 张行

    2001-01-01

    目的:观察苦参碱对K562细胞hTERT-mRNA表达及端粒酶活性的影响作用,并明确两者的相关性。方法:以0.1~2mg/ml苦参碱处理K562细胞48h后,RT-PCR检测其hTERT-mRNA表达,同时行TRAP-PCR-ELISA端粒酶活性检测。结果:K562细胞为一强端粒酶活性细胞株,在浓度分别为0.1、05、10、20mg/ml苦参碱作用后,K562细胞hTERT-mRNA表达明显受抑,同时伴有端粒酶活性下降,抑制率分别为0.%、13.%、91.7%和98.6%。结论:苦参碱可降低K562细胞hTERT-mRNA表达,同时伴端粒酶活性下降;端粒酶活性强度与hTERT-mRNA表达有关。%Objective:This study was designed to investigate the effects of matrine on hTERT-mRNA expression and telomerase activity in K562 cells and their relationship. Methods: The hTERT-mRNA expression was determined by RT-PCR assay in untreated and matrine-treated K562 cells, and telomerase activity was analyzed by TRAP-PCR-ELISA assay. Results: The hTERT-mRNA expression of K562 cells was significantly inhibited when treatment with matrine, while telomerase activity was decreased. Conclusion: Matrine can inhibit the hTERT-mRNA expression and telomerase activity of K562 cells. Telomerase activity might be related to the expression of hTERT-mRNA.

  18. 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

  19. Activity of ladanein on leukemia cell lines and its occurrence in Marrubium vulgare.

    Science.gov (United States)

    Alkhatib, Racha; Joha, Sami; Cheok, Meyling; Roumy, Vincent; Idziorek, Thierry; Preudhomme, Claude; Quesnel, Bruno; Sahpaz, Sevser; Bailleul, François; Hennebelle, Thierry

    2010-01-01

    Three methoxylated flavones isolated from Marrubium peregrinum - ladanein, scutellarein-5,7,4'-trimethyl ether, and scutellarein-5,6,7,4'-tetramethyl ether - were assayed for their cytotoxicity towards a recently developed dasatinib-resistant murine leukemia cell line (DA1-3b/M2 (BCR-ABL)), together with the structurally related non-methylated flavone scutellarein. The most active compound, ladanein, was looked for in 20 common Lamiaceae species by a quick HPLC screening. Among the possible positive results, the most interesting source was found to be Marrubium vulgare, which led to the isolation and identification of ladanein for the first time in this species. Ladanein also displayed moderate (20-40 microM) activities against K562, K562R (imatinib-resistant), and 697 human leukemia cell lines but was toxic neither to MOLM13 nor to human peripheral blood mononuclear cells. This work provides a common natural source for the hemi-synthesis of future ladanein-derived flavones and the study of their antileukemic activity.

  20. H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

    Science.gov (United States)

    Dong, Yaqiong; Xiong, Min; Duan, Lianning; Liu, Ze; Niu, Tianhui; Luo, Yuan; Wu, Xinpin; Xu, Chengshan; Lu, Chengrong

    2014-08-01

    Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

  1. Effect of magnetic nanoparticles of Fe3O4 and 5-bromotetrandrine on reversal of multidrug resistance in K562/A02 leukemic cells

    Directory of Open Access Journals (Sweden)

    Jian Cheng

    2009-09-01

    Full Text Available Jian Cheng1*, Weiwei Wu1*, Bao-an Chen1, Feng Gao1, Wenlin Xu2, Chong Gao1, Jiahua Ding1, Yunyu Sun1, Huihui Song1, Wen Bao1, Xinchen Sun3, Cuirong Xu1, Wenji Chen1, Ningna Chen1, Lijie Liu4, Guohua Xia1, Xiaomao Li5, Xuemei Wang61Department of Hematology, 3Department of Oncology, The Afiliated Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China; 2Department of Hematology, The First People’s Hospital of Zhengjiang, Zhenjiang, People’s Republic of China; 4Institution of Physiology, 6State Key Lab of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, People’s Republic of China; 5Department of Physics, University of Saarland, Saarbruechen, Germany; *These authors have contributed equally to this workAbstract: This study aims to evaluate the multidrug resistance (MDR reversal activity by magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4 and 5-bromotetrandrine (BrTet MDR cell line K562/A02 solitarily or symphysially. The proliferation of K562 and K562/A02 cells and the cytotoxicity on peripheral blood mononuclear cells (PMBCs were evaluated by MTT assay. Cellular accumulation of daunorubicin (DNR was analyzed by flow cytometry. Real-time polymerase chain reaction and Western blotting analyses were performed to examine the mRNA and protein levels of mdr1, respectively. The results showed that the combination of MNPs-Fe3O4 and BrTet with effective concentrations significantly increased cytotoxicity against MDR cell line K562/A02. Both BrTet and MNPs-Fe3O4 increased the intracellular DNR accumulation in the K562/A02 cell line, and downregulated the level of mdr1 gene and expression of P-glycoprotein. Furthermore, the combination did not have significant cytotoxicity in PMBCs. We propose that MNPs-Fe3O4 conjugated with DNR and BrTet probably have synergetic effects on MDR reversal.Keywords: magnetic nanoparticles of Fe3O4, 5-bromotetrandrine, multidrug resistance K562/A02

  2. Carbon monoxide induced erythroid differentiation of K562 cells mimics the central macrophage milieu in erythroblastic islands.

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    Shlomi Toobiak

    Full Text Available Growing evidence supports the role of erythroblastic islands (EI as microenvironmental niches within bone marrow (BM, where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule--CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N₂A served as control for CO atmosphere (COA. Under both atmospheres cells proliferation ceased: in N₂A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N₂A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor's population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.

  3. PESV干预K562细胞PI3K/Akt信号通路凋亡调控的研究%Study on PI3K/Akt messenger passage and apoptosis controlling in PESV-preconditioned K562 cells

    Institute of Scientific and Technical Information of China (English)

    张蕾; 杨文华; 于文俊; 郝征; 张佳

    2012-01-01

    [Objective] To investigate the effect of PESV on the expression of massage protein PI3K / Akt, apoptosis regulators Bcl-2 and Bad expression in K562 cells. [Methods] In vitro cultured K562 cells preconditioned with PESV for different times were determined for cell apoptosis rate with cytoflowmeter, the level of P13K and p-AKT protein with Western blot and Bcl-2, Bad mRNA level by realtime fluorescence quantitative RT-PCR. [Results] Compared with the control group, the apoptosis rate of PESV treated K562 cells was significantly increaseds, the expression of PI3K and p-Akt and anti-apoptosis-related gene Bcl-2 mRNA was decreased; the expression of pro-apoptotic gene Bad mRNA was increased. [Conclusion] PESV may regulate the expression of Bcl-2 and Bad, inhibit the proliferation of K562 cells and promote their apoptosis by reducing the expression of PI3K and Akt signaling protein.%[目的]探讨PESV对K562细胞PI3K/Akt信号蛋白及凋亡调控因子Bcl-2和Bad表达的影响.[方法]将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,Western blot检测PI3K及p-Akt蛋白水平变化,实时荧光定量RT-PCR检测Bcl-2、Bad mRNA水平变化.[结果]与对照组相比,PESV处理后K562细胞凋亡率显著增加,PI3K及p-Akt表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.[结论]PESV可能通过降低PI3K、Akt信号蛋白表达,调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.

  4. Experimental study on K562 cell apoptosis induced by steroid saponins extracted from the flowers of Cestrum nocturnum Linn%夜香树花甾体皂苷诱导K562细胞凋亡机制研究

    Institute of Scientific and Technical Information of China (English)

    赵世元; 黄之虎; 叶海洪; 钟振国; 张明艳; 李彩萍

    2013-01-01

    目的 研究夜香树花甾体皂苷对人白血病细胞K562的增殖抑制作用和凋亡诱导影响.方法 用MTT法检测夜香树花甾体皂苷对K562细胞的增殖抑制作用;Wright's染色观察细胞形态学改变;应用流式细胞仪检测细胞周期和凋亡率.Annexin V/PI双标记法、TUNEL法检测细胞凋亡特点;采用Western blotting法检测Caspase-3、线粒体和胞浆中细胞色素C表达.结果 夜香树花甾体皂苷对K562细胞生长具有显著的抑制作用,并呈剂量-时间依赖性,夜香树花甾体皂苷可阻滞K562细胞周期于G0/G1期,经夜香树花甾体皂苷处理后的K562细胞Annexin V+/P-细胞显著增加,TUNEL细胞涂片可见强烈荧光效应.夜香树花甾体皂苷可诱导K562细胞线粒体细胞色素C释放和Caspase-3活化.结论 夜香树花甾体皂苷可有效抑制K562细胞增殖,诱导K562细胞凋亡,与作用剂量和作用时间呈正相关,这一过程可能与夜香树花甾体皂苷损伤线粒体和激活Caspase-3有关.

  5. 人参多糖对人白血病细胞株K562细胞信号转导的调控%Effect of GPS on JAK2-STAT5, NF-κB signal transduction pathways in K562 cells

    Institute of Scientific and Technical Information of China (English)

    谢冰松; 李静; 陈地龙; 王亚平; 王建伟

    2009-01-01

    目的:研究人参多糖(GPS)对人红白血病细胞株(K562)信号转导的调控.方法:采用细胞体外培养技术,免疫细胞化学、Western Blot检测GPS诱导后K562细胞蛋白酪氨酸激酶JAK2、核因子NF-κB p65和信号传导及转录激活因子5(STAT5)的表达,免疫沉淀法检测GPS对K562细胞JAK2磷酸化的影响,RT-PCB技术检测GPS诱导后K562细胞c-myc,p53表达的变化.结果:免疫细胞化学和Western Blot检测GPS诱导后K562细胞,JAK2蛋白表达明显增强,随着剂量的加大、时间的延长更加明显[(12.33±2.05)vs(88.18±13.57)];NF-κB p65[(79.48±9.56)vs(6.98±1.45)]和STAT5[(71.56±16.28)vs(10.23±2.16)]蛋白表达明显减弱;GPs(40 mg/L)作用K562细胞6 d后,加入GM-CSF继续刺激5~10 min,JAK2酪氨酸磷酸化增强,而刺激20 min后JAK2酪氨酸磷酸化明显减弱.GPS(40 mg/L)诱导K562细胞3,6 d后.c-myc mRNA明显减弱[(196±12)vs(136±11)],而p53 mRNA无明显变化[(202±16)vs(181±16)].结论:GPS激活了酪氨酸蛋白激酶JAK2,促进其磷酸化,同时抑制STAT5,NF-κB信号转导途径,下调原癌基因c-myc表达,促进K562细胞凋亡与分化.

  6. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    Directory of Open Access Journals (Sweden)

    Mignogna Giuseppina

    2006-03-01

    Full Text Available Abstract In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively.

  7. SENYAWA BIOAKTIF RIMPANG JAHE (Zingiber officinale Roscue MENINGKATKAN RESPON SITOLITIK SEL NK TERHADAP SEL KANKER DARAH K-562 IN VITRO [Ginger Root Bioactive Compounds Increased Cytolitic Response of Natural Killer (NK Cells Against Leucemic Cell Line K-562 In Vitro

    Directory of Open Access Journals (Sweden)

    Fransiska Rungkat Zakaria 2

    2006-08-01

    Full Text Available Natural killer (NK cell, a kind of lymphocyte cells, plays an important role in attacking infectious, immature, and cancer cell. Its function could be modulated by food bioactive compounds. This experiment was conducted to investigate the effects of ginger root bioactive compounds such as oleoresin, gingerol, and shogaol on cytolitic response of NK cell in vitro. Lymphocyte cells were isolated by centrifugation on ficoll-hypaque density (1,77 ?0,001 g/ml method. Leukemic cells line K-562 as target cells(TC labelled by [3H]-timidin, together with lymphocyte as effector cell (EC were cultured in two ratio levels of EC : TC equal to 1:50 and 1:100, and two culture conditions, for 4 hours, respectively. Paraquate dichloride (1,1-dimethyl-4,4-bipyridilium dichloride 3 mM was used to induce stress oxidative circumstance. Cytolytic capacity of NK cells was determined by percentage of TC lysed by NK cells, in normal and oxidative stress conditions. Statistical analysis showed that the effects of ginger bioactive compounds on cytolytic response of NK cell depended on the culture conditions, as shown by cultures in the presence of oleoresin, and gingerol, but not shogaol. In the lymphocyte culture without stress oxidative, oleoresin, gingerol and shogaol compounds increased significantly cytolytic response of NK cells cultured at a ratio of TC : EC equal to 1:50, with the highest increament of 65 % at oleoresin concentration of 50 ?g/ml. However, in culture at a ratio of TC : EC equals to 1:100, only oleoresin at a concentration of 50 ?g/ml increased significantly cytolytic response of NK cells with the highest increament of 8 %. Shogaol did not affect significantly NK cells cytolytic response. Under stress oxidative conditions, shogaol increased significantly cytolytic response of NK cells cultured at a ratio of TC:EC equal to 1:50, but the highest increament of 56 % , was by oleoresin at concentration of 50 ?g/ml. Meanwhile, oleoresin and gingerol did

  8. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    Science.gov (United States)

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene.

  9. The expression of PML in chronic myeloid leukemia and effect on cell proliferation

    Institute of Scientific and Technical Information of China (English)

    吴洁

    2013-01-01

    Objective To investigate whether PML is expressed differently in chronic myeloid leukemia (CML) patients and healthy controls,then explore the effect of PML on proliferation in leukemia cell lines K562.Methods Realtime PCR was used to detect the PML expression in

  10. Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

    Science.gov (United States)

    Pessoto, Felipe S.; Yokomizo, Cesar H.; Prieto, Tatiana; Fernandes, Cleverton S.; Silva, Alan P.; Kaiser, Carlos R.; Basso, Ernani A.; Nantes, Iseli L.

    2015-01-01

    A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics. PMID:26075034

  11. Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

    Directory of Open Access Journals (Sweden)

    Felipe S. Pessoto

    2015-01-01

    Full Text Available A series of thiosemicarbazone (TSC p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics.

  12. Leukemia

    Science.gov (United States)

    Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, the bone marrow produces abnormal white blood cells. ...

  13. Survivin反义寡核苷酸对5-FU诱导人红白血病细胞系K562凋亡的影响%Survivin antisense oligodeoxy-nucleotid enhances 5-FU- induced apoptosis of leukemic cell line K562

    Institute of Scientific and Technical Information of China (English)

    靳秋月; 王瑞珉; 谢红; 陈立军

    2006-01-01

    [目的]探讨survivin反义寡核苷酸(Antisense Oligodeoxy-nucleotid,ASODN)对5-FU(5-氟尿嘧啶)诱导人红白血病细胞系K562细胞凋亡的影响.[方法]体外培养K562细胞,合成survivin ASODN并经脂质体转染至K562细胞内,MTT法观察survivin ASODN组、5-FU组及5-FU联合survivin ASODN的细胞毒作用,Hoechst33342/PI双荧光染色观察各组细胞核形态,镜下计算各组细胞凋亡率.[结果]400、600、800、1000nmol/L survivin ASODN处理K562细胞44h后,IC50为800 nmol/L;与单独使用survivin ASODN或5-FU相比,5-FU联合800 nmol/L survivin ASODN后细胞生长明显受到抑制(P<0.01);Hoechest33342/PI双荧光染色可观察到survivin ASODN组、5-FU组及5-FU联合survivin ASODN组均出现明显核固缩、凝集等细胞凋亡表现.镜下细胞计数,survivin ASODN组、5-FU组及5-FU联合survivin ASODN组细胞凋亡率分别为54.55%、53.85%、86.70%.[结论]Survivin ASODN可增强K562细胞对5-FU的敏感性.

  14. RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%~ 72.5%) than in supernatant system (33.1%~ 46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

  15. Development of a multi-step leukemogenesis model of MLL-rearranged leukemia using humanized mice.

    Directory of Open Access Journals (Sweden)

    Kunihiko Moriya

    Full Text Available Mixed-lineage-leukemia (MLL fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/- (NOG mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification. We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

  16. Midazolam Induces Cellular Apoptosis in Human Cancer Cells and Inhibits Tumor Growth in Xenograft Mice

    Science.gov (United States)

    Mishra, Siddhartha Kumar; Kang, Ju-Hee; Lee, Chang Woo; Oh, Seung Hyun; Ryu, Jun Sun; Bae, Yun Soo; Kim, Hwan Mook

    2013-01-01

    Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosis-inducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-XL and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties. PMID:24008365

  17. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

    Science.gov (United States)

    Aimola, Idowu A.; Inuwa, Hajiya M.; Nok, Andrew J.; Mamman, Aisha I.; Bieker, James J.

    2017-01-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer PMID:26879870

  18. Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

    Science.gov (United States)

    Ayello, Janet; Hochberg, Jessica; Flower, Allyson; Chu, Yaya; Baxi, Laxmi V; Quish, William; van de Ven, Carmella; Cairo, Mitchell S

    2017-02-01

    Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p cell numbers (p cells to enhance B-NHL targeting in vitro and in vivo. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  19. Antagonistic effects of black tea against gamma radiation-induced oxidative damage to normal lymphocytes in comparison with cancerous K562 cells.

    Science.gov (United States)

    Ghosh, Debjani; Dey, Subrata Kumar; Saha, Chabita

    2014-11-01

    The potential of naturally occurring antioxidants to reduce the cellular oxidative damage induced by ionizing radiation has been studied for more than a decade for their pharmacological application during cancer treatment. It is already known that radioprotective efficacy of phytochemicals might influence various end points of radiation damage. Flavonoids are well-known natural radioprotectors, and their biological effects depend upon their chemical structure. In the present study, radioprotective effect of black tea rich in flavonoids was evaluated against gamma radiation-induced oxidative damage on normal lymphocytes and compared with erythroleukemic K562 cells. Pre-treatment with black tea extract (BTE) significantly reduced radiation-induced loss of cell viability, generation of reactive oxygen species, mitochondrial dysfunction, activation of caspase-3 and apoptosis in normal lymphocytes compared to K562 cells. BTE also regulates the activity of endogenous antioxidant enzymes. The changes in the mRNA expression of bax, bcl2, p53 and Nrf2 were also followed to evaluate regulation of radiation-induced apoptosis by BTE. These findings suggest that black tea may have the potential of a natural radioprotective agent which can be used as adjunct with radiation during cancer treatment.

  20. MAPK/ERK信号通路调节K562细胞中mdr1基因的诱导性表达%The mitogen-activated protein kinase pathway regulates the induced expression of mdr1 gene in K562 cells

    Institute of Scientific and Technical Information of China (English)

    罗文娟; 许文林; 吕旭晶; 邱志远; 陈巧云; 王法春

    2009-01-01

    Objective To investigate the effect of mitogen-activated protein kinase(MAPK)pathway on the transcriptional expression of mdr1 gene induced by doxorubicin ( DOX)and study the transcription regulation of mdr1 gene.Methods K562 cells were treated with DOX(0.01 μg/ml)with the initial concentration of 0.01 μg/ml for 24 hours,then change the culture media without DOX.K562 cells were cultured until the its status wag recovered.Subsequently the cells were treated with DOX(0.02μg/ml)for 24 hours again.The concentration of DOX was increaged until 0.05 μg/ml by following the protocol above.K562 cells were collected at the concentration of 0.01 μg/ml,0.03μg/ml and 0.05μS/ml DOX.Expression of mdr1 gene were examined by reverse transcription-polymerase chain reaction(RT-PCR).Pglycoprotein(P-gP)wag detected by flow cytometry.Western blot wag performed to detect ERK and P-ERk.K562 cells were pretreated with MAPK inhibitor PD98059 for 1 hour.and then DOX was added.RT-PCR and FCM were used to detect the expression of mdr1 mRNA and P-gp.Results When K562 cells were exposured to DOX.the phosphorylation of ERK wag increaged.the mdr1 gene wag highly expressed as well as its corresponding protein P-gp.When the concentration of DOX was 0.05μg/ml,the expression of mdr1 gene and P-gp were increased over 5 fold.When K562 cells were pretreated with MAPK inhibitor PD98059,the expression of mdr1 gene induced by DOX(the concentration was 0.03 μg/ml and 0.05 μg/m1)was effectively inhibited by(74.1±0.11)%and(70.2±0.14)%respectively.Conclusions DOX could induce the expression of mdr1 gene in K562 cells accompanied by the activation of MAPK/ERK pathway.The block of activation of ERK could inhibit the induced expression of mdr1 gene.%目的 观察多柔比星(doxorubicin,DOX)诱导K562细胞mdr1基因表达过程中丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(ERK)信号通路的作用,探讨mdr1基因的转录调控机制.方法

  1. Targeting 6-phosphogluconate dehydrogenase in the oxidative PPP sensitizes leukemia cells to antimalarial agent dihydroartemisinin.

    Science.gov (United States)

    Elf, S; Lin, R; Xia, S; Pan, Y; Shan, C; Wu, S; Lonial, S; Gaddh, M; Arellano, M L; Khoury, H J; Khuri, F R; Lee, B H; Boggon, T J; Fan, J; Chen, J

    2017-01-12

    The oxidative pentose phosphate pathway (PPP) is crucial for cancer cell metabolism and tumor growth. We recently reported that targeting a key oxidative PPP enzyme, 6-phosphogluconate dehydrogenase (6PGD), using our novel small-molecule 6PGD inhibitors Physcion and its derivative S3, shows anticancer effects. Notably, humans with genetic deficiency of either 6PGD or another oxidative PPP enzyme, glucose-6-phosphate dehydrogenase, exhibit non-immune hemolytic anemia upon exposure to aspirin and various antimalarial drugs. Inspired by these clinical observations, we examined the anticancer potential of combined treatment with 6PGD inhibitors and antimalarial drugs. We found that stable knockdown of 6PGD sensitizes leukemia cells to antimalarial agent dihydroartemisinin (DHA). Combined treatment with DHA and Physcion activates AMP-activated protein kinase, leading to synergistic inhibition of human leukemia cell viability. Moreover, our combined therapy synergistically attenuates tumor growth in xenograft nude mice injected with human K562 leukemia cells and cell viability of primary leukemia cells from human patients, but shows minimal toxicity to normal hematopoietic cells in mice as well as red blood cells and mononucleocytes from healthy human donors. Our findings reveal the potential for combined therapy using optimized doses of Physcion and DHA as a novel antileukemia treatment without inducing hemolysis.

  2. Analgesic effects of lappaconitine in leukemia bone pain in a mouse model

    Directory of Open Access Journals (Sweden)

    Xiao-Cui Zhu

    2015-05-01

    Full Text Available Bone pain is a common and severe symptom in cancer patients. The present study employed a mouse model of leukemia bone pain by injection K562 cells into tibia of mouse to evaluate the analgesic effects of lappacontine. Our results showed that the lappaconitine treatment at day 15, 17 and 19 could effectively reduce the spontaneous pain scoring values, restore reduced degree in the inclined-plate test induced by injection of K562 cells, as well as restore paw mechanical withdrawal threshold and paw withdrawal thermal latency induced by injection of K562 cells to the normal levels. Additionally, the molecular mechanisms of lappaconitine’s analgesic effects may be related to affect the expression levels of endogenous opioid system genes (POMC, PENK and MOR, as well as apoptosis-related genes (Xiap, Smac, Bim, NF-κB and p53. Our present results indicated that lappaconitine may become a new analgesic agent for leukemia bone pain management.

  3. β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS.

    Directory of Open Access Journals (Sweden)

    Zhiying Yu

    Full Text Available β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl-β-elemene (DX1, 13-(cis-3,5-dimethyl-1-piperazinyl-β-elemene (DX2, 13-(4-ethyl-1-piperazinyl-β-elemene (DX3, 13-(4-isopropyl-1-piperazinyl-β-elemene (DX4 and 13-piperazinyl-β-elemene (DX5, were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2O(2, a decrease of mitochondrial membrane potential (MMP, and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2O(2 production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2O(2-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP. The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2O(2 which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.

  4. 黄芪多糖和丁酸钠联合用药对K562细胞胎儿血红蛋白合成的作用%Combined Use of Astragalus Polysaccharide with Sodium Butyrate in Induction of Fetal Hemoglobin Synthesis on K562 Cells

    Institute of Scientific and Technical Information of China (English)

    曹祥; 钱新华; 徐梅佳; 陈佳; 郭丽珊

    2011-01-01

    目的 探讨黄芪多糖(APS)和丁酸钠(NaB)联合用药对人K562细胞胎儿血红蛋白(HbF)合成的作用,为临床联合用药治疗β-珠蛋白生成障碍性贫血(β-地贫)提供实验依据.方法 以K562细胞为模型,低剂量APS和NaB联合用药诱导的细胞为实验组,低剂量APS、NaB单药和常规剂量 NaB单药诱导的细胞分别为阳性对照组1、2、3,未加药组细胞为对照组4,采用联苯胺染色和Western blot技术分析药物作用K562细胞96 h后红系分化和HbF表达.结果 1.实验组对细胞生长的抑制作用显著弱于对照组3(P<0.05).2.APS和NaB联合作用K562细胞的最佳剂量组合为APS 2.50 g·L-1+NaB 250 μmol·L-1.3.联苯胺染色结果显示实验组联苯胺染色阳性率于48 h显著升高,96 h达高峰,与各对照组比较差异均有统计学意义(F=966.630,P<0.05),作用可维持至144 h.4.Western blot结果显示实验组和对照组3诱导K562细胞后HbF合成分别增加至对照组4的(1.82±0.16)倍和(1.57±0.08)倍(F=26.569,P<0.05),实验组HbF表达水平显著高于对照组3(P<0.05).结论 APS和 NaB低剂量联合用药诱导HbF表达增强,作用维持时间长,细胞毒性低,有望成为β-地贫的一种新的治疗方案.%Objective To provide an experimental basis for clinical combination therapy of β - thalassemia through investigating the effects of combined use of low - dose astragalus polysaccharide(APS) and sodium butyrate(NaB) in the induction of fetal hemoglobin(HbF)synthesis on K562 cells.Methods K562 cells were chosen as the cell model, cells treated with low - dose APS combined with NaB were taken as the experimental group,while low -dose APS,low -dose NaB and regular-dose NaB were set up as the positive control group 1,2,3 and untreated cells as the control group 4.Benzidine staining and Western blot were used to analyze erythroid differentiation and HbF expression in K562 cells after getting treated with drugs for 96 h.Results 1.The K562 cell inhibiting

  5. Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions.

    Science.gov (United States)

    Finotti, Alessia; Bianchi, Nicoletta; Fabbri, Enrica; Borgatti, Monica; Breveglieri, Giulia; Gasparello, Jessica; Gambari, Roberto

    2015-01-01

    Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.

  6. Inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    李杰

    2012-01-01

    Objective To explore the inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia (CML) cells and its possible mechanism. Methods The effects of rapamycin at various concentrations on cell proliferation of CML cell line K562 cells were analyzed by MTT. The expressions

  7. Experiment list: SRX069159 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available al_provider=ATCC || cell=K562 || cell organism=Human || cell description=leukemia || cell karyotype=cancer || cell lineage=The contin...uous cell line K-562 was established by Lozzio and Lozzi

  8. Experiment list: SRX069222 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available al_provider=ATCC || cell=K562 || cell organism=Human || cell description=leukemia || cell karyotype=cancer || cell lineage=The contin...uous cell line K-562 was established by Lozzio and Lozzi

  9. Experiment list: SRX037116 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available al_provider=ATCC || cell=K562 || cell organism=Human || cell description=leukemia || cell karyotype=cancer || cell lineage=The contin...uous cell line K-562 was established by Lozzio and Lozzi

  10. Identification and functional analysis of acute myeloid leukemia susceptibility associated single nucleotide polymorphisms at non-protein coding regions of RUNX1.

    Science.gov (United States)

    Xu, Xin; Ren, Xiuyu; Wang, Haiying; Zhao, Yao; Yi, Zhengjun; Wang, Kaifeng; Zhang, Shizhuang; Wang, Lin; Samuelson, David J; Hu, Zhenbo

    2016-01-01

    Little is known about the susceptibility to acute myeloid leukemia. We aim to search non-protein coding regions of key hematopoiesis transcription factors for genetic variations associated with acute myeloid leukemia susceptibility. We genotyped SNPs of RUNX1 P1 promoter, P2 promoter, +23 enhancer, intron 5.2 enhancer, PU.1 promoter, CEBPA promoter, and CEBPE promoter from acute myeloid leukemia patients and healthy controls. Rs2249650 and rs2268276 at RUNX1 intron 5.2 enhancer were found to be associated with acute myeloid leukemia susceptibility. Artificial reporters containing different rs2249650 and rs2268276 alleles showed differential activities in the K562 cell line, a human immortalized myeloid leukemia line. Rs2249650 contributes to reporter activities more than rs2268276. Gel shift assay is consistent with the luciferase assay. Supershift assay indicated that one potential binding protein was PU.1. To sum up, rs2268276 and especially rs2249650 may be qualified as new acute myeloid leukemia susceptibility-associated SNPs.

  11. RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Houcai; Yu, Jing; Zhang, Lixia; Xiong, Yuanyuan; Chen, Shuying; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wei, Hui; Rao, Qing; Wang, Min; Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn

    2014-04-18

    Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.

  12. Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights.

    Science.gov (United States)

    Harrison, Nicholas R; Laroche, Fabrice J F; Gutierrez, Alejandro; Feng, Hui

    2016-01-01

    Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

  13. IL-15 improves the cytotoxicity of cytokine-induced killer cells against leukemia cells by upregulating CD3+CD56+ cells and downregulating regulatory T cells as well as IL-35.

    Science.gov (United States)

    Tao, Qianshan; Chen, Tianping; Tao, Lili; Wang, Huiping; Pan, Ying; Xiong, Shudao; Zhai, Zhimin

    2013-01-01

    Cytokine-induced killer (CIK) cells are usually generated from peripheral blood mononuclear cells with the stimulation of IL-2 in vitro. Unlike the conventional IL-2-stimulated CIK cells (IL-2-CIK cells), we investigated the characteristics and potential mechanism of IL-15-stimulated CIK cells (IL-15-CIK cells) in this study. Compared with IL-2-CIK cells, the percentage of CD3CD56 cells was significantly increased in IL-15-CIK cells, but the expression of regulatory T (Treg) cells and IL-35 was significantly decreased in IL-15-CIK cells. Meanwhile, the in vitro cytotoxicity against human myeloid leukemia cells K562 of IL-15-CIK cells was significantly augmented compared with IL-2-CIK cells. These data suggest that IL-15 may improve the cytotoxicity of CIK cells against leukemia cells by upregulating CD3CD56 cells and downregulating Treg cells and IL-35.

  14. 三磷酸腺苷生物发光法在白血病细胞株K562化疗药物敏感实验中应用探讨%A study of the application of an ATP-bioluminescence assay in chemosensitivity test of leukemic cell line K562

    Institute of Scientific and Technical Information of China (English)

    储德勇; 李炜如; 廖清奎

    2003-01-01

    目的:探讨三磷酸腺苷生物发光法(ATP-BLA法)在白血病细胞药敏实验中的可行性.方法:采用ATP-BLA法,用液体闪烁记数仪检测ATP标准品以及白血病细胞株K562在不同细胞浓度、药物浓度、培养时间下的荧光发光值.结果:ATP标准品浓度、K562细胞浓度的对数与荧光发光值的对数具极好的线性关系(P<0.001);药敏实验时,结束细胞培养的时间以第4 d为宜,药物浓度的选择以1×PPC为最佳.结论:ATP-BLA法在白血病药敏试验中是可行的,有一定的临床应用价值,值得进一步研究.

  15. 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.

  16. Resveratrol Increases Anti-Proliferative Activity of Bestatin Through Downregulating P-Glycoprotein Expression Via Inhibiting PI3K/Akt/mTOR Pathway in K562/ADR Cells.

    Science.gov (United States)

    Wang, Li; Wang, Changyuan; Jia, Yongming; Liu, Zhihao; Shu, Xiaohong; Liu, Kexin

    2016-05-01

    Multidrug resistance (MDR) is a major obstacle in the clinical therapy of hematological malignancies. P-glycoprotein (P-gp) overexpression results in reduction of intracellular drug concentration with a consequence that the cytotoxicity of anti-tumor drugs is decreased, which leads to MDR in K562/ADR cells. In this study, we found that resveratrol enhanced the anti-proliferative activity of bestatin in K562/ADR cells. Co-treatment with resveratrol, IC50 values of bestatin in K562/ADR cells significantly decreased and activation of caspase-3 and caspase-8 increased, which indicated that resveratrol potentiated bestatin-induced apoptosis. Resveratrol increased the intracellular concentration of bestatin through inhibiting P-gp function and downregulating P-gp expression at mRNA and protein levels, which increased anti-proliferative activity of bestatin in K562/ADR cells. Resveratrol decreased the phosphorylation of Akt and mTOR but did not affect the phosphorylations of JNK or ERK1/2. These results demonstrated that resveratrol could increase the anti-proliferative activity of bestatin through downregulating P-gp expression via suppressing the PI3K/Akt/mTOR signaling pathway.

  17. Vasoactive intestinal peptide signaling axis in human leukemia

    Institute of Scientific and Technical Information of China (English)

    Glenn; Paul; Dorsam; Keith; Benton; Jarrett; Failing; Sandeep; Batra

    2011-01-01

    The vasoactive intestinal peptide (VIP) signaling axis constitutes a master "communication coordinator" between cells of the nervous and immune systems.To date,VIP and its two main receptors expressed in T lymphocytes,vasoactive intestinal peptide receptor (VPAC)1 and VPAC2,mediate critical cellular functions regulating adaptive immunity,including arresting CD4 T cells in G 1 of the cell cycle,protection from apoptosis and a potent chemotactic recruiter of T cells to the mucosa associated lymphoid compartment of the gastrointestinal tissues.Since the discovery of VIP in 1970,followed by the cloning of VPAC1 and VPAC2 in the early 1990s,this signaling axis has been associated with common human cancers,including leukemia.This review highlights the present day knowledge of the VIP ligand and its receptor expression profile in T cell leukemia and cell lines.Also,there will be a discussion describing how the anti-leukemic DNA binding transcription factor,Ikaros,regulates VIP receptor expression in primary human CD4 T lymphocytes and T cell lymphoblastic cell lines (e.g.Hut-78).Lastly,future goals will be mentioned that are expected to uncover the role of how the VIP signaling axis contributes to human leukemogenesis,and to establish whether the VIP receptor signature expressed by leukemic blasts can provide therapeutic and/or diagnostic information.

  18. 下调PPP2R5C表达对K562细胞TP53相关通路基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    沈琦[1,2; 张新友[1; 李扬秋[2,3

    2016-01-01

    目的:利用基因芯片技术研究RNA干扰下调PPP2R5C对K562细胞中TP53通路相关基因表达的影响。方法:Affymetrix U133 plus 2.0基因表达谱芯片检测核转染PPP2R5C-siRNA991和SC对照组的K562细胞,应用Genespring GX 11.0软件分析差异基因相关信号通路变化情况。结果:基因芯片分析TP53信号通路紧密相关的22个基因,发现全部基因发生了差异表达,其中上调基因有8个,下调基因有14个。明显上调的基因有EP300、CCND1、BRCA2和USP7,而明显下调的基因则有MDM4、ESR1、ATM、CDK2、CDKN1A、TP53和CDKN2A。结论:下调K562细胞PPP2R5C基因时,在一定程度上选择性调节与细胞增殖密切相关的TP53信号通路相关基因的表达,从而抑制K562细胞增殖,促进了K562细胞凋亡。

  19. [BMMSC from blastic phase CML down-regulate leukemia cell apoptosis].

    Science.gov (United States)

    Wang, Ying; Han, Yu-Xiang; Niu, Zhi-Yun; Wang, Xing-Zhe; Hua, Huan; Shang, Yin-Tao; Wang, Fu-Xu; Zhang, Xue-Jun; Luo, Jian-Min

    2014-10-01

    The purpose of this study was to investigate the effect of bone marrow mesenchymal stem cells (BMMSC) from patients with chronic myeloid leukemia (CML) in blastic phase (Bp) on K562 cells and the primary CML-Bp cells, and to explore its potential mechanisms. K562 cells and primary CML-Bp cells were co-cultured with BMMSC of different groups; the cell proliferation was detected by MTT method, the cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry, the expression levels of Caspase-8, Caspase-9, and activated Caspase-3 in cells were measured by Western blot. The results showed that the CML-Bp BMMSC could enhance the survival rate of K562 cells treated with adviamycin (ADM) and display protective effect on K562 cells and primary CML-Bp mononuctear cells, inhibited ADM-induced leukimia cell apoptosis (P < 0.05); as compared with CML-chronic phase (CML-Cp) BMMSC and normal BMMSC, the CML-Bp BMMSC showed the highest protective effect on leukemic cells, the mitochondrial membrane potential of co-cultured cells slightly droped (P < 0.05). In the CML-Bp BMMSC cultured with K562 cells, the expression level of caspase-3 was more down-regulated than that in K562 alone plus ADM group, while the expression of caspase-9 significantly increased (P < 0.05). It is concluded that the CML-Bp BMMSC down-regulates ADM-induced leukemia cell appoptosis, its mechanism may relate with the inhibition of mitochondrial membrane potential drop, the stabilization of unactive expression of caspase-9 and down-regulation of caspase-3 expression.

  20. 携SH2-Caspase8融合基因重组腺病毒的构建及对K562细胞增殖的影响%Construction of recombinant adenovirus carrying SH2-Caspase8 fusion gene and its effect on proliferation in K562 cells

    Institute of Scientific and Technical Information of China (English)

    刘双凤; 胡晶; 曹唯希; 史静; 彭智; 王海霞; 李亚娟; 冯文莉

    2011-01-01

    Objective To construct the recombinant adenovirus carrying SH2-Caspase8 fusion gene and its mutant to observe its inhibitory effect on proliferation of K562 cells. Methods SH2-Caspase8 fusion gene was amplified by RT-PCR and splicing PCR, and cloned into the shuttle plasmid pAdTrack-CMV of adenovirus. The shuttle plasmid pAdT-SC-EGFP was constructed and identified by double-enzyme digestion and DNA sequencing, and transformed into ultra-competent pAdEasy-BJ5183 after Pme Ⅰ digestion. Defective replication recombinant adenovirus plasmid pAdE-SC-EGFP and its mutant were generated by homologous recombination and further transfected into AD293 cells to package recombinant adenovirus after Pac Ⅰ digestion. Recombinant adenovirus plasmid AdE-SC-EGFP was identified by PCR and Western blotting, respectively, and amplified for the measurement of its titers. Results PCR, enzyme digestion and DNA sequencing showed that the shuttle and recombinant plasmids of adenovirus, pAdT-SC-EGFP and pAdE-SC-EGFP, were successfully constructed. PCR and EGFP expression confirmed that the recombinant plasmid of adenovirus, pAdE-SC-EGFP, was successfully packaged. After amplification, its titer was 1.5 ×l012pfu/ml. Western blot analysis displayed the expression of target protein. MTT and colony formation test could inhibit the proliferation of K562 cells. Conclusion Recombinant adenovirus plasmid, AdE-SC-EGFP, carrying SH2-Caspase8 fusion gene and its mutant, has been successfully constructed, and can significantly inhibit the proliferation of BCR-ABL positive K562 cells in vitro.%目的构建并鉴定携SH2 -Caspase8融合基因的重组腺病毒AdE-SC-EGFP及其突变体,观察其对K562细胞增殖的抑制作用。方法采用RT-PCR、重叠PCR扩增SH2-Caspase8融合基因,克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-SC-EGFP,进行酶切和测序鉴定。将PmeⅠ酶切后的穿梭质粒转化pAdEasy-BJ5183感受态,通过细菌内同源重组产

  1. Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

    Science.gov (United States)

    de Ravel, Marc Rolland; Alameh, Ghina; Melikian, Maxime; Mahiout, Zahia; Emptoz-Bonneton, Agnès; Matera, Eva-Laure; Lomberget, Thierry; Barret, Roland; Rocheblave, Luc; Walchshofer, Nadia; Beltran, Sonia; El Jawad, Lucienne; Mappus, Elisabeth; Grenot, Catherine; Pugeat, Michel; Dumontet, Charles; Le Borgne, Marc; Cuilleron, Claude Yves

    2015-02-26

    A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/β-pregnane-3,20-dione or 5β-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/β-pregnane-3,20-dione, among which the 5β-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 μM versus 1.2 and 10.6 μM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5β-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 μM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

  2. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  3. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15 mRNA in Various Human Cell Types

    Directory of Open Access Journals (Sweden)

    Sang Mee Hwang

    2013-01-01

    Full Text Available CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC, two peripheral bloods (PB, 12 granulocyte products, natural killer (NK-92, B-lymphocyte (CO88BV59-1, K-562 leukemia cell line, human embryonic stem cell (hESC, and human fibroblasts (HF. HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

  4. Biology and relevance of human acute myeloid leukemia stem cells.

    Science.gov (United States)

    Thomas, Daniel; Majeti, Ravindra

    2017-03-23

    Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research. © 2017 by The American Society of Hematology.

  5. SIRT1 inhibition impairs non-homologous end joining DNA damage repair by increasing Ku70 acetylation in chronic myeloid leukemia cells.

    Science.gov (United States)

    Zhang, Wenjun; Wu, Haixia; Yang, Meng; Ye, Shiguang; Li, Liang; Zhang, Hong; Hu, Jiong; Wang, Xuguang; Xu, Jun; Liang, Aibin

    2016-03-22

    Most chemotherapeutic agents for leukemia are DNA damaging agents. However, DNA lesions can be repaired by activities of DNA repair systems. Increasing evidence have shown that enhanced DNA damage repair capacity contributes to chemotherapy resistance in leukemia cells. Thus, targeting DNA repair mechanisms is a promising strategy for novel leukemia treatment. SIRT1 expressions were downregulated by lentivirus-delivered SIRT1 shRNA in myeloid leukemia cells. SIRT1 mRNA and protein levels were analyzed by real-time PCR and Western blot, respectively. Flow cytometry was carried out to analyze cell cycle progression, apoptosis and DNA damage repair efficiency. DNA damage levels were assessed by alkaline comet assay, and H2AX phosphorylation was analyzed by immunoblotting and immunofluorescence. A mouse leukemia model was established by transplanting lentivirus-infected K562 cells containing SIRT1 shRNA into sublethally irradiated NOD/SCID mice, and tumorigenesis was evaluated by detecting tumor weights and mice survival. SIRT1 expressions were upregulated in myeloid leukemic patients. Downregulation of SIRT1 by RNAi promoted etoposide-induced DNA damage in myeloid leukemia cells accompanied by reduced NHEJ activity, and increased Ku70 acetylation. Furthermore, SIRT1 knockdown resulted in cell cycle arrest, induction of apoptosis and reduction of K562 cell proliferation accompanied by enhanced p53 and FOXO1 acetylation in K562 cells after etoposide treatment. Importantly, SIRT1 downregulation reduced the tumorigenesis ability of K562 cells in mouse xenografts following chemotherapy treatment. These results revealed that SIRT1 promotes the NHEJ repair pathway by deacetylating Ku70 in K562 cells, suggesting that SIRT1 is a novel therapeutic target for treating myeloid leukemia.

  6. 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.

  7. Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

    Science.gov (United States)

    Cole, Alicia; Wang, Zezhou; Coyaud, Etienne; Voisin, Veronique; Gronda, Marcela; Jitkova, Yulia; Mattson, Rachel; Hurren, Rose; Babovic, Sonja; Maclean, Neil; Restall, Ian; Wang, Xiaoming; Jeyaraju, Danny V; Sukhai, Mahadeo A; Prabha, Swayam; Bashir, Shaheena; Ramakrishnan, Ashwin; Leung, Elisa; Qia, Yi Hua; Zhang, Nianxian; Combes, Kevin R; Ketela, Troy; Lin, Fengshu; Houry, Walid A; Aman, Ahmed; Al-Awar, Rima; Zheng, Wei; Wienholds, Erno; Xu, Chang Jiang; Dick, John; Wang, Jean C Y; Moffat, Jason; Minden, Mark D; Eaves, Connie J; Bader, Gary D; Hao, Zhenyue; Kornblau, Steven M; Raught, Brian; Schimmer, Aaron D

    2015-06-08

    From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

  8. Cytotoxicity of (-)-vitisin B in human leukemia cells.

    Science.gov (United States)

    Wu, Shing-Sheng; Chen, Lih-Geeng; Lin, Ren-Jye; Lin, Shyr-Yi; Lo, Yueh-E; Liang, Yu-Chih

    2013-07-01

    Vitis thunbergii var. taiwaniana (VTT) is an indigenous Taiwanese wild grape and is used as a folk medicine in Taiwan. VTT is rich in polyphenols, especially quercetin and resveratrol derivatives, which were demonstrated to exhibit inhibitory activities against carcinogenesis and prevent some neurodegenerative diseases. (-)-Vitisin B is one of the resveratrol tetramers extracted from VTT. In this study, we investigated the mechanisms of (-)-vitisin B on the induction of apoptosis in human HL-60 promyelocytic leukemia cells. First, (-)-vitisin B significantly inhibited cell proliferation through inducing cell apoptosis. This effect appeared to occur in a time- and dose-dependent manner. Cell-cycle distribution was also examined, and we found that (-)-vitisin B significantly induced a sub-G1 population in a dose-dependent manner. In addition, (-)-vitisin B exhibited stronger inhibitory effects on cell proliferation than resveratrol. Second, (-)-vitisin B dose dependently induced apoptosis-related protein expressions, such as the cleavage form of caspase-3, caspase-8, caspase-9, poly(ADP ribose) polymerase, and the proapoptotic Bax protein. Third, (-)-vitisin B treatment also resulted in increases in c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. Moreover, the (-)-vitisin B-induced FasL expression and caspase-3 activation could be reversed by a JNK inhibitor. These results suggest that (-)-vitisin B-induced apoptosis of leukemia cells might be mediated through activation of JNK and Fas death-signal transduction.

  9. Synthesis and evaluation of the cytotoxic activity of novel ethyl 4-[4-(4-substitutedpiperidin-1-yl)]benzyl-phenylpyrrolo[1,2-a]quinoxaline-carboxylate derivatives in myeloid and lymphoid leukemia cell lines.

    Science.gov (United States)

    Desplat, Vanessa; Vincenzi, Marian; Lucas, Romain; Moreau, Stéphane; Savrimoutou, Solène; Pinaud, Noël; Lesbordes, Jordi; Peyrilles, Elodie; Marchivie, Mathieu; Routier, Sylvain; Sonnet, Pascal; Rossi, Filomena; Ronga, Luisa; Guillon, Jean

    2016-05-01

    Leukemia is the most common blood cancer, and its development starts at diverse points, leading to distinct subtypes that respond differently to therapy. This heterogeneity is rarely taken into account in therapies, so it is still essential to look for new specific drugs for leukemia subtypes or even for therapy-resistant cases. Among heterocyclic compounds that attracted a lot of attention because of its wide spread biological activities, the pyrrolo[1,2-a]quinoxaline heterocyclic framework has been identified as interesting scaffolds for antiproliferative activity against various human cancer cell lines. In the present study, novel ethyl 4-[4-(4-substitutedpiperidin-1-yl)]benzyl-phenylpyrrolo[1,2-a]quinoxaline-carboxylate derivatives 1a-l have been designed and synthesized. Their cytotoxicities were evaluated against five different leukemia cell lines, including Jurkat and U266 (lymphoid cell lines), and K562, U937, HL60 (myeloid cell lines), as well as normal human peripheral blood mononuclear cells (PBMNCs). Then, apoptosis study was performed with the more interesting compounds. The new pyrrolo[1,2-a]quinoxaline series showed promising cytotoxic potential against all leukemia cell lines tested, and some compounds showed better results than the reference compound A6730. Some compounds, such as 1a, 1e, 1g and 1h are promising because of their high activity against leukemia and their low activity against normal hematopoietic cells. Structure-activity relationships of these new synthetic compounds 1a-l are here also discussed.

  10. Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo

    National Research Council Canada - National Science Library

    Budhraja, Amit; Gao, Ning; Zhang, Zhuo; Son, Young-Ok; Cheng, Senping; Wang, Xin; Ding, Songze; Hitron, Andrew; Chen, Gang; Luo, Jia; Shi, Xianglin

    2012-01-01

    In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo...

  11. Genomic loss of mismatched human leukocyte antigen and leukemia immune escape from haploidentical graft-versus-leukemia.

    Science.gov (United States)

    Vago, Luca; Toffalori, Cristina; Ciceri, Fabio; Fleischhauer, Katharina

    2012-12-01

    Recent developments in cell processing and immunosuppressive strategies has allowed the safe infusion of high numbers of donor T cells in the context of clinical haploidentical hematopoietic stem cell transplantation (HSCT). Haploidentical T cells display an intrinsic ability to recognize and eliminate residual patient leukemic cells, largely due to alloreactivity against the patient-specific human leukocyte antigen (HLA) molecules encoded on the mismatched haplotype. However, recent evidence has shown that leukemia, like many other tumors displaying pronounced genomic instability, is frequently able to evade this potent graft-versus-leukemia effect by undergoing de novo genomic mutations, which result in the permanent loss of only those HLA molecules targeted by haploidentical donor T-cell alloreactivity. This review summarizes the recent clinical and experimental evidence regarding this phenomenon, and its therapeutic and clinical consequences.

  12. Molecular association of 2-(n-alkylamino)-1,4-naphthoquinone derivatives: Electrochemical, DFT studies and antiproliferative activity against leukemia cell lines

    Science.gov (United States)

    Patil, Rishikesh; Bhand, Sujit; Konkimalla, V. Badireenath; Banerjee, Priyabrata; Ugale, Bharat; Chadar, Dattatray; Saha, Sourav Kr.; Praharaj, Prakash Priyadarshi; Nagaraja, C. M.; Chakrovarty, Debamitra; Salunke-Gawali, Sunita

    2016-12-01

    Molecular structures and their molecular association of 2-(n-alkylamino)-1,4-naphthoquinone, viz., LH-3; propyl, LH-4; butyl and LH-8; octyl derivatives were studied by single crystal X-ray diffraction studies. Synthesis and characterization of 2-octylamino-1,4-naphthoquinone; LH-8 was discussed. The molecule of LH-3 crystallizes in orthorhombic space group P21/c, while the LH-4 and LH-8 molecule crystallizes in triclinic space group P-1. LH-3, LH-4 and LH-8 showed intermolecular N-H⋯O and C-H⋯O interactions, LH-3 showed unique C(3)-H(3)⋯O(1) interaction. Interchain π-π stacking, slipped π-π stacking and C⋯O close contacts was respectively observed in LH-3, LH-4 and LH-8. Electrochemical studies were performed on first eight members of homologous series of 2-(n-alkylamino)-1,4-naphthoquinone (LH-1 to LH-8) by cyclic voltammetry. Naphthoquinone to naphthosemiquinone reversible redox couple was observed in all compounds ∼ E1/2 = -0.657 ± 0.05 V. HOMO-LUMO band gap was determined for the neutral form as well as the monoanionic radical form viz. naphthosemiquinone form of selected derivatives by DFT studies. It has been observed that the electron density is delocalized in the naphthoquinone ring in both neutral as well as one electron reduced form of compounds. Antiproliferative activity of LH-1 to LH-8 was evaluated against two cancer cell lines, THP1(acute monocytic leukemia) and K562(human immortalized myelogenous leukemia cell line) cells. It was observed that, in THP1 cells, compounds LH-2 and LH-3 are very active while LH-1, LH-4 and LH-6 were moderately active and LH-5, LH-7 and LH-8 were totally inactive. Contrastingly, in K562 cells all of the compounds were moderately active.

  13. PTEN基因对慢性粒细胞白血病Survivin、Xiap、Smac调控的研究%Regulation of wild type PTEN gene on Survivin, Xiap and Smac in chronic leukemia cells

    Institute of Scientific and Technical Information of China (English)

    成志勇; 万建设; 王亚丽; 梁丽青; 梁文同; 穆敬; 芦希; 潘崚

    2011-01-01

    Objective To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation,apoptosis and the possible regulations of apoptosis-related molecules Survivin,Xiap and Smac gene in human chronic myeloid leukemia(CML)and cell line K562 cells.Methods(1)The recombinant adenovirus containing green fluorescent protein(GFP)and PTEN(Ad-PTEN-GFP)or empty vector(AdGFP)was transfected into K562 cells.The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry(FCM).PTEN,Survivin,Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR(FQ-PCR)while PTEN protein levels analyzed by Western blot.(2)The expression levels of PTEN,Survivin,Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia(CML)patients in chronic phase(CML-CP),10 CML patients in blast crises(CML-BC)and 10 normal control marrow mononuclear cells(MMNC).Results The growth of K562 cells was suppressed markedly.And the maximal growth inhibition rate was 38.6% after the tranfection of PTEN.Survivin,Xiap,Smac mRNA expression levels were down-regulated by around 6.14,7.44 and 2.95 folds respectively(0.0700 ±0.0059,0.0089 ±0.0006,0.0600 ±0.0039 vs 0.4370 ± 0.0790,0.0661 ± 0.0072,0.1580 ± 0.0078 vs 0.4530 ± 0.0810,0.0700 ± 0.0079.0.1770 ±0.0085,all P < 0.01).The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control.But Survivin,Xiap,Smae mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control.Conclusion The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin,Xiap and Smac genes.%目的 探讨肿瘤抑制基因PTEN对人慢性粒细胞白血病(CML)中生存素(Survivin)、X连锁凋亡抑制蛋白(Xiap)、线粒体促凋亡蛋白(Smac)调控的作用.方法(1)将携带有野生型PTEN和绿

  14. Activity of elaeochytrin A from Ferula elaeochytris on leukemia cell lines.

    Science.gov (United States)

    Alkhatib, Racha; Hennebelle, Thierry; Joha, Sami; Idziorek, Thierry; Preudhomme, Claude; Quesnel, Bruno; Sahpaz, Sevser; Bailleul, François

    2008-12-01

    Phytochemical investigation of the roots of Ferula elaeochytris made it possible to isolate two sesquiterpene esters, 6-anthraniloyljaeschkeanadiol (elaeochytrin A) and 4beta-hydroxy-6alpha-(p-hydroxybenzoyloxy)dauc-9-ene (elaeochytrin B), as well as eight known compounds: 6-angeloyljaeschkeanadiol, teferidin, ferutinin, 6-(p-hydroxybenzoyl)epoxyjaeschkeanadiol, 6-(p-hydroxybenzoyl)lancerotriol, 5-caffeoylquinic acid, 1,5-dicaffeoylquinic acid and sandrosaponin IX. The cytotoxic activities of all compounds were investigated on K562R (imatinib-resistant) human chronic myeloid leukaemia and DA1-3b/M2(BCR-ABL) (dasatinib-resistant) mouse leukemia cell line. Elaeochytrin A was the most active compound on both cell lines (IC(50)=12.4 and 7.8microM, respectively). It was also tested on non-resistant human promyelocytic leukemia cells (HL60, IC(50)=13.1microM) and was not toxic to normal peripheral blood mononuclear cells up to 100microM.

  15. ETV6 mutations in early immature human T cell leukemias

    Science.gov (United States)

    Van Vlierberghe, Pieter; Ambesi-Impiombato, Alberto; Perez-Garcia, Arianne; Haydu, J. Erika; Rigo, Isaura; Hadler, Michael; Tosello, Valeria; Della Gatta, Giusy; Paietta, Elisabeth; Racevskis, Janis; Wiernik, Peter H.; Luger, Selina M.; Rowe, Jacob M.; Rue, Montserrat

    2011-01-01

    Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ∼5–10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors. PMID:22162831

  16. Apoptosis induction in human leukemic cells by a novel protein Bengalin, isolated from Indian black scorpion venom: through mitochondrial pathway and inhibition of heat shock proteins.

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    Gupta, Shubho Das; Gomes, Antony; Debnath, Anindita; Saha, Archita; Gomes, Aparna

    2010-01-27

    Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC(50) values of 3.7 and 4.1 microg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.

  17. A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

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    Cui X Tracy

    2011-05-01

    Full Text Available Abstract An investigation of the electrochemical activity of human white blood cells (WBC for biofuel cell (BFC applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient, a B lymphoblastoid cell line (BLCL, and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents.

  18. Autologous and allogeneic typing of human leukemia cells: definition of surface antigens restricted to lymphocytic leukemia cells.

    OpenAIRE

    Naito, K.; Yamaguchi, H; Horibe, K; Shiku, H.; Takahashi, T.; Suzuki, S; Yamada, K.

    1983-01-01

    Serum from a patient (CO) with acute lymphoblastic leukemia was reactive in immunoadherence assays with autologous leukemia cells but not with autologous blood lymphocytes or bone marrow cells during complete remission. Extensive absorption tests with an array of leukemia cells and normal cells were performed in order to define the specificity of the reaction. The autologous leukemia reactivity was either completely or partially absorbed with acute lymphoblastic leukemia cells obtained from 1...

  19. Animals Models of Human T Cell Leukemia Virus Type I Leukemogenesis.

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    Niewiesk, Stefan

    2016-01-01

    Infection with human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in a minority of infected individuals after long periods of viral persistence. The various stages of HTLV-I infection and leukemia development are studied by using several different animal models: (1) the rabbit (and mouse) model of persistent HTLV-I infection, (2) transgenic mice to model tumorigenesis by HTLV-I specific protein expression, (3) ATL cell transfers into immune-deficient mice, and (4) infection of humanized mice with HTLV-I. After infection, virus replicates without clinical disease in rabbits and to a lesser extent in mice. Transgenic expression of both the transactivator protein (Tax) and the HTLV-I bZIP factor (HBZ) protein have provided insight into factors important in leukemia/lymphoma development. To investigate factors relating to tumor spread and tissue invasion, a number of immune-deficient mice based on the severe combined immunodeficiency (SCID) or non-obese diabetic/SCID background have been used. Inoculation of adult T cell leukemia cell (lines) leads to lymphoma with osteolytic bone lesions and to a lesser degree to leukemia development. These mice have been used extensively for the testing of anticancer drugs and virotherapy. A recent development is the use of so-called humanized mice, which, upon transfer of CD34(+)human umbilical cord stem cells, generate human lymphocytes. Infection with HTLV-I leads to leukemia/lymphoma development, thus providing an opportunity to investigate disease development with the aid of molecularly cloned viruses. However, further improvements of this mouse model, particularly in respect to the development of adaptive immune responses, are necessary.

  20. Transformation of human fetal thymus and spleen lymphocytes by human t-cell leukemia virus type Ι

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    Akagi,Tadaatsu

    1985-04-01

    Full Text Available Co-cultivation of human thymus and spleen lymphocytes, which were obtained from 26-week and 27-week fetuses, with a lethally-irradiated human cord T-cell line harboring human T-cell leukemia virus type Ι(HTLV-Ι resultes in the establishment of T-cell lines positive for adult T-cell leukemia-associated antigens and producing HTLV-Ι. These cell lines had the phenotype of a helper/inducer subset of peripheral T-cells as evidenced by the reactivity with monoclonal antibodies to human T-cells.

  1. STUDY ON EFFECTS OF QUERCETIN ON PML GENE AND PROTEIN IN LEUKEMIA CELL LINES

    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 effect of quercetin on PML gene and protein expression and localization in leukemia cell lines. MethodsCell morphology was assayed by Wright,s stain and fluorescence stain, and PML Mrna expression by RT-PCR, PML protein localization by immuno fluorescence.ResultsNB4 and HL-60 cells differentiated morphologically after treatment with all trans retinoic acid (ATRA) while K562 cells did not differentiate. Typical apoptosis was found in each cell line after treatment with quercetin. Immuno fluorescence analysis showed, after treatment with ATRA, the fusion protein disappeared in NB4 cells and PML protein relocated, while HL-60 and K562 cells had no difference from control cells. After treatment with quercetin, the fusion protein disappeared in NB4 cells, PML protein relocated, then degraded. In HL-60 cells and K562 cells, PML protein also located and then degraded. The expression of PML Mrna was not changed in all three cell lines after treatment with ATRA or quercetin. ConclusionPML plays the role of differentiation and apoptosis inducer in leukemia cells at the translational level. PML in POD plays the role of apoptosis inducer and the growth control of leukemia cells.

  2. Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells

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    Hadi Karami

    2014-05-01

    Full Text Available Purpose: Up-regulation of Mcl-1, a known anti-apoptotic protein, is associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effect of Mcl-1 small interference RNA (siRNA on the proliferation and apoptosis of HL-60 acute myeloid leukemia (AML cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western blotting, respectively. Trypan blue assay was performed to assess tumor cell proliferation after siRNA transfection. The cytotoxic effect of Mcl-1 siRNA on leukemic cells was measured using MTT assay. Apoptosis was detected using ELISA cell death assay. Results: Mcl-1 siRNA clearly lowered both Mcl-1 mRNA and protein levels in a time-dependent manner, leading to marked inhibition of cell survival and proliferation. Furthermore, Mcl-1 down-regulation significantly enhanced the extent of HL-60 apoptotic cells. Conclusion: Our results suggest that the down-regulation of Mcl-1 by siRNA can effectively trigger apoptosis and inhibit the proliferation of leukemic cells. Therefore, Mcl-1 siRNA may be a potent adjuvant in AML therapy.

  3. Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia

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    Zou Yong

    2012-07-01

    Full Text Available Abstract Background We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized. Methods This study examined the methylation status at histone H3 lysine 4 (H3K4 and histone H3 lysine 9 (H3K9 in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI, Trichostatin A (TSA, and 5-aza-2’-deoxycytidine (5-Aza on the cells. Results We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases. Conclusions This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

  4. Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.

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    Michele Cea

    Full Text Available Aberrant histone deacetylase (HDAC activity is frequent in human leukemias. However, while classical, NAD(+-independent HDACs are an established therapeutic target, the relevance of NAD(+-dependent HDACs (sirtuins in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527 and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

  5. Anti-leukemia activity of in vitro-expanded human gamma delta T cells in a xenogeneic Ph+ leukemia model.

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    Gabrielle M Siegers

    Full Text Available Gamma delta T cells (GDTc lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph(+ cell lines in vitro. We have generated a fluorescent and bioluminescent Ph(+ cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy.

  6. Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

    Science.gov (United States)

    Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

    2013-01-01

    The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

  7. Inhibition of phosphotyrosine phosphatase 1B causes resistance in BCR-ABL-positive leukemia cells to the ABL kinase inhibitor STI571.

    Science.gov (United States)

    Koyama, Noriko; Koschmieder, Steffen; Tyagi, Sandhya; Portero-Robles, Ignacio; Chromic, Jörg; Myloch, Silke; Nürnberger, Heike; Rossmanith, Tanja; Hofmann, Wolf-Karsten; Hoelzer, Dieter; Ottmann, Oliver Gerhard

    2006-04-01

    Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of BCR-ABL-mediated transformation in vitro and in vivo. To investigate whether PTP1B modulates the biological effects of the abl kinase inhibitor STI571 in BCR-ABL-positive cells, we transfected Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cell-derived K562 cells with either wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B (K562/D181A), or empty vector (K562/mock). Cells were cultured with or without STI571 and analyzed for its effects on proliferation, differentiation, and apoptosis. In both K562/mock and K562/PTP1B cells, 0.25 to 1 mumol/L STI571 induced dose-dependent growth arrest and apoptosis, as measured by a decrease of cell proliferation and an increase of Annexin V-positive cells and/or of cells in the sub-G(1) apoptotic phase. Western blot analysis showed increased protein levels of activated caspase-3 and caspase-8 and induction of poly(ADP-ribose) polymerase cleavage. Low concentrations of STI571 promoted erythroid differentiation of these cells. Conversely, K562/D181A cells displayed significantly lower PTP1B-specific tyrosine phosphatase activity and were significantly less sensitive to STI571-induced growth arrest, apoptosis, and erythroid differentiation. Pharmacologic inhibition of PTP1B activity in wild-type K562 cells, using bis(N,N-dimethylhydroxamido)hydroxooxovanadate, attenuated STI571-induced apoptosis. Lastly, comparison of the STI571-sensitive Ph+ acute lymphoblastic leukemia cell line SupB15 with a STI571-resistant subline revealed significantly decreased PTP1B activity and enhanced BCR-ABL phosphorylation in the STI571-resistant SupB15 cells. In conclusion, functional PTP1B is involved in STI571-induced growth and cell cycle arrest, apoptosis, and differentiation, and attenuation of PTP1B function may contribute to resistance towards STI571.

  8. Wogonoside induces growth inhibition and cell cycle arrest via promoting the expression and binding activity of GATA-1 in chronic myelogenous leukemia cells.

    Science.gov (United States)

    Li, Hui; Hui, Hui; Xu, Jingyan; Yang, Hao; Zhang, Xiaoxiao; Liu, Xiao; Zhou, Yuxin; Li, Zhiyu; Guo, Qinglong; Lu, Na

    2016-06-01

    GATA-1, a zinc finger transcription factor, has been demonstrated to play a key role in the progression of leukemia. In this study, we investigate the effects of wogonoside, a naturally bioactive flavonoid derived from Scutellaria baicalensis Georgi, on cell growth and cell cycle in chronic myeloid leukemia (CML) cells, and uncover its underlying mechanisms. The experimental design comprised CML cell lines K562, imatinib-resistant K562 (K562r) cells, and primary CML cells, treated in vitro or in vivo, respectively, with wogonoside; growth and cell cycle were then evaluated. We found that wogonoside could induce growth inhibition and G0/G1 cell cycle arrest in both normal and K562r cells. Wogonoside promotes the expression of GATA-1 and facilitates the binding to methyl ethyl ketone (MEK) and p21 promoter, thus inhibiting MEK/extracellular signal-regulated kinase signaling and cell cycle checkpoint proteins, including CDK2, CDK4, cyclin A, and cyclin D1, and increasing p21 expression. Furthermore, in vivo studies showed that administration of wogonoside decreased CML cells and prolonged survival in NOD/SCID mice with CML cell xenografts. In conclusion, these results clearly revealed the inhibitory effect of wogonoside on the growth in CML cells and suggested that wogonoside may act as a promising drug for the treatment of imatinib-resistant CML.

  9. Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias.

    Science.gov (United States)

    Schwarting, R; Castello, R; Moldenhauer, G; Pezzutto, A; von Hoegen, I; Ludwig, W D; Parnes, J R; Dörken, B

    1992-11-01

    S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.

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

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    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.

  11. Novel human polyomaviruses, Merkel cell polyomavirus and human polyomavirus 9, in Japanese chronic lymphocytic leukemia cases

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    Imajoh Masayuki

    2012-06-01

    Full Text Available Abstract Background Chronic lymphocytic leukemia (CLL is the rarest adult leukemia in Japan, whereas it is the most common leukemia in the Western world. Recent studies from the United States and Germany suggest a possible etiological association between Merkel cell polyomavirus (MCPyV and CLL, although no data have been reported from Eastern countries. To increase the volume of relevant data, this study investigated the prevalence and DNA loads of MCPyV and human polyomavirus 9 (HPyV9, another lymphotropic polyomavirus, in Japanese CLL cases. Findings We found that 9/27 CLL cases (33.3 % were positive for MCPyV using quantitative real-time polymerase chain reaction analysis. The viral DNA loads ranged from 0.000017 to 0.0012 copies per cell. All cases were negative for HPyV9. One MCPyV-positive CLL case was evaluated by mutational analysis of the large T (LT gene, which indicated the presence of wild-type MCPyV without a nucleotide deletion. DNA sequence analysis of the entire small T (ST gene and the partial LT gene revealed that a Japanese MCPyV isolate, designated CLL-JK, had two nucleotide gaps when compared with the reference sequence of the North American isolate MCC350. Conclusions This study provides the first evidence that MCPyV is present in a subset of Japanese CLL cases with low viral DNA loads. MCPyV and HPyV9 are unlikely to contribute directly to the development of CLL in the majority of Japanese cases. MCPyV isolated from the Japanese CLL cases may constitute an Asian group and its pathogenicity needs to be clarified in future studies.

  12. Imatinib induces H2AX phosphorylation and apoptosis in chronic myelogenous leukemia cells in vitro via caspase-3/Mst1 pathway

    Institute of Scientific and Technical Information of China (English)

    Yan-jun ZHANG; Lian-ning DUAN; Cheng-rong LU; Yan CAO; Yuan LUO; Rong-feng BAO; Shu YAN; Mei XUE; Feng ZHU; Zhe WANG

    2012-01-01

    Aim:Histone H2AX is a novel tumor suppressor and its phosphorylation at the C terminus (Ser139 and Tyr142)is required for tumor cell apoptosis.The aim of the present study was to elucidate the mechanisms underlying imatinib-induced C-terminal phosphorylation of H2AX in chronic myelogenous leukemia cells in vitro.Methods:BCR-ABL-positive K562 cells were used.Microscopy,Western blotting and flow cytometry were used to study the signaling pathways that regulate imatinib-induced H2AX phosphorylation and the apoptotic mechanisms.Results:Treatment of K562 cells with imatinib (1-8 μmol/L)induced phosphorylation of H2AX at Ser139 and Tyr142 in time-and dose-dependent manners.In contrast,imatinib at the same concentrations did not affect H2AX acetylation at Lys 5,and the acetylated H2AX maintained a higher level in the cells.Meanwhile,imatinib (1-8 μmol/L)activated caspase-3 and its downstream mammalian STE20-like kinase 1 (Mst1),and induced apoptosis of K562 cells.The caspase-3 inhibitor Z-VAD (40 μmol/L)reduced imatinibinduced H2AX phosphorylation at Ser139 and Tyr142 and blocked imatinib-induced apoptosis of K562 cells.Imatinib (4 μmol/L)induced expression of Williams-Beuren syndrome transcription factor (WSTF),but not wild-type p53-induced phosphatase 1 (Wip1)in K562 cells.Conclusion:The caspase-3/Mst1 pathway is required for H2AX C-terminal phosphorylation at Ser139 and Tyr142 and subsequent apoptosis in Bcr-Abl-positive K562 cells induced by imatinib.

  13. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    Science.gov (United States)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  14. GENE EXPRESSION OF NOVEL RETROVIRUS ASSOCIATED WITH HUMAN ACUTE MULOID LEUKEMIA

    Institute of Scientific and Technical Information of China (English)

    许晓华; 徐荣臻; 王世炯; 郑树; 朱宁希; 周旋

    2003-01-01

    Objective: To explore the potentiality of retroviral etiology in human acute myeloid leukemia(AML). Methods: The expression of clone 6#11 in leukemic cell samples from 19 AML cases and peripheral blood mononuclear cells (PBMNCs) from 20 controls was studied by means of Northern blot and reversal transcription polymerase chain reaction (RT-PCR). Results: Northern blot and RT-PCR analyses showed that the expression level of clone 6#11 was significantly higher in AML patients than that in control. Conclusion: Northern blot and RT-PCR analyses revealed that the expression of novel retrovirus were associated with acute myeloid leukemia.

  15. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

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    Chen YJ

    2016-07-01

    Full Text Available Yu-Jen Chen,1–4 Li-Wen Fang,5 Wen-Chi Su,6,7 Wen-Yi Hsu,1 Kai-Chien Yang,1 Huey-Lan Huang8 1Department of Medical Research, 2Department of Radiation Oncology, Mackay Memorial Hospital, 3Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 4Institute of Pharmacology, Taipei Medical University, Taipei, 5Department of Nutrition, I-Shou University, Kaohsiung, 6Research Center for Emerging Viruses, China Medical University Hospital, 7Graduate Institute of Clinical Medical Science, China Medical University, Taichung, 8Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, Republic of China Abstract: Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML, chronic myeloid leukemia (CML, and acute lymphoblastic leukemia (ALL cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of

  16. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

    Science.gov (United States)

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  17. Autophagy is associated with cucurbitacin D-induced apoptosis in human T cell leukemia cells.

    Science.gov (United States)

    Nakanishi, Tsukasa; Song, Yuan; He, Cuiying; Wang, Duo; Morita, Kentaro; Tsukada, Junichi; Kanazawa, Tamotsu; Yoshida, Yasuhiro

    2016-04-01

    We previously reported that the inflammasome inhibitor cucurbitacin D (CuD) induces apoptosis in human leukemia cell lines. In the present study, we investigated the effects of co-treatment with an additional Bcl-xL inhibitor, Z36. Treatment with Z36 induced cell death in leukemia cell lines, with MT-4 cells exhibiting the lowest sensitivity to Z36. Co-treatment of cells with Z36 and CuD resulted in a greater degree of cell death for Hut78 and Jurkat cells than treatment with CuD alone. In contrast, co-treatment of MT-4 cells with Z36 and CuD had a suppressive effect on cell death. The autophagy inhibitor 3-methyladenine (3-MA) suppressed the growth of leukemia cell lines HuT78, Jurkat, MT-1, and MT-4. CuD-induced cell death was enhanced by 3-MA in Jurkat cells, but inhibited in MT-4 cells. Western blotting results revealed cleavage of poly(ADP ribose) polymerase (PARP), supporting CuD-induced cell death; 3-MA enhanced CuD-Z36-induced PARP cleavage. Taken together, our results indicate that autophagy negatively regulates chemical-induced cell death of leukemia cells, and that controlling autophagy could be beneficial in the development of more effective chemotherapies against leukemia.

  18. Therapeutic activity of two xanthones in a xenograft murine model of human chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Berthou Christian

    2010-12-01

    Full Text Available Abstract Background We previously reported that allanxanthone C and macluraxanthone, two xanthones purified from Guttiferae trees, display in vitro antiproliferative and proapoptotic activities in leukemic cells from chronic lymphocytic leukemia (CLL and leukemia B cell lines. Results Here, we investigated the in vivo therapeutic effects of the two xanthones in a xenograft murine model of human CLL, developed by engrafting CD5-transfected chronic leukemia B cells into SCID mice. Treatment of the animals with five daily injections of either allanxanthone C or macluraxanthone resulted in a significant prolongation of their survival as compared to control animals injected with the solvent alone (p = 0.0006 and p = 0.0141, respectively. The same treatment of mice which were not xenografted induced no mortality. Conclusion These data show for the first time the in vivo antileukemic activities of two plant-derived xanthones, and confirm their potential interest for CLL therapy.

  19. STUDY ON EFFECTS OF RED ORPIMENT AND ATRA ON PML GENE AND PROTEIN IN LEUKEMIA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate PML gene and protein expression and localization in leukemia cell lines. Methods Cell morphology was assayed by Wright's stain and fluorescence stain, and PML mRNA ex- pression by RT-PCR, PML protein localization by immuno-fluorescence. Results NB4 and HL-60 cells dif- ferentiated morphologically after treatment with anti-retinoic acid ( ATRA ) while K562 cells did not differenti- ate. Typical apoptosis was found in each cell line after treatment with red orpiment. Immuno-fluorescence analysis showed, after treatment with ATRA, the fusion protein disappeared in NB4 cells and the PML protein relocated, while HL-60 and K562 cells had no difference from control cells. After treatment with red orpiment , the fusion protein disappeared in NB4 cells, then degraded, which was also seen in HL-60 cells and K562 cells. The expression of PML mRNA was not changed in all three cell lines after treatment with ATRA or red orpi- ment. Conclusion PML plays the role of differentiation and apoptosis inducer in leukemia cells at the trans- lational level. PML in POD plays the role of apoptosis inducer and the growth control of leukemia cells.

  20. Development of K562 cell clones expressing β-globin mRNA carrying the β039 thalassaemia mutation for the screening of correctors of stop-codon mutations

    Science.gov (United States)

    Salvatori, Francesca; Cantale, Vera; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing

  1. Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.

    Science.gov (United States)

    Salvatori, Francesca; Cantale, Vera; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2009-07-09

    Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0

  2. Cyclo-oxygenase 2 inhibitor, nabumetone, inhibits proliferation in chronic myeloid leukemia cell lines.

    Science.gov (United States)

    Vural, Filiz; Ozcan, Mehmet Ali; Ozsan, Güner Hayri; Ateş, Halil; Demirkan, Fatih; Pişkin, Ozden; Undar, Bülent

    2005-05-01

    The anti-tumor effect of cyclo-oxygenase (COX) inhibitors has been documented in several studies. COX2 inhibitors have attracted more attention because of the fewer side-effects and the more prominent anti-tumor effects. However, experience with these drugs in hematological malignancies is limited. In our study, a potent COX2 inhibitor, nabumetone (NBT), was investigated for its anti-proliferative and apoptotic effects in K-562 and Meg-01 chronic myeloid leukemia blastic cell lines as a single agent or in combination with adriamycin (ADR) and interferon alpha (IFN-a). In these cell lines, a dose-dependent inhibition of proliferation was observed with NBT. We observed no significant apoptotic effect of NBT. However, NBT potentiated the apoptotic effect of ADR in the K-562 cell line. Bcl-2 expression was reduced by NBT (11% vs. 2%). The combination of NBT with IFN did not have any significant effect on the K-562 cell line. We suggest that NBT inhibits proliferation and potentiates the apoptotic effect of ADR in chronic myeloid leukemia cell lines.

  3. Discovery of 3H-Benzo[4,5]thieno[3,2-d]pyrimidin-4-ones as Potent, Highly Selective, and Orally Bioavailable Inhibitors of the Human Protooncogene Proviral Insertion Site in Moloney Murine Leukemia Virus (PIM) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Zhi-Fu; Hasvold, Lisa A.; Leverson, Joel D.; Han, Edward K.; Guan, Ran; Johnson, Eric F.; Stoll, Vincent S.; Stewart, Kent D.; Stamper, Geoff; Soni, Nirupama; Bouska, Jennifer J.; Luo, Yan; Sowin, Thomas J.; Lin, Nan-Horng; Giranda, Vincent S.; Rosenberg, Saul H.; Penning, Thomas D.; (Abbott)

    2010-02-19

    Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with subnanomolar to low single-digit nanomolar K{sub i} values and exhibit excellent selectivity against a panel of diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b (PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells with submicromolar EC{sub 50} values. For example, compound 14j inhibited the growth of K562 cells with an EC{sub 50} value of 1.7 {micro}M and showed K{sub i} values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3, respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based. The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes, good permeability, modest protein binding, and no CYP inhibition below 20 {micro}M concentration.

  4. Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs

    DEFF Research Database (Denmark)

    Pallesen, Lone Tjener; Pedersen, Lise Refstrup Linnebjerg; Petersen, Torben Ellebæk

    2008-01-01

    expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions...

  5. Down-regulation of wt1 expression in leukemia cell lines as part of apoptotic effect in arsenic treatment using two compounds.

    Science.gov (United States)

    Glienke, Wolfgang; Chow, Kai U; Bauer, Nina; Bergmann, Lothar

    2006-08-01

    Arsenic trioxide (As2O3) induces remission in patients with acute promyelocytic leukemia (APL). To better understand molecular mechanisms of arsenic actions, this study investigated the effect of two different arsenic compounds on gene expression of apoptosis and cellular proliferation related genes. The Wilms' tumor gene (wt1) is up-regulated in acute myeloid leukemia (AML) and a variety of leukemia cell lines. The expression of wt1 in these cells is proposed to have an anti-apoptotic effect. HL-60 and K562 were treated with arsenic trioxide (As2O3) and sodium arsenite (NaAsO2) at concentrations between 0 - 10 microM for up to 48 h. The induction of apoptosis was accompanied by down-regulation of hTERT and wt1 mRNA and protein expression but up-regulation of par-4. Low concentrations of 0.1 microM arsenic induced expression of the anti-apoptotic bcl-2 gene in both cell lines HL-60 and K562. There were no major differences encountered between compounds. After arsenic treatment of the leukemia cell lines HL-60 and K562 the up-regulation of par-4 may contribute to the induction of apoptosis rather than down-regulation of bcl-2. The therapeutic effect of arsenic is the induction of apoptosis by modulating the gene expression profile of pro- and anti-apoptotic genes including the wt1 gene.

  6. Frequency of chromosomally-integrated human herpesvirus 6 in children with acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Annie Gravel

    Full Text Available INTRODUCTION: Human herpesvirus 6 (HHV-6 is a ubiquitous pathogen infecting nearly 100% of the human population. Of these individuals, between 0.2% and 1% of them carry chromosomally-integrated HHV-6 (ciHHV-6. The biological consequences of chromosomal integration by HHV-6 remain unknown. OBJECTIVE: To determine and compare the frequency of ciHHV-6 in children with acute lymphoblastic leukemia to healthy blood donors. METHODOLOGY: A total of 293 DNA samples from children with pre-B (n=255, pre-pre-B (n=4, pre-T (n=26 and undetermined (n=8 leukemia were analyzed for ciHHV-6 by quantitative TaqMan PCR (QPCR using HHV-6 specific primers and probe. As control, DNA samples from 288 healthy individuals were used. Primers and probe specific to the cellular GAPDH gene were used to estimate integrity and DNA content. RESULTS: Out of 293 DNA samples from the leukemic cohort, 287 contained amplifiable DNA. Of these, only 1 (0.35% contained ciHHV-6. Variant typing indicates that the ci-HHV-6 corresponds to variant A. None of the 288 DNA samples from healthy individuals contained ciHHV-6. CONCLUSION: The frequency of ciHHV-6 in children with acute lymphoblastic leukemia is similar (p=0.5 to that of healthy individuals. These results suggest that acute lymphoblastic leukemia does not originate as a consequence to integration of HHV-6 within the chromosomes.

  7. Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-O-methyl-ascochlorin in leukemia cells.

    Science.gov (United States)

    Shin, Jae-Moon; Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji; Bae, Young-Seuk; Chang, Young-Chae

    2016-05-01

    4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.

  8. Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia.

    Science.gov (United States)

    Stringaris, Kate; Sekine, Takuya; Khoder, Ahmad; Alsuliman, Abdullah; Razzaghi, Bonnie; Sargeant, Ruhena; Pavlu, Jiri; Brisley, Gill; de Lavallade, Hugues; Sarvaria, Anushruthi; Marin, David; Mielke, Stephan; Apperley, Jane F; Shpall, Elizabeth J; Barrett, A John; Rezvani, Katayoun

    2014-05-01

    The majority of patients with acute myeloid leukemia will relapse, and older patients often fail to achieve remission with induction chemotherapy. We explored the possibility that leukemic suppression of innate immunity might contribute to treatment failure. Natural killer cell phenotype and function was measured in 32 consecutive acute myeloid leukemia patients at presentation, including 12 achieving complete remission. Compared to 15 healthy age-matched controls, natural killer cells from acute myeloid leukemia patients were abnormal at presentation, with downregulation of the activating receptor NKp46 (P=0.007) and upregulation of the inhibitory receptor NKG2A (P=0.04). Natural killer cells from acute myeloid leukemia patients had impaired effector function against autologous blasts and K562 targets, with significantly reduced CD107a degranulation, TNF-α and IFN-γ production. Failure to achieve remission was associated with NKG2A overexpression and reduced TNF-α production. These phenotypic and functional abnormalities were partially restored in the 12 patients achieving remission. In vitro co-incubation of acute myeloid leukemia blasts with natural killer cells from healthy donors induced significant impairment in natural killer cell TNF-α and IFN-γ production (P=0.02 and P=0.01, respectively) against K562 targets and a trend to reduced CD107a degranulation (P=0.07). Under transwell conditions, the inhibitory effect of AML blasts on NK cytotoxicity and effector function was still present, and this inhibitory effect was primarily mediated by IL-10. These results suggest that acute myeloid leukemia blasts induce long-lasting changes in natural killer cells, impairing their effector function and reducing the competence of the innate immune system, favoring leukemia survival.

  9. Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells.

    Science.gov (United States)

    Chen, Yu-Jen; Chou, Cheng-Jen; Chang, Tun-Tschu

    2009-04-01

    MMH01 is a compound isolated from Antrodia cinnamomea. MMH01 markedly inhibited growth of human leukemia U937 and pancreatic cancer BxPC3 cells. It resulted in distinct patterns of cell cycle distribution in U937 (G2/M, sub-G1 and polyploidy) and BxPC3 cells (G0/G1 and sub-G1). The modes of cell death in U937 cells include apoptosis and mitotic catastrophe, whereas apoptosis-associated events or necrosis in BxPC3 cells. Neither mitochondrial membrane permeabilization nor caspase dependence was noted. Proteins involving mitotic catastrophe-associated cell death such as cyclin B1 and checkpoint kinase 2 were activated in U937 cells. Only slight to moderate viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. In conclusion, MMH01 possesses cytotoxicity against human leukemia and pancreatic cancer cells.

  10. Analogue peptides for the immunotherapy of human acute myeloid leukemia.

    Science.gov (United States)

    Hofmann, Susanne; Mead, Andrew; Malinovskis, Aleksandrs; Hardwick, Nicola R; Guinn, Barbara-Ann

    2015-11-01

    The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies.

  11. Human T-cell leukemia virus type I (HTLV-I infection and the onset of adult T-cell leukemia (ATL

    Directory of Open Access Journals (Sweden)

    Matsuoka Masao

    2005-04-01

    Full Text Available Abstract The clinical entity of adult T-cell leukemia (ATL was established around 1977, and human T-cell leukemia virus type 1 (HTLV-I was subsequently identified in 1980. In the 25 years since the discovery of HTLV-I, HTLV-I infection and its associated diseases have been extensively studied, and many of their aspects have been clarified. However, the detailed mechanism of leukemogenesis remains unsolved yet, and the prognosis of ATL patients still poor because of its resistance to chemotherapy and immunodeficiency. In this review, I highlight the recent progress and remaining enigmas in HTLV-I infection and its associated diseases, especially ATL.

  12. Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo.

    Science.gov (United States)

    Budhraja, Amit; Gao, Ning; Zhang, Zhuo; Son, Young-Ok; Cheng, Senping; Wang, Xin; Ding, Songze; Hitron, Andrew; Chen, Gang; Luo, Jia; Shi, Xianglin

    2012-01-01

    In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. ©2011 AACR.

  13. Aberrant expression of CKLF-like MARVEL transmembrane member 5 (CMTM5) by promoter methylation in myeloid leukemia.

    Science.gov (United States)

    Niu, Jihong; Li, Henan; Zhang, Yao; Li, Jinlan; Xie, Min; Li, Lingdi; Qin, Xiaoying; Qin, Yazhen; Guo, Xiaohuan; Jiang, Qian; Liu, Yanrong; Chen, Shanshan; Huang, Xiaojun; Han, Wenling; Ruan, Guorui

    2011-06-01

    CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Human AQP5 plays a role in the progression of chronic myelogenous leukemia (CML.

    Directory of Open Access Journals (Sweden)

    Young Kwang Chae

    Full Text Available Aquaporins (AQPs have previously been associated with increased expression in solid tumors. However, its expression in hematologic malignancies including CML has not been described yet. Here, we report the expression of AQP5 in CML cells by RT-PCR and immunohistochemistry. While normal bone marrow biopsy samples (n = 5 showed no expression of AQP5, 32% of CML patient samples (n = 41 demonstrated AQP5 expression. In addition, AQP5 expression level increased with the emergence of imatinib mesylate resistance in paired samples (p = 0.047. We have found that the overexpression of AQP5 in K562 cells resulted in increased cell proliferation. In addition, small interfering RNA (siRNA targeting AQP5 reduced the cell proliferation rate in both K562 and LAMA84 CML cells. Moreover, by immunoblotting and flow cytometry, we show that phosphorylation of BCR-ABL1 is increased in AQP5-overexpressing CML cells and decreased in AQP5 siRNA-treated CML cells. Interestingly, caspase9 activity increased in AQP5 siRNA-treated cells. Finally, FISH showed no evidence of AQP5 gene amplification in CML from bone marrow. In summary, we report for the first time that AQP5 is overexpressed in CML cells and plays a role in promoting cell proliferation and inhibiting apoptosis. Furthermore, our findings may provide the basis for a novel CML therapy targeting AQP5.

  15. RHAMNAZIN INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF HUMAN JURKAT LEUKEMIA CELLS IN VITRO.

    Science.gov (United States)

    Philchenkov, A A; Zavelevych, M P

    2015-01-01

    Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  16. Cytotoxic and apoptotic effects of prenylflavonoid artonin B in human acute lymphoblastic leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Chun-chung LEE; Chun-nan LIN; Guey-mei JOW

    2006-01-01

    Aim: To investigate the anticancer effects and molecular mechanism of artonin B on the human acute lymphoblastic leukemia CCRF-CEM cells compared with other prenylflavonoid compounds. Methods: The effects of four prenylflavonoids on the growth of CCRF-CEM and HaCa cells were studied by 3-(4,5)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis were detected through Hoechst 33258 staining. The effect of artonin B on the cell cycle of CCRF-CEM cells were studied by propidium iodide method. The change in mitochondrial membrane potential was detected by rohdamine 123 staining. The cytochrome c release and caspase 3 activity were checked by immunoassay kits, respectively. The expression of Bcl-2 family proteins was detected by Western blot. Results: Our data revealed that artonin B strongly induced human CCRF-CEM leukemia cell death in a dose- and time-dependent manner by MTT assay, but not on normal epithelia cells (HaCa cells). Artonin B-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by Hoechst 33258 staining. The induction of human CCRF-CEM leukemia cancer cell death was caused by an induction of apoptosis through mitochondrial membrane potential change, cytochrome c release, sub-G1 proportion increase, downregulation of Bcl-2 expression, upregulation of Bax and Bak expression and activation of caspase 3 pathways. Conclusion: These results clearly demonstrated that artonin B is able to inhibit proliferation by induction of hypoploid cells and cell apoptosis. Moreover, the anticancer effects of artonin B were related to mitochondrial pathway and caspase 3 activation in human CCRF-CEM leukemia cells.

  17. Significance of murine retroviral mutagenesis for identification of disease genes in human acute myeloid leukemia.

    Science.gov (United States)

    Erkeland, Stefan J; Verhaak, Roel G W; Valk, Peter J M; Delwel, Ruud; Löwenberg, Bob; Touw, Ivo P

    2006-01-15

    Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia.

  18. 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.

  19. 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.

  20. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

    Science.gov (United States)

    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  1. SLAM family predicting the initiation potential of human acute lymphoblastic leukemia in NOD/SCID mice

    Institute of Scientific and Technical Information of China (English)

    WANG Na; ZHOU Jian-feng; HUANG Liang; XIAO Fei; LIU Jin-ping; WANG Di; GENG Zhe; WANG Jin; MA Shu-yan; SHU Li-li; CHEN Tai-ping

    2011-01-01

    Background The SLAM family recently has been reported to show an important biological role in lymphocyte development and immunological function, and it is efficient to highly purify hematopoietic stem cells using a simple combination of SLAM family members. To elucidate the presence of this family on acute lymphoblastic leukemia (ALL),as well as its relationship with the leukemia-initiating potential, we analyzed the expression pattern of this family members on human ALL progenitor cells, combined with serial xenotransplantation assay.Methods Expression analysis was carried out by flow cytometry. We combined the expression pattern of human CD150,CD244 and CD48 with serial xenotransplantation of B-ALL progenitor cells to indicate their relationship.Results CD48 and CD244 were expressed on most B-ALL progenitor cells, the percentage being (93.08±6.46)% and (63.37±29.31)%, respectively. Interestingly, the proportion of CD150+ cells declined obviously in engrafted cases ((24.94±7.32)%) compared with non-engrafted cases ((77.54±5.93)%, P <0.01), which indicated that only blast cells with low percentage of CD150+ population were able to reconstitute leukemia into primary, secondary and tertiary NOD/SCID mice.Conclusions SLAM family members are present on B-ALL progenitor cells and the leukemia-initiating potential of leukemic blasts is correlated negatively with the proportion of CD150+ cells, the percentage of which can serve as a useful predictor for engraftment success of B-ALL to immune deficient mice.

  2. microRNA-1271靶向调控白血病细胞CYLD蛋白的表达%microRNA-1271 regulates the expression of CYLD in leukemia cells

    Institute of Scientific and Technical Information of China (English)

    张茜; 王媛媛; 刘丽萍; 倪芳

    2015-01-01

    Objective To explore the regulatory effect of microRNA-1271 on the expression of its target gene CYLD in leukemia cells. Methods microRNA-1271 expression levels in normal peripheral blood mononuclear cells( PB-MC) and leukemia cells were determined by quantitative real-time PCR. microRNA-1271 targeting CYLD 3′-UTR was predicted by TargetScan. 3′-UTR of CYLD was inserted into the dual luciferase reporter vector psi-CHECK2. The reporter activity was evaluated by the dual Luciferase Reporter Assay System after the luciferase promoter vector and microRNA were co-transferred into the HEK293A cell line. K562 cell lines were transfected with microRNA-1271 inhibitors ( anti-microRNA-1271 ) or a negative control microRNA ( anti-microRNA-NC ) , the expression of CYLD protein in the above transfected K562 cells were determined by Western blot. Results microRNA-1271 was up-regulated in human leukemia cell lines and primary leukemia cells compared to normal human PBMCs. The re-sults of dual luciferase assays validate CYLD as a specific target gene of microRNA-1271 . Inhibition of microRNA-1271 resulted in the upregulation of CYLD protein expression in K562 cell line. Conclusion microRNA-1271 is overexpressed in leukemia cells, the microRNA-1271 abnormal overexpression may play a key role in leukemia due to the down-regulation of CYLD.%目的探讨人白血病细胞中微小RNA(microRNA)-1271对CYLD蛋白表达的调控作用。方法 qRT-PCR检测microRNA-1271在不同的人白血病细胞系、临床初诊未治的几种类型原代白血病细胞、正常人外周血单个核细胞中的表达差异,利用 Targetscan 信息学预测软件预测 microRNA-1271靶向CYLD基因,构建携带靶基因野生型及突变型3’非翻译区(3’UTR)(缺失了整段预测的microRNA-1271结合序列)的双荧光素酶报告基因质粒,采用脂质体 Lipo-fectamine 3000包裹双荧光素酶重组质粒及 microRNA-1271模拟物(mimic)或阴性对照,共转染HEK293A细胞,应用

  3. Nanoassemblies from amphiphilic cytarabine prodrug for leukemia targeted therapy.

    Science.gov (United States)

    Liu, Jing; Zhao, Dujuan; He, Wenxiu; Zhang, Huiyuan; Li, Zhonghao; Luan, Yuxia

    2017-02-01

    The anti-leukemia effect of cytarabine (Ara-C) is severely restricted by its high hydrophilic properties and rapid plasma degradation. Herein, a novel amphiphilic small molecular prodrug of Ara-C was developed by coupling a short aliphatic chain, hexanoic acid (HA) to 4-NH2 of the parent drug. Based on the amphiphilic nature, the resulting bioconjugate (HA-Ara) could spontaneously self-assemble into stable spherical nanoassemblies (NAs) with an extremely high drug loading (∼71wt%). Moreover, folate receptor (FR)-targeting NAs with high grafting efficient folic acid - bovine serum albumin (FA-BSA) conjugate immobilized on the surface (NAs/FA-BSA) was prepared. The results of MTT assays on FR-positive K562 cells and FR-negative A549 cells demonstrated higher cytotoxicity of HA-Ara NAs than the native drug. Especially, the IC50 values revealed that NAs/FA-BSA was 3 and 2-fold effective than non-targeted NAs after 24 and 48h treatment with K562 cells, respectively indicating FR-mediated enhanced anti-tumor efficacy. In vitro cellular uptake, larger accumulation of HA-Ara NAs were observed in comparative with the free FITC and the results further confirmed the selective uptake of NAs/FA-BSA in folate receptor enriched cancer cells. Above all, self-assembled HA-Ara NAs exhibited potential superiority for Ara-C delivery and FA-modified NAs would be an excellent candidate for targeting leukemia therapy.

  4. METHYLATION PATTERN OF LRP15 GENE IN LEUKEMIA

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis.Methods The methylation of LRP15 promoter and first exon of bone marrow mononuclear cells in 73 patients with AL, 10 with chronic leukemia (CL), 9 with hematological benign diseases, and 20 healthy transplantation donors was analyzed by using methylation specific polymerase chain reaction. The methylation of LRP15 gene promoter and first exon in COS7, K562, and HL60 cell lines was also assayed.Results No LRP15 gene promoter methylation was detected in COS7 cell line. LRP15 gene promoter was methylated in K562 and HL60 cell lines. No deletion of LRP15 gene was detected in all samples. In nearly all French-American-British leukemia subtypes, we found that frequency of LRP15 methylation in adult patients with AL was 71.23%( 52/73 ). There was no detectable methylation in any of the 20 healthy donors and 8 chronic myeloid leukemia patients.The difference in frequency of LRP15 methylation between AL patients and healthy donors or CL patients ( 10. 00%,1/10) was significant (P < 0. 01 ). Hypermethylation of LRP15 gene was found in 57. 14% (16/28) of newly diagnosed AL patients, 83.33% of relapsed AL patients respectively, which was significantly different ( P < 0. 05). We also demonstrated LRP15 methylation in 55.56% (5/9) adults with benign hematological diseases.Conclusions LRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.

  5. Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Marrero, Maria Teresa; Estevez, Sara; Negrin, Gledy; Quintana, Jose [Departamento de Bioquimica, Unidad Asociada al Consejo Superior de Investigaciones Cientificas, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria (Spain); Lopez, Mariana; Perez, Francisco J.; Triana, Jorge [Departamento de Quimica, Universidad de Las Palmas de Gran Canaria, Instituto Canario de Investigacion del Cancer, 35017 Las Palmas de Gran Canaria (Spain); Leon, Francisco [Instituto de Productos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Avda. Astrofisico F. Sanchez 3, 38206 La Laguna, Tenerife (Spain); Estevez, Francisco, E-mail: festevez@dbbf.ulpgc.es [Departamento de Bioquimica, Unidad Asociada al Consejo Superior de Investigaciones Cientificas, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria (Spain)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Ayanin diacetate as apoptotic inducer in leukemia cells. Black-Right-Pointing-Pointer Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x{sub L}. Black-Right-Pointing-Pointer The intrinsic and the extrinsic pathways are involved in the mechanism of action. Black-Right-Pointing-Pointer Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G{sub 2}-M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the overexpression of Bcl-x{sub L}. Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

  6. Matrine suppresses cell growth of human chronic myeloid leukemia cells via its inhibition of the interleukin-6/Janus activated kinase/signal transducer and activator of transcription 3 signaling cohort.

    Science.gov (United States)

    Ma, Lingdi; Zhu, Zhichao; Jiang, Lijia; Sun, Xiao; Lu, Xuzhang; Zhou, Min; Qian, Sixuan; Jianyong, Li

    2015-01-01

    Matrine, alkaloid isolated from Sophora flavescens, is known to be pleiotropic by exerting anti-inflammatory, anti-oxidation, as well as anti-cancer effects. However, the precise molecular targets or pathways responsible for its activities still remain unclear. The present study aimed to determine the underlying mechanisms of matrine in inhibiting the chronic myeloid leukemia cells (CML). It was observed that matrine treatment significantly suppressed CML cells proliferation, induced apoptosis and resulted in the accumulation of cells in the G0/G1 phase, accompanied by a significant decrease in Bcl-xL, Cyclin D1, and c-Myc expression. Western blot analyses revealed that matrine treatment resulted in the down-regulation in phospho-STAT3 and phospho-JAK2 without significantly effects on STAT3 and JAK2 protein levels. Matrine significantly reduced the expression of IL-6, a potent upstream activating factor of STAT3. These results strongly suggested the IL-6/JAK/STAT3 pathway play an important role in matrine's anti-leukemia effects in K562 cells.

  7. Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Shao-Qing Kuang

    Full Text Available The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL. We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2'-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.

  8. Effects of inorganic and organic arsenic compounds on growth and apoptosis of human T-lymphoblastoid leukemia cells.

    Science.gov (United States)

    Hikita, Eri; Arai, Mariko; Tanaka, Sachiko; Onda, Kenji; Utsumi, Hiroya; Yuan, Bo; Toyoda, Hiroo; Hirano, Toshihiko

    2011-12-01

    To investigate the effects of inorganic and organic arsenic compounds on human T-lymphoblastoid leukemia cells. Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5¬diphenyltetrazolium bromide (MTT) assay. Apoptotic cell morphology was examined by cell staining with Hoechst 33342. Cellular caspase-3/7 activities were measured after arsenic treatment. The inhibitory concentration by 50% (IC(50)) values of As(2)O(3) towards MOLT-4 and daunorubicin- resistant MOLT-4/DNR cell proliferation were 0.87 and 0.92 μM, while the values for arsenic acid were 69.1 and 116.6 μM, respectively. These arsenic compounds also inhibited mitogen-induced proliferation of human peripheral blood mononuclear cells. Six organic arsenic compounds did not inhibit leukemia cell proliferation. As(2)O(3) and arsenic acid induced apoptotic cell morphology and increased caspase-3/7 activity in the leukemia cells. Ascorbic acid and buthionine sulfoxide enhanced, while N-acetyl-L-cysteine abated, the suppressive effects of inorganic arsenic compounds on leukemia cell proliferation. As(2)O(3) and arsenic acid inhibit proliferation and induce apoptosis in MOLT-4 and daunorubicine-resistant MOLT-4/DNR cells via glutathione-depletion and subsequent caspase-3/7 activation. Organic arsenic compounds have no inhibitory activity on the leukemia cell proliferation. Inorganic arsenic compounds are suggested as useful agents for treatment of T-lymphoblastoid leukemia.

  9. An animal model of adult T-cell leukemia: humanized mice with HTLV-1-specific immunity.

    Science.gov (United States)

    Tezuka, Kenta; Xun, Runze; Tei, Mami; Ueno, Takaharu; Tanaka, Masakazu; Takenouchi, Norihiro; Fujisawa, Jun-ichi

    2014-01-16

    Human T-cell leukemia virus type 1 (HTLV-1) is causally associated with adult T-cell leukemia (ATL), an aggressive T-cell malignancy with a poor prognosis. To elucidate ATL pathogenesis in vivo, a variety of animal models have been established; however, the mechanisms driving this disorder remain poorly understood due to deficiencies in each of these animal models. Here, we report a novel HTLV-1-infected humanized mouse model generated by intra-bone marrow injection of human CD133(+) stem cells into NOD/Shi-scid/IL-2Rγc null (NOG) mice (IBMI-huNOG mice). Upon infection, the number of CD4(+) human T cells in the periphery increased rapidly, and atypical lymphocytes with lobulated nuclei resembling ATL-specific flower cells were observed 4 to 5 months after infection. Proliferation was seen in both CD25(-) and CD25(+) CD4 T cells with identical proviral integration sites; however, a limited number of CD25(+)-infected T-cell clones eventually dominated, indicating an association between clonal selection of infected T cells and expression of CD25. Additionally, HTLV-1-specific adaptive immune responses were induced in infected mice and might be involved in the control of HTLV-1-infected cells. Thus, the HTLV-1-infected IBMI-huNOG mouse model successfully recapitulated the development of ATL and may serve as an important tool for investigating in vivo mechanisms of ATL leukemogenesis and evaluating anti-ATL drug and vaccine candidates.

  10. Identification of a novel human tRNA(Ser(CGA)) functional in murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmitz, A; Pedersen, F S

    2000-01-01

    (CGA)) was detected in cell lines of human, monkey and mouse origin. The UCG codon is the most rarely used codon in human genes. The cloned human tRNA(Ser(CGA)) gene encodes an 85 nucleotide, intron-less tRNA, contains a consensus split intragenic promoter and is located at region p21.3-22.2 on chromosome 6......We have identified a human tRNA(Ser) isoacceptor matching the UCG codon. The tRNA was discovered via its ability to act in reverse transcription of a murine leukemia virus vector containing a complementary tRNA primer binding site (Lund et al., Nucleic Acids Res., 28 (2000) 791-799). The tRNA(Ser....... The integrity and functionality of the cloned tRNA(Ser(CGA)) gene was verified by in vitro transcription analysis in HeLa nuclear extracts....

  11. The human CD38 monoclonal antibody daratumumab shows antitumor activity and hampers leukemia-microenvironment interactions in chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Matas-Céspedes, Alba; Vidal-Crespo, Anna; Rodriguez, Vanina

    2017-01-01

    Purpose: To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ chronic lymphocytic leukemia (CLL) subtype. Experimental Design: The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blo...

  12. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  13. Rhamnazin inhibits proliferation and induces apoptosis of human jurkat leukemia cells in vitro

    Directory of Open Access Journals (Sweden)

    А. А. Philchenkov

    2015-12-01

    Full Text Available Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  14. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    Science.gov (United States)

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S.; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam

    2016-01-01

    ABSTRACT The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV. IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level

  15. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

    Science.gov (United States)

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam; Neil, James C

    2017-03-01

    The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most

  16. Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells.

    Science.gov (United States)

    Conti, Anastasia; Rota, Federica; Ragni, Enrico; Favero, Chiara; Motta, Valeria; Lazzari, Lorenza; Bollati, Valentina; Fustinoni, Silvia; Dieci, Giorgio

    2016-06-10

    Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies.

  17. Epidemiology, treatment, and prevention of human T-cell leukemia virus type 1-associated diseases.

    Science.gov (United States)

    Gonçalves, Denise Utsch; Proietti, Fernando Augusto; Ribas, João Gabriel Ramos; Araújo, Marcelo Grossi; Pinheiro, Sônia Regina; Guedes, Antônio Carlos; Carneiro-Proietti, Anna Bárbara F

    2010-07-01

    Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus to be discovered, is present in diverse regions of the world, where its infection is usually neglected in health care settings and by public health authorities. Since it is usually asymptomatic in the beginning of the infection and disease typically manifests later in life, silent transmission occurs, which is associated with sexual relations, breastfeeding, and blood transfusions. There are no prospects of vaccines, and screening of blood banks and in prenatal care settings is not universal. Therefore, its transmission is active in many areas such as parts of Africa, South and Central America, the Caribbean region, Asia, and Melanesia. It causes serious diseases in humans, including adult T-cell leukemia/lymphoma (ATL) and an incapacitating neurological disease (HTLV-associated myelopathy/tropical spastic paraparesis [HAM/TSP]) besides other afflictions such as uveitis, rheumatic syndromes, and predisposition to helminthic and bacterial infections, among others. These diseases are not curable as yet, and current treatments as well as new perspectives are discussed in the present review.

  18. Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with imatinib mesylate against chronic myelogenous leukemia cell lines

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    Xin P

    2017-04-01

    Full Text Available Pengliang Xin, Chuntuan Li, Yan Zheng, Qunyi Peng, Huifang Xiao, Yuanling Huang, Xiongpeng Zhu Department of Haematology, First Hospital of Quanzhou Affiliated to Fujian Medical University, Licheng, Quanzhou, Fujian Province, China Background: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway is a therapy target of cancer. We aimed to confirm the effect of dual PI3K/mTOR inhibitor NVP-BEZ235 on proliferation, apoptosis, and autophagy of chronic myelogenous leukemia (CML cells and sensitivity of tyrosine kinase inhibitor in vitro.Methods: Two human CML cell lines, K562 and KBM7R (T315I mutant strain, were used. The proliferation of CML cells was detected by MTS (Owen’s reagent assay. Cell cycle and apoptosis assay were examined by flow cytometric analysis. The phosphorylation levels and the expression levels were both evaluated by Western blot analysis. NVP-BEZ235 in combination with imatinib was also used to reveal the effect on proliferation and apoptosis.Results: NVP-BEZ235 significantly inhibited the proliferation in a time- and dose-dependent manner, and the half-maximal inhibitory concentration values of NVP-BEZ235 inhibiting the proliferation of K562 and KBM7R were 0.37±0.21 and 0.43±0.27 µmol/L, respectively, after 48 h. Cell apoptosis assay showed that NVP-BEZ235 significantly increased the late apoptotic cells. Cell cycle analysis indicated that the cells were mostly arrested in G1/G0 phase after treatment by NVP-BEZ235. In addition, results also found that, after treatment by NVP-BEZ235, phosphorylation levels of Akt kinase and S6K kinase significantly reduced, and the expression levels of cleaved caspase-3 significantly increased; meanwhile, the expression levels of caspase-3, B-cell lymphoma-2, cyclin D1, and cyclin D2 significantly decreased, and the ratio of LC3II/LC3I was significantly increased with increased LC3II expression level. Moreover, imatinib in combination with NVP-BEZ235

  19. The Possible Association between Constitutive Heterochromatin Polymorphism and Human Leukemias

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    Abolfazl Movafagh

    2007-01-01

    Full Text Available Objective: Polymorphism of the size of heterochromatin region of chromosomes has been well documented in human genome and it consists of DNA sequences that are not transcribed. The prime aim of the present study was to evaluate the heterochromatin polymorphism associated with chromosomes in leukemic patients.Materials and Methods: The study was conducted on 35 consecutive leukemic patients and 34 healthy individuals in Modaress and Taleghani hospitals, Tehran, Iran between 2004-2006. By applying Barium Hydroxide saline Giemsa (BSC method with certain alterations, the variant heterochromatin polymorphism of chromosomes 1, 9 and 16 on bone marrow and peripheral blood lymphocyte cultures were evaluated. Chi-square and Fisher’s exact tests were used for statistical analysis with SPSS software.Results: Constitutive heterochromatin polymorphism of chromosomes 1 and 9 in leukemic patients revealed statistical significant differences when compared with chromosomes of healthy controls (p=0.0005 and (p=0.006 respectively. The differences were not significant for chromosome 16, it was 11.4% in leukemic patients and 0% in the control group (p=0.05. The frequency of partial and complete inversions did not show any significant differences between the leukemic patients and the control group.Conclusion: The constitutive heterochromatin polymorphism blocks may provide an opportunity to serve as a marker for the detection and characterization of the chromosomes in leukemic patients.

  20. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin(+)) and leukemia stem cell population (CD34(+)CD38(-)Lin(-/low)). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7μM) and G2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Apigenin decreases cell viability and telomerase activity in human leukemia cell lines.

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    Jayasooriya, R G P T; Kang, Sang-Hyuck; Kang, Chang-Hee; Choi, Yung Hyun; Moon, Dong-Oh; Hyun, Jin-Won; Chang, Weon-Young; Kim, Gi-Young

    2012-08-01

    Recent studies have shown that apigenin (4',5,7-trihydroxyflavone inhibits human malignant cancer cell growth through cell cycle arrest and apoptosis. However, the underlying relationship between apoptosis and telomerase activity in response to apigenin exposure is not well understood. In this study, we found that apigenin significantly induces direct cytotoxicity in human leukemia cells (U937, THP-1 and HL60) through activation of the caspase pathway. As we presumed, treatment with apigenin was found to increase the level of intracellular reactive oxygen species (ROS), whereas pretreatment with antioxidants, N-acetyl-cysteine (NAC) or glutathione (GSH), completely attenuated ROS generation. Surprisingly, these antioxidants did not promote recuperation from apigenin-induced cell death. We further showed that apigenin downregulates telomerase activity in caspase-dependent apoptosis and observed that apigenin dosing results in downregulation of telomerase activity by suppression of c-Myc-mediated telomerase reverse transcriptase (hTERT) expression. In addition, treatment of apigenin-dosed cells with the two antioxidants did not restore telomerase activity. Taken together, this data suggests that ROS is not essential for suppression of apigenin-mediated apoptosis associated with the activation of caspases and regulation of telomerase activity via suppression of hTERT. We conclude that apigenin has a direct cytotoxic effect and the loss of telomerase activity in leukemia cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.

    Science.gov (United States)

    Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Ishikawa, J; Horikawa, Y; Hashimoto, K; Moriyama, Y; Tsujimura, T; Nishiura, T

    1995-07-15

    Thrombopoietin (TPO) is a newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto-oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c-mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

  3. Effect of dioxin on normal and leukemic human hematopoietic cells

    Energy Technology Data Exchange (ETDEWEB)

    Lambertenghi-Deliliers, G.; Soligo, D. [Univ. degli Studi, Milan (Italy). Dipt. die Ematologia, Ospedale Maggiore Policlinico IRCCS; Fracchiolla, N.S. [Ospedale Maggiore Policlinico IRCCS, Milan (Italy). Dipt. di Ematologia; Servida, F. [Fondazione Matarelli, Milan (Italy); Bertazzi, P.A. [Istituti Clinici di Perfezionamento, Milan (Italy). Dipt. di Medicina del Lavoro

    2004-09-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) arises from chlorination of phenolic substrates or from partial combustion of organic materials in the presence of chlorine sources. TCDD has a large number of biological effects such as long-lasting skin disease, cardiovascular disease, diabete and cancer. TCDD is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes the receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of some genes including cytokines and growth factors. In this study, we analyzed the effect of escalating doses of TCDD on human CD34{sup +} progenitor cells from the leukapheresis of normal donors stimulated with G-CSF as well as the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia). The possible specific modulation of gene expression induced by the TCDD exposure was then tested by means of microarray analyses.

  4. The dog as a model for comparative studies of lymphoma and leukemia in humans 

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    Aleksandra Pawlak

    2013-05-01

    Full Text Available Dogs have accompanied humankind for thousands of years. They share the same environment, and thus are exposed to the same environmental factors such as air pollution, tobacco smoke, and various chemicals. Recent development of veterinary care has led to a significant extension of dogs’ lifespan and allowed the diagnosis and treatment of a growing number of different diseases in this species. Among all diseases in dogs, cancer is considered the main cause of mortality, with lymphoproliferative disorders accounting for up to 30�0of all canine cancers. Some of them, such as non-Hodgkin lymphoma (NHL and lymphocytic leukemia, are very similar in the etiology, pathogenesis and response to treatment to the diseases occurring in humans. Due to anatomical and physiological similarities to humans, the dog is a useful model for the study of new therapeutic strategies for humans. Studies on the canine neoplasia are currently limited by the lack of well-characterized and widely available cell lines; thus, recently obtained canine NHL cell lines may become a valuable model for such studies. Investigation of their sensitivity to the antiproliferative effects of different factors should allow the creation of a database similar to the existing classification of human leukemias and lymphomas. This should enable quick and accurate diagnosis and selection of appropriate treatment based on phenotypic analysis and histopathological examination of clinical samples. The cooperation between human and veterinary oncologists gives the opportunity to use the dog as a model for the study of certain types of cancers presenting a challenge for modern medicine.

  5. MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

    Science.gov (United States)

    Xishan, Zhu; Ziying, Lin; Jing, Du; Gang, Liu

    2015-01-01

    Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

  6. Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

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    Houman Kahroba

    2016-07-01

    Full Text Available Background Human leukemia inhibitory factor (hLIF is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG used for induction. Single step immobilized metal affinity chromatography (IMAC used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

  7. Heterogeneous nuclear expression of the promyelocytic leukemia (PML) protein in normal and neoplastic human tissues.

    Science.gov (United States)

    Gambacorta, M.; Flenghi, L.; Fagioli, M.; Pileri, S.; Leoncini, L.; Bigerna, B.; Pacini, R.; Tanci, L. N.; Pasqualucci, L.; Ascani, S.; Mencarelli, A.; Liso, A.; Pelicci, P. G.; Falini, B.

    1996-01-01

    The RING-finger promyelocytic leukemia (PML) protein is the product of the PML gene that fuses with the retinoic acid receptor-alpha gene in the t(15; 17) translocation of acute promyelocytic leukemia. Wild-type PML localizes in the nucleus with a typical speckled pattern that is a consequence of the concentration of the protein within discrete subnuclear domains known as nuclear bodies. Delocalization of PML from nuclear bodies has been documented in acute promyelocytic leukemia cells and suggested to contribute to leukemogenesis. In an attempt to get new insights into the function of the wild-type PML protein and to investigate whether it displays an altered expression pattern in neoplasms other than acute promyelocytic leukemia, we stained a large number of normal and neoplastic human tissues with a new murine monoclonal antibody (PG-M3) directed against the amino-terminal region of PML. As the PG-M3 epitope is partially resistant to fixatives, only cells that overexpress PML are detected by the antibody in microwave-heated paraffin sections. Among normal tissues, PML was characteristically up-regulated in activated epithelioid histiocytes and fibroblasts in a variety of pathological conditions, columnar epithelium in small active thyroid follicles, well differentiated foamy cells in the center of sebaceous glands, and hypersecretory endometria (Arias-Stella). Interferons, the PML of which is a primary target gene, and estrogens are likely to represent some of the cytokines and/or hormones that may be involved in the up-regulation of PML under these circumstances. In keeping with this concept, we found that PML is frequently overexpressed in Hodgkin and Reed-Sternberg cells of Hodgkin's disease, a tumor of cytokine-producing cells. Among solid tumors, overexpression of PML was frequently found in carcinomas of larynx and thyroid (papillary), epithelial thymomas, and Kaposi's sarcoma, whereas carcinomas of the lung, thyroid (follicular), breast, and colon were

  8. Mouse xenograft modeling of human adult acute lymphoblastic leukemia provides mechanistic insights into adult LIC biology

    Science.gov (United States)

    Dey, Aditi; Castleton, Anna Z.; Schwab, Claire; Samuel, Edward; Sivakumaran, Janani; Beaton, Brendan; Zareian, Nahid; Zhang, Christie Yu; Rai, Lena; Enver, Tariq; Moorman, Anthony V.; Fielding, Adele K.

    2014-01-01

    The distinct nature of acute lymphoblastic leukemia (ALL) in adults, evidenced by inferior treatment outcome and different genetic landscape, mandates specific studies of disease-initiating mechanisms. In this study, we used NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches to elucidate leukemia-initiating cell (LIC) biology in primary adult precursor B (pre-B) ALL to optimize disease modeling. In contrast with xenografting studies of pediatric ALL, we found that modification of the NSG host environment using preconditioning total body irradiation (TBI) was indispensable for efficient engraftment of adult non-t(4;11) pre-B ALL, whereas t(4;11) pre-B ALL was successfully reconstituted without this adaptation. Furthermore, TBI-based xenotransplantation of non-t(4;11) pre-B ALL enabled detection of a high frequency of LICs (<1:6900) and permitted frank leukemic engraftment from a remission sample containing drug-resistant minimal residual disease. Investigation of TBI-sensitive stromal-derived factor-1/chemokine receptor type 4 signaling revealed greater functional dependence of non-t(4;11) pre-B ALL on this niche-based interaction, providing a possible basis for the differential engraftment behavior. Thus, our studies establish the optimal conditions for experimental modeling of human adult pre-B ALL and demonstrate the critical protumorogenic role of microenvironment-derived SDF-1 in regulating adult pre-B LIC activity that may present a therapeutic opportunity. PMID:24825861

  9. Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human

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    Burny Arsène

    2007-03-01

    Full Text Available Abstract In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV, belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1. This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

  10. In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice

    NARCIS (Netherlands)

    M. von Bonin (Malte); M. Wermke (Martin); K.N. Cosgun (Kadriye Nehir); C. Thiede; M. Bornhäuser (Martin); G. Wagemaker (Gerard); C. Waskow (Claudia)

    2013-01-01

    textabstractHuman cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface

  11. KRAS (G12D Cooperates with AML1/ETO to Initiate a Mouse Model Mimicking Human Acute Myeloid Leukemia

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    Shanmin Zhao

    2014-01-01

    Full Text Available Background/Aims: It has been demonstrated that KRAS mutations represent about 90% of cancer-associated mutations, and that KRAS mutations play an essential role in neoplastic transformation. Cancer-associated RAS mutations occur frequently in acute myeloid leukemia (AML, suggesting a functional role for Ras in leukemogenesis. Methods: We successfully established a mouse model of human leukemia by transplanting bone marrow cells co-transfected with the K-ras (G12D mutation and AML1/ETO fusion protein. Results: Mice transplanted with AML/ETO+KRAS co-transduced cells had the highest mortality rate than mice transplanted with AML/ETO- or KRAS-transduced cells (115d vs. 150d. Upon reaching a terminal disease stage, EGFP-positive cells dominated their spleen, lymph nodes, peripheral blood and central nervous system tissue. Immunophenotyping, cytologic analyses revealed that AML/ETO+KRAS leukemias predominantly contained immature myeloid precursors (EGFP+/c-Kit+/Mac-1-/Gr-1-. Histologic analyses revealed that massive leukemic infiltrations were closely packed in dense sheets that effaced the normal architecture of spleen and thymus in mice transplanted with AML1/ETO + KRAS co-transduced cells. K-ras mRNA and protein expression were upregulated in bone marrow cells of the K-ras group and AML1/ETO + Kras group. The phosphorylation of MEK/ERK was significantly enhanced in the AML1/ETO + Kras group. The similar results of the AML1/ETO + Nras group were consistent with those reported previously. Conclusion: Co-transduction of KrasG12D and AML1/ETO induces acute monoblastic leukemia. Since expression of mutant K-ras alone was insufficient to induce leukemia, this model may be useful for investigating the multi-step leukemogenesis model of human leukemia.

  12. Identification of small molecules that support human leukemia stem cell activity ex vivo.

    Science.gov (United States)

    Pabst, Caroline; Krosl, Jana; Fares, Iman; Boucher, Geneviève; Ruel, Réjean; Marinier, Anne; Lemieux, Sébastien; Hébert, Josée; Sauvageau, Guy

    2014-04-01

    Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.

  13. 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.

  14. The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

    Science.gov (United States)

    Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

    2012-09-01

    We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

  15. Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

    Energy Technology Data Exchange (ETDEWEB)

    Hosono, Makoto; Takaori-Kondo, Akifumi; Zheng-Sheng, Yao; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Imada, Kazunori; Okuma, Minoru; Uchiyama, Takashi; Konishi, Junji

    1995-10-01

    Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered {sup 125}I- and {sup 111}In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using {sup 111}In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL.

  16. Induction of apoptosis in human myeloid leukemia cells by remote exposure of resistive barrier cold plasma.

    Science.gov (United States)

    Thiyagarajan, Magesh; Anderson, Heather; Gonzales, Xavier F

    2014-03-01

    Cold atmospheric plasma (CAP), an ambient temperature ionized gas, is gaining extensive interest as a promising addition to anti-tumor therapy primarily due to the ability to generate and control delivery of electrons, ions, excited molecules, UV photons, and reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) to a specific site. The heterogeneous composition of CAP offers the opportunity to mediate several signaling pathways that regulate tumor cells. Consequently, the array of CAP generated products has limited the identification of the mechanisms of action on tumor cells. The aim of this work is to assess the cell death response of human myeloid leukemia cells by remote exposure to CAP generated RNS by utilizing a novel resistive barrier discharge system that primarily produces RNS. The effect of variable treatments of CAP generated RNS was tested in THP-1 cell (human monocytic leukemia cell line), a model for hematological malignancy. The number of viable cells was evaluated with erythrosine-B staining, while apoptosis and necrosis was assessed by endonuclease cleavage observed by agarose gel electrophoresis and detection of cells with the exclusionary dye propidium iodide and fluorescently labeled annexin-V by flow cytometry and fluorescent microscopy. Our observations indicate that treatment dosage levels of 45 s of exposure to CAP emitted RNS-induced apoptotic cell death and for higher dosage conditions of ≥50 s of exposure to CAP induced necrosis. Overall the results suggest that CAP emitted RNS play a significant role in the anti-tumor potential of CAP.

  17. Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance.

    Science.gov (United States)

    Di Cagno, Raffaella; De Angelis, Maria; Lavermicocca, Paola; De Vincenzi, Massimo; Giovannini, Claudio; Faccia, Michele; Gobbetti, Marco

    2002-02-01

    Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination

  18. A prototype nonpeptidyl, hydrazone class, thrombopoietin receptor agonist, SB-559457, is toxic to primary human myeloid leukemia cells.

    Science.gov (United States)

    Kalota, Anna; Gewirtz, Alan M

    2010-01-07

    Biologic characterization of SB-559457 (SB), a nonpeptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity toward human leukemia cells. Antiproliferative effects followed by significant, nonapoptotic, cell death within 72 hours occurred in 24 of 26 acute myeloid leukemia, 0 of 6 acute lymphoblastic leukemia, and 3 of 6 chronic myeloid leukemia patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB-, but not in rhTpo-, treated cells. Expression profiling of cells exposed to SB versus rhTpo revealed statistically significant, more than 2-fold changes in GAPDH and REDD1 gene expression, confirmed by quantitative reverse-transcribed polymerase chain reaction. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB-induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful antileukemic agents by virtue of their combined thrombopoietic and antileukemic effects.

  19. Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

    Directory of Open Access Journals (Sweden)

    Fato Romana

    2011-04-01

    Full Text Available Abstract Background We previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells. Methods We tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours. Results A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50 included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50. Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential. Conclusion Our study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

  20. Suppression of HSP27 increases the anti-tumor effects of quercetin in human leukemia U937 cells

    Science.gov (United States)

    CHEN, XI; DONG, XIU-SHUAI; GAO, HAI-YAN; JIANG, YONG-FANG; JIN, YING-LAN; CHANG, YU-YING; CHEN, LI-YAN; WANG, JING-HUA

    2016-01-01

    Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti-cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV-G-NR-U6-shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis-associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti-cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia. PMID:26648539

  1. Suppression of HSP27 increases the anti‑tumor effects of quercetin in human leukemia U937 cells.

    Science.gov (United States)

    Chen, Xi; Dong, Xiu-Shuai; Gao, Hai-Yan; Jiang, Yong-Fang; Jin, Ying-Lan; Chang, Yu-Ying; Chen, Li-Yan; Wang, Jing-Hua

    2016-01-01

    Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti‑cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV‑G‑NR‑U6‑shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis‑associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti‑cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia.

  2. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation

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    Jia-Wei Hsu

    2011-01-01

    Full Text Available Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  3. High concentrations of L-ascorbic acid specifically inhibit the growth of human leukemic cells via downregulation of HIF-1α transcription.

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    Hiroshi Kawada

    Full Text Available We examined the antileukemic effects of high concentrations of L-ascorbic acid (high AA on human leukemic cells. In vitro, high AA markedly induced apoptosis in various leukemic cell lines by generating hydrogen peroxide (H2O2 but not in normal hematopoietic stem/progenitor cells. High AA significantly repressed leukemic cell proliferation as well as neoangiogenesis in immunodeficient mice. We then noted that in leukemic cells, HIF-1α transcription was strongly suppressed by high AA and correlated with the transcription of VEGF. Our data indicate that exposure to high AA markedly increased the intracellular AA content of leukemic cells and inhibited the nuclear translocation of NF-κB, which mediates expression of HIF-1α. We next generated K562 cells that overexpressed HIF-1α (K562-HIF1α cells and assessed the mechanistic relationship between inhibition of HIF-1α transcription and the antileukemic effect of high AA. The ability of high AA to induce apoptosis was significantly lower in K562-HIF1α cells than in K562 cells in vitro. We found that expression of HIF-1α-regulated antiapoptotic proteins of the Bcl-2 family, such as Mcl-1, Bcl-xL, and Bcl-2, was significantly suppressed by high AA in K562 cells, but was sustained at higher levels in K562-HIF1α cells, regardless of high AA exposure. Moreover, repression of cell proliferation and neoangiogenesis by high AA was completely abrogated in mice receiving transplants of K562-HIF1α cells. These results indicate that, along with H2O2 generation, downregulation of HIF-1α transcription plays a crucial role in growth inhibition of human leukemic cells by high AA.

  4. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

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    Mueller Nancy

    2005-10-01

    Full Text Available Abstract Background Human T-cell leukemia virus type I (HTLV-I causes adult T-cell leukemia (ATL after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. Results The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. Conclusion These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.

  5. Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

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    Ward Michael

    2009-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

  6. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zehai; LIU Xinyue; ZOU Ping

    2007-01-01

    This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concen- trations (12.5-200 μmol/L) for different time lengths (12-48 h). The proliferation of the U937 leu- kemic cells was determined by MTT assay. Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resvera-trol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

  7. LEUKEMIA INHIBITORY FACTOR IN FERTILE AND INFERTILE HUMAN REPRODUCTIVE TRACT IN VIVO

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    M. Ghaffari

    2000-01-01

    Full Text Available Maternal leukemia inhibitory factor (LIF is required for successful implanta¬tion in mice, but little is known about its role and expression in human reproduc¬tion. Here we report on the pattern of LIF mRNA expression in 30 samples of previously fertile and 11 infertile human endometrium, 10 samples of previously fertile post-menopausal endometrium and 10 uterine (Fallopian tubes from pre¬viously fertile women using the reverse transcriptase-polymerase chain reaction (RT-PCR. All samples were removed with informed patient consent and Ethical Sheffield university Committee approval. Pieces of each sample were processed for electron microscopy to confirm tissue normality and stage of cycle. LIF mRNA was expressed throughout most of the secretory phase (from about day 18 of the cycle and menstruation phase (days 1-4 of cycles in fertile women. However it was not expressed during the proliferative phase. In addition LIF mRNA was absent from the uterine tube at all stages of the cycle and from the postmeno¬pausal and infertile tissue. These results suggest that LIF is expressed in a men¬strual cycle-dependent manner in fertile human endometrium and its expression is likely to be under hormonal control and is not dependent on pregnancy. In addition, our results showed lack of LIF production in infertile women, which may suggest a role for LIF in fertility.

  8. Leukemia inhibitory factor favours neurogenic differentiation of long-term propagated human midbrain precursor cells

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Widmer, Hans R; Zimmer, Jens;

    2009-01-01

    , human embryonic (5 weeks post-conception) ventral mesencephalic (VM) precursor cells were propagated as neural tissue-spheres (NTS) in epidermal growth factor (EGF; 20 ng/ml) and fibroblast growth factor 2 (FGF2; 20 ng/ml). After more than 325 days, the NTS were transferred to media containing either...... EGF+FGF2, EGF+FGF2+heparin or leukemia inhibitory factor (LIF; 10 ng/ml)+FGF2+heparin. Cultures were subsequently propagated for more than 180 days with NTS analyzed at various time-points. Our data show for the first time that human VM neural precursor cells can be long-term propagated as NTS...... in the presence of EGF and FGF2. A positive effect of heparin was found only after 150 days of treatment. After switching into different media, only NTS exposed to LIF contained numerous cells positive for markers of newly formed neurons. Besides of demonstrating the ability of human VM NTS to be long...

  9. 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...

  10. No evidence of murine leukemia virus-related viruses in live attenuated human vaccines.

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    William M Switzer

    Full Text Available BACKGROUND: The association of xenotropic murine leukemia virus (MLV-related virus (XMRV in prostate cancer and chronic fatigue syndrome reported in previous studies remains controversial as these results have been questioned by recent data. Nonetheless, concerns have been raised regarding contamination of human vaccines as a possible source of introduction of XMRV and MLV into human populations. To address this possibility, we tested eight live attenuated human vaccines using generic PCR for XMRV and MLV sequences. Viral metagenomics using deep sequencing was also done to identify the possibility of other adventitious agents. RESULTS: All eight live attenuated vaccines, including Japanese encephalitis virus (JEV (SA-14-14-2, varicella (Varivax, measles, mumps, and rubella (MMR-II, measles (Attenuvax, rubella (Meruvax-II, rotavirus (Rotateq and Rotarix, and yellow fever virus were negative for XMRV and highly related MLV sequences. However, residual hamster DNA, but not RNA, containing novel endogenous gammaretrovirus sequences was detected in the JEV vaccine using PCR. Metagenomics analysis did not detect any adventitious viral sequences of public health concern. Intracisternal A particle sequences closest to those present in Syrian hamsters and not mice were also detected in the JEV SA-14-14-2 vaccine. Combined, these results are consistent with the production of the JEV vaccine in Syrian hamster cells. CONCLUSIONS: We found no evidence of XMRV and MLV in eight live attenuated human vaccines further supporting the safety of these vaccines. Our findings suggest that vaccines are an unlikely source of XMRV and MLV exposure in humans and are consistent with the mounting evidence on the absence of these viruses in humans.

  11. 免疫磁珠分选白血病KG1a细胞中CD34+CD38-干细胞及其特性研究%Isolation and characteristic of CD34 + CD38-stem cells in leukemia cell line KG1a using magnetic activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    王国征; 李慧; 吴远彬; 李达; 贺艳杰; 周雪云

    2013-01-01

    目的 从白血病KGla细胞中分选CD34+ CD38-干细胞并研究其生物学特性.方法 免疫磁珠法分选CD34+ CD38-细胞,流式细胞术分析细胞表面膜抗原、细胞周期,甲基纤维素培养体系观察其克隆性;以HL60、K562、CD34+ CD38+细胞为对照,甲基偶氮唑蓝法检测柔红霉素对CD34+ CD38-细胞的抑制作用;BALB/c裸鼠皮下接种,观察体内成瘤能力.结果 分选的CD34+ CD38-细胞纯度达95%以上,(69.03 +3.25)%处于Go期,克隆形成率为(38.64±2.68)%,明显抵抗柔红霉素;不同浓度柔红霉素作用后,CD34+ CD38-、CD34+ CD38+、HL60、K562细胞的活性差异有统计学意义(F =961.136,P=0.000);CD34+ CD38-在裸鼠皮下成瘤率显著高于CD34+ CD38+细胞(P<0.05).结论 免疫磁珠法分选白血病干细胞简单易行,分选的细胞符合白血病干细胞生物学特性.%Objective To isolate the CD34+ CD38- stem cells from human acute leukemia cell line KGla and to research its biological characteristics. Methods CD34+ CD38- cells were isolated by magnetic activated cell sorting( MACS) ; the cell surface membrane antigens and cell cycle were analyzed by flow cytometry; its clonality was observed with methyl cellulose system. HL60,K562 and D34+ CD38+ cells were selected as control,and the methyl thiazolyl tetrazolium(MTT) assay was taken to observe the depressant effect of rubidomycin to CD34+ CD38- cells. BALB/c nude mice were inoculated subcutaneous-ly to observe the tumorigenicity. Results The purity of CD34+ CD38- cells were above 95% and(69.03 ± 3. 25) % cells in the G0 phase. The cloning efficiency of CD34+ CD38- cells were( 38. 64 ± 2. 68) % , and the CD34+ CD38- cells were obviously resistant to rubidomycin. There were statistic difference of cytoactive among CD34+ CD38-, CD34+ CD38 + , HL60, and K562 cells under giving the same concentrations of rubidomycin circumstances (F = 961. 136,P= 0.000). The tumorigenesis ability of CD34+ CD38- cells in nude mice was

  12. The clinical significance of γ-catenin in acute myeloid leukemia

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    Xu J

    2016-06-01

    Full Text Available Jiadai Xu,1 Wei Wu,1 Wenyi Shen,1 Peng Liu2 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 2Department of Hematology, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China Abstract: Dysregulation of γ-catenin may function as an oncogenic factor in various malignancies. We investigated γ-catenin expression in acute myeloid leukemia (AML and explored its role in the pathogenesis of AML. γ-Catenin was significantly overexpressed in AML patients compared to healthy donors. The γ-catenin expression in AML patients with lower white blood cells (<30×109/L was significantly higher than those with higher white blood cells (≥30×109/L. The expression levels of γ-catenin in AML patients with mutated CEBPα were significantly higher than those with unmutated CEBPα. AML patients with lower γ-catenin levels were more likely to achieve complete remission compared with patients who have higher γ-catenin levels. In K562 cells, γ-catenin knockdown suppressed cellular proliferation, while the cellular migration was greatly enhanced. Moreover, knocking down of γ-catenin enhanced the cytotoxicity of decitabine in K562 cells. Our investigation has indicated a potential role of γ-catenin in the pathogenesis of AML. Keywords: γ-catenin, acute myeloid leukemia, AML, decitabine, prognosis, bone marrow, shRNA

  13. Polyphenols isolated from Allium cepa L. induces apoptosis by suppressing IAP-1 through inhibiting PI3K/Akt signaling pathways in human leukemic cells.

    Science.gov (United States)

    Han, Min Ho; Lee, Won Sup; Jung, Ji Hyun; Jeong, Jae-Hun; Park, Cheol; Kim, Hye Jung; Kim, GonSup; Jung, Jin-Myung; Kwon, Taeg Kyu; Kim, Gi-Young; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun

    2013-12-01

    Allium cepa Linn is commonly used as supplementary folk remedy for cancer therapy. Evidence suggests that Allium extracts have anti-cancer properties. However, the mechanisms of the anti-cancer activity of A. cepa Linn are not fully elucidated in human cancer cells. In this study, we investigated anti-cancer effects of polyphenols extracted from lyophilized A. cepa Linn (PEAL) in human leukemia cells and their mechanisms. PEAL inhibited cancer cell growth by inducing caspase-dependent apoptosis. The apoptosis was suppressed by caspase 8 and 9 inhibitors. PEAL also up-regulated TNF-related apoptosis-inducing ligand (TRAIL) receptor DR5 and down-regulated survivin and cellular inhibitor of apoptosis 1 (cIAP-1). We confirmed these findings in other leukemic cells (THP-1, K562 cells). In addition, PEAL suppressed Akt activity and the PEAL-induced apoptosis was significantly attenuated in Akt-overexpressing U937 cells. In conclusion, our data suggested that PEAL induced caspase-dependent apoptosis in several human leukemic cells including U937 cells. The apoptosis was triggered through extrinsic pathway by up-regulating DR5 modulating as well as through intrinsic pathway by modulating IAP family members. In addition, PEAL induces caspase-dependent apoptosis at least in part through the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. This study provides evidence that PEAL might be useful for the treatment of leukemia.

  14. Comparison of effects of the ethanolic extracts of brazilian propolis on human leukemic cells as assessed with the MTT assay.

    Science.gov (United States)

    Franchi, Gilberto C; Moraes, Cleber S; Toreti, Viviane C; Daugsch, Andreas; Nowill, Alexandre E; Park, Yong K

    2012-01-01

    Propolis is a resinous product collected by honey bees. It was also reported that propolis has a wide variety of biological actions, including antimicrobial activity and antioxidant, anti-inflammatory, and suppressive effects of dioxin toxicity activities. The aim of this study was to compare the in vitro cytotoxic activities of green propolis (G12) and red propolis (G13) in human leukemia cells. These cells were incubated with different concentrations of propolis and 48 hours after the IC(50) was calculated for each cell. The results showed that the red propolis has cytotoxic effect in vitro higher than green propolis. Red propolis was showed to be cytostatic in K562 cells and caused the same amount of apoptosis as its control Gleevec. In conclusion, these results showed that red propolis is more cytotoxic than the green propolis in a variety of human cell lines of leukemia. Red propolis may contain drugs capable of inhibiting cancer cell growth. Therefore, further isolation of respective chemical ingredients from the red propolis (G13) for identification of the activities is necessary.

  15. Comparison of Effects of the Ethanolic Extracts of Brazilian Propolis on Human Leukemic Cells As Assessed with the MTT Assay

    Directory of Open Access Journals (Sweden)

    Gilberto C. Franchi

    2012-01-01

    Full Text Available Propolis is a resinous product collected by honey bees. It was also reported that propolis has a wide variety of biological actions, including antimicrobial activity and antioxidant, anti-inflammatory, and suppressive effects of dioxin toxicity activities. The aim of this study was to compare the in vitro cytotoxic activities of green propolis (G12 and red propolis (G13 in human leukemia cells. These cells were incubated with different concentrations of propolis and 48 hours after the IC50 was calculated for each cell. The results showed that the red propolis has cytotoxic effect in vitro higher than green propolis. Red propolis was showed to be cytostatic in K562 cells and caused the same amount of apoptosis as its control Gleevec. In conclusion, these results showed that red propolis is more cytotoxic than the green propolis in a variety of human cell lines of leukemia. Red propolis may contain drugs capable of inhibiting cancer cell growth. Therefore, further isolation of respective chemical ingredients from the red propolis (G13 for identification of the activities is necessary.

  16. Preferential response of acute myeloid leukemias with translocation involving chromosome 17 to human recombinant granulocyte colony-stimulating factor.

    Science.gov (United States)

    Pébusque, M J; Lafage, M; Lopez, M; Mannoni, P

    1988-07-01

    Induction of proliferation and differentiation in response to the addition of recombinant human granulocyte colony-stimulating factor (G-CSF) was studied by both suspension and semisolid cultures in a series of acute myeloid leukemias (AML). Induction of proliferation by G-CSF alone was observed in six of 27 cases of AML. All acute promyelocytic leukemias with the specific chromosomal translocation t(15;17) and one case of myelomonocytic leukemia with balanced chromosomal translocation involving chromosome 17 at band q12q21 were induced to proliferate strongly by the G-CSF. However, contrary to the long-term proliferative effect observed with granulocyte/macrophage colony-stimulating factor (GM-CSF), G-CSF activity can be characterized by its capability to initiate and promote the growth of responding AML cells but not to sustain long-term proliferation. Finally, no terminal differentiation was found, as assessed by morphology, cytochemistry, and cell surface marker analysis. These results indicate that G-CSF may be sufficient to provide a specific signal for induction of a transient proliferation in AML without induction of terminal differentiation. The cells with the highest response are clonal leukemia cells, all bearing a translocation involving the chromosome region 17q12q21 in which the G-CSF gene has been recently located.

  17. Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism

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    Velez-Pardo Carlos

    2011-06-01

    Full Text Available Abstract Background Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia. Results It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM or 100:1 (300 μM: 3 μM, respectively. Conclusion We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia.