Home About us Contact
|
Home About us Contact | ||
|
|
||
K562 Cells (k562 + cell)
Selected AbstractsCARDIOTOXIN III INDUCES APOPTOSIS IN K562 CELLS THROUGH A MITOCHONDRIAL-MEDIATED PATHWAYCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 7 2005Sheng-Huei Yang SUMMARY 1.,Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells. 2.,Cardiotoxin III was found to inhibit the growth of K562 cells in a time- and dose-dependent manner, with an IC50 value of 1.7 ,g/mL, and displayed several features of apoptosis, including apoptotic body formation, an increase in the sub-G1 population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. 3.,Investigation of the mechanism of CTX III-induced apoptosis revealed that treatment of K562 cells with CTX III resulted in the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into the cytosol and activation of caspase-9 and caspase-3 and the subsequent cleavage of the caspase-3 substrate PARP; however, CTX III did not generate reactive oxygen species (ROS). 4.,Taken together, the results indicate that CTX III induces apoptosis in K562 cells through an ROS-independent mitochondrial dysfunction pathway. [source] Photodynamic Action of Benzo[a]pyrene in K562 CellsPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2007Daza De Moraes Vaz Batista Filgueira Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA,protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA. [source] Binding Features of BCL2-Targeted Oligodeoxynucleotides with K562 CellsCHEMINFORM, Issue 6 2005A. M. Timofeev Abstract For Abstract see ChemInform Abstract in Full Text. [source] Induction of apoptosis by A3 adenosine receptor agonist N6 -(3-iodobenzyl)-adenosine-5,- N -methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanismACTA PHYSIOLOGICA, Issue 2 2010P. Mlejnek Abstract Aim:, The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N6 -(3-iodobenzyl)-adenosine-5,- N -methylcarboxamide (IB-MECA) was studied. Methods:, To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. Results:, We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. Conclusion:, Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR. [source] Induction of G2/M phase arrest and apoptosis by a novel indoloquinoline derivative, IQDMA, in K562 cellsDRUG DEVELOPMENT RESEARCH, Issue 9 2006Yi-Hsiung Lin Abstract The indoloquinoline, IQDMA (N,-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine), was identified as a novel antineoplastic agent with broad spectrum of antitumor activities against several human cancer cells. IQDMA-induced G2/M arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27, and down-regulation of Cdk1and Cdk2. IQDMA had no effect on the levels of cyclin A, cyclin B1, cyclin D3, or Cdc25C. IQDMA also increased apoptosis, as characterized by apoptotic body formation, increase of the sub G1 population and poly (ADP-ribose) polymerase (PARP) cleavage. Further mechanistic analysis demonstrated that IQDMA upregulated FasL protein expression, and kinetic studies showed the sequential activation of caspases-8, -3, and -9. Both caspase-8 and caspase-3 inhibitors, but not a caspase-9-specific inhibitor, suppressed IQDMA-induced cell death. These molecular alterations provide an insight into IQDMA-caused growth inhibition, G2/M arrest, and apoptotic death of K562 cells. Drug Dev. Res. 67:743,751, 2006. © 2006 Wiley-Liss, Inc. [source] Lectin-aided separation of circulating tumor cells and assay of their response to an anticancer drug in an integrated microfluidic deviceELECTROPHORESIS, Issue 18 2010Li Li Abstract Metastasis caused by the entry of circulating tumor cells (CTCs) into the bloodstream or lymphatic vessels is a major factor contributing to death in cancer patients. Separation of CTCs and studies on CTC,drug interactions are very important for prognostic and therapeutic implications of metastatic cancer. In this study, an integrated microfluidic device for CTC separation through the combination of lectin and microstructure is presented. This microfluidic device and lectin concanavalin A were utilized for the separation of K562 cells in whole blood samples. The results showed that the separation efficiency can reach 84%, which is much higher than that of an experiment without concanavalin A treatment. To further demonstrate the feasibility of this microfluidic device application in sequential studies after target cells were separated, the interactions of K562 cells and an anticancer drug, cytarabine, were also examined. After 6,h on-chip treatment with cytarabine, the viabilities of K562 cells were 85.29, 77.05, and 40% for drug concentration levels of 0.25, 0.5, and 1.0,g/L, respectively. This system can facilitate the rapid and efficient in vitro investigation of CTC separation and CTC-related studies. [source] Carbamylated erythropoietin increases frataxin independent from the erythropoietin receptorEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 6 2010Brigitte Sturm Eur J Clin Invest 2010; 40 (6): 561,565 Abstract Background, Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin. Recently we showed in a clinical pilot study in Friedreich's ataxia patients that recombinant human erythropoietin (rhuEPO) significantly increases frataxin-expression. In this in vitro study, we investigated the role of the erythropoietin receptor (EPO-R) in the frataxin increasing effect of rhuEPO and if nonerythropoietic carbamylated erythropoietin (CEPO), which cannot bind to the classical EPO-R increases frataxin expression. Materials and methods, In our experiments human erythroleukaemic K562 cells (+ EPO-R), human monocytic leukemia THP-1 cells (, EPO-R) and isolated primary lymphocytes from healthy control and FRDA patients were incubated with different concentrations of rhuEPO or CEPO. Frataxin-expression was detected by an electrochemical luminescence immunoassay (based on the principle of an ELISA). Results, We show that rhuEPO increases frataxin-expression in K562 cells (expressing EPO-R) as well as in THP-1 cells (without EPO-R expression). These results were confirmed by the finding that CEPO, which cannot bind to the classical EPO-R increased frataxin expression in the same concentration range as rhuEPO. In addition, we show that both EPO derivatives significantly increase frataxin-expression in vitro in control and Friedreich's ataxia patients primary lymphocytes. Conclusion, Our results provide a scientific basis for further studies examining the effectiveness of nonerythropoietic derivatives of erythropoietin for the treatment of Friedreich's ataxia patients. [source] Up-regulation of leukocyte CXCR4 expression by sulfatide: An L-selectin-dependent pathway on CD4+ T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2007Pascal Duchesneau Abstract CXCR4 plays significant roles in immune and inflammatory responses and is important for selective recruitment of leukocytes. We previously showed that CXCR4 surface expression of human lymphocytes was affected by sulfatide, an in vivo ligand for L-selectin. Increased CXCR4 expression was shown to promote biologically relevant functions such as integrin-dependent adhesion and transmigration. Here, we show that sulfatide-induced CXCR4 up-regulation also occurs on other leukocyte subsets in humans and mice. B cells and CD4+CD25+ T cells had the highest CXCR4 up-regulation after sulfatide stimulation. Transfection of L-selectin was sufficient for K562 cells to acquire sulfatide-induced CXCR4 up-regulation, while analysis of L-selectin knockout mice revealed that this response was critically L-selectin dependent only for CD4+ T cells, suggesting an alternative pathway in CD8+ T cells and B cells. Sulfatide triggered several intracellular signaling events in CD4+ T cells, but only tyrosine kinase activation, including members of the Src family, were essential for L-selectin to CXCR4 signaling. CXCR4 up-regulation was rapid, enhanced CXCL12-induced signaling and increased chemotaxis toward CXCL12, and therefore has potentially important roles in vivo. Thus, the response to CXCL12 depends in part on tissue expression of sulfatide and, specifically in CD4+ T cells, also depends on the surface level of L-selectin. [source] Characterization of sequence variations in human histone H1.2 and H1.4 subtypesFEBS JOURNAL, Issue 14 2005Bettina Sarg In humans, eight types of histone H1 exist (H1.1,H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC,GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA,AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC,GTC shift, indicating that this is a relatively frequent polymorphism. The AAA,AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions. [source] Growth inhibition of mammalian cells by eosinophil cationic proteinFEBS JOURNAL, Issue 1 2002Takashi Maeda Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC50 values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were,estimated to be ,,1,5 µm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic. [source] Role of Chk1 and Chk2 in Ara-C-induced differentiation of human leukemia K562 cellsGENES TO CELLS, Issue 2 2005Kazuchika Takagaki Human chronic myelogenous leukemia K562 cells are relatively resistant to the anti-metabolite cytosine arabinoside (Ara-C) and, when treated with Ara-C, they differentiate into erythrocytes without undergoing apoptosis. In this study we investigated the mechanism by which Ara-C induces K562 cells to differentiate. We first observed that Ara-C-induced differentiation of these cells is completely inhibited by the radiosensitizing agent caffeine, an inhibitor of ATM and ATR protein kinases. We next found that Ara-C activates Chk1 and Chk2 in the cells, and that the activation of Chk1, but not of Chk2, was almost completely inhibited by caffeine. Proteasome-mediated degradation of Cdc25A and phosphorylation of Cdc25C were induced by Ara-C treatment, presumably due to the activation of Chk2 and Chk1, respectively. To directly observe the effects of checkpoint kinase activation in Ara-C-induced differentiation, we suppressed Chk1 or Chk2 with the Chk1-specific inhibitor Gö6976, by generating cell lines stably over-expressing dominant-negative forms of Chk2, or by siRNA-mediated knock-down of the Chk1 or the Chk2 gene. The results suggest that Ara-C-induced erythroid differentiation of K562 cells depends on both Chk1 and Chk2 pathways. [source] Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53-independent apoptosis of chronic myeloid leukemia cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Jin-Hwang Liu Abstract Bcr-abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr-abl and induces apoptosis of CML cells. As reported previously, p53wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib-induced p53 independent proapoptotic mechanism. We found imatinib up-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73; expression of pml and bax; formation of PML-nuclear body (NB); and co-localization of TAp73/PML-NB in p53-nonfunctioning K562 and p53mutant Meg-01 CML cells, but not in BCR-ABL - HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib-induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML-NB co-localization, and the subsequent apoptosis. Conversely, interferon ,-2a (IFN,), which increased phosphrylated TAp73 and TAp73/PML-NB co-localization, increased additively apoptosis with imatinib. The imatinib-induced TAp73/PML-NB co-localization was accompanied by co-immpunoprecipitation of TAp73 with pml. The imatinib-induced co-localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53mutant and one with p53wild. A novel p53-independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML-NB and probably with chk2 and bax being involved is hereby evident in some imatinib-treated CML cells. © 2009 UICC [source] Cytotoxic T lymphocyte mediated recognition of human pancreatic cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2002Matthias Peiper Abstract T lymphocytes play an important role in tumor rejection and their response to human malignant melanoma has been well documented. In contrast, the existence of cytotoxic T lymphocytes (CTL) to pancreatic cancer remains unclear. Tumor-associated lymphocytes (TAL) and peripheral blood monocytes (PBMC) were isolated from pancreatic cancer patients. Tumor-specific CTL were generated from TAL and PBMC using solid-phase anti-CD3, low-dose IL-2 (50 IU/ml) and repetitive autologous tumor stimulation. The specificity of CTL was tested in standard cytotoxicity assays using autologous tumor cells, autologous fibroblasts when available, several allogeneic pancreatic tumor cells and the NK-sensitive cell line K562. Anti-HLA-Class I MAb, W6/32, was used to demonstrate that tumor-specific CTL were HLA-Class I restricted. HLA-molecules of human pancreatic cancer cells were washed out using acid elution. Eight consecutive, histologically confirmed pancreatic cancer specimen as well as peripheral blood mononuclear cells were analyzed. CTL were capable of lysing autologous tumor cells significantly after 3 stimulations with autologous tumor cells. T cell mediated recognition was HLA-Class I restricted as shown by incubation with MAb anti-HLA-Class I. In case of HLA-A2 positivity, incubation of tumor cells in cytotoxicity assays resulted in significant inhibition. Autologous fibroblasts or K562 cells were lysed significantly less. HLA-Class I molecule elution resulted in significantly lower recognition of these cells by CTL. These results show for the first time in a larger series the possibility of generating CTL in human pancreatic cancer. The identification of new tumor associated antigens or tumor antigens will be crucial for establishing new treatment strategies. © 2002 Wiley-Liss, Inc. [source] Novel indoloquinoline derivative, IQDMA, inhibits STAT5 signaling associated with apoptosis in K562 cellsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2008Sheng-Huei Yang Abstract N,-(11H-indolo[3,2-c]quinolin-6-yl)- N,N -dimethylethane-1,2-diamine (IQDMA), an indoloquinoline derivative, synthesized in our laboratory, has been demonstrated to be an effective antitumor agent in human leukemia cells. In the present study, treatment with IQDMA inhibited phosphorylation of epidermal growth factor receptor (EGFR), Src, Bcr-Abl, and Janus-activated kinase (JAK2) in a time-dependent manner. IQDMA also degraded JAK2 protein. Moreover, signal transducer and activator of transcription 5 (STAT5) signaling were also blocked by IQDMA. However, IQDMA did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt and extracellular signal-regulated kinase 1/2. Furthermore, IQDMA upregulated the expression of p21 and p27 and downregulated the expression of cyclin D1, myeloid cell leukemia-1(Mcl-1), Bcl-XL, and vascular endothelial growth factor (VEGF). Taken together, these results indicate that IQDMA causes significant induction of apoptosis in K562 cells via downregulation of EGFR, Src, Bcr-Abl, JAK2, and STAT5 signaling and modulation of p21, p27, cyclin D1, Mcl-1, Bcl-XL, and VEGF proteins. Thus, IQDMA appears to be a potential therapeutic agent for treating leukemia K562 cells. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:396,404, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20254 [source] Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell linesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Jianghong Wu Abstract Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1,5 ,M) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5,10 ,M) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2,5 ,M) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy. J. Cell. Biochem. 82: 200,214, 2001. © 2001 Wiley-Liss, Inc. [source] Apoptosis inducing activity of viscin, a lipophilic extract from Viscum album L.JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2005K. Urech Detection of antiproliferative activity and bioactivity-guided fractionation of viscin, a lipophilic extract from Viscum album L., led to the isolation of betulinic acid, oleanolic acid and ursolic acid as active components. Viscin, betulinic acid, oleanolic acid and ursolic acid inhibited growth and induced apoptotic cell death in Molt4, K562 and U937 leukaemia cells. The growth inhibitory effect of viscin was more pronounced in Molt4 and U937 cells (IC50 (concentration that inhibited cell proliferation by 50%): 118 ± 24 and 138 ± 24 ,g mL,1) than in K562 cells (IC50: 252 ± 37 ,g mL,1). Oleanolic acid was the least effective in all cell lines (7.5,45.5% inhibition at 10 ,g mL,1) and ursolic acid the most active in Molt4 and U937 cells (81.8 and 97.8% inhibition, respectively, at 5 ,g mL,1). A dose-dependent loss of membrane phospholipid asymmetry associated with apoptosis was induced in all cell lines as shown in flow cytometry by the externalization of phosphatidylserine and morphological changes in cell size and granularity. There were differences in individual cell lines' response towards the apoptosis-inducing effect of viscin, betulinic acid, oleanolic acid and ursolic acid. The triterpenoids ,-amyrin, ,-amyrinacetate, lupeol, lupeolacetate, ,-sitosterol and stigmasterol, and the fatty acids oleic acid, linoleic acid, palmitic acid and stearic acid were also present in the lipophilic extract. [source] Chronic Alcohol Consumption Is Associated With an Increased Cytotoxic Profile of Circulating Lymphocytes That May Be Related With the Development of Liver InjuryALCOHOLISM, Issue 5 2010Francisco Javier Laso Background:, Apoptosis has recently emerged as a key component of acute and chronic liver diseases and it could be related to alcoholic liver disease. In the present study, we attempted to analyze the cytotoxic profile of circulating lymphocytes in chronic alcoholic patients grouped according to ethanol intake status and presence of liver disease. Methods:, We investigate the phenotypic and functional behavior of different compartments of peripheral blood (PB) cytotoxic T and natural killer (NK) cells in chronic alcoholic patients without liver disease and active ethanol intake (AWLD group; n = 22), and in subjects with alcohol liver cirrhosis (ALC group; n = 22). Results:, AWLD patients showed an expansion of both CD4+/CD8+ cytotoxic T cells and NK/T cells, in association with an enhanced cytolytic activity against K562 cells and a higher ability to induce in vitro expression of the pro-apoptotic protein APO2.7 in HepG2 cells. Conversely, ethanol intake in ALC patients was associated with decreased NK cell numbers, a reduced cytotoxic activity against K562 cells without significant changes in the expression of APO2.7, and a pro-fibrotic profile of cytokine secretion. Conclusions:, Overall, our results suggest that alcoholic patients display different phenotypical and functional changes in circulating PB cytotoxic lymphocytes according to the presence of alcoholic liver disease, which could be related to the development and progress of liver injury. [source] Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cellsMOLECULAR CARCINOGENESIS, Issue 7 2010Yang Li Abstract We examined whether betulin, a naturally abundant compound, has anticancer functions in human cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, and breast cancer MCF-7 cells with IC50 values ranging from 10 to 15,µg/mL. While betulin exhibited only moderate anticancer activity in other human cancer cells such as hepatoma SK-HEP-1 cells, prostate carcinoma PC-3, and lung carcinoma NCI-H460, with IC50 values ranging from 20 to 60,µg/mL, it showed minor growth inhibition in human erythroleukemia K562 cells (IC50,>,100,µg/mL). We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model. Betulin (10,µg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30,min after treatment with betulin. Betulin, unlike its chemical derivative betulinic acid, did not directly trigger mitochondrial cytochrome c release in isolated mitochondria. Importantly, Bax and Bak were rapidly translocated to the mitochondria 30,min after betulin treatment. The sequential activation of caspase-9 and caspase-3/-7 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed behind those mitochondrial events. Furthermore, specific downregulation of either caspase-9, Bax, or Bak by siRNA effectively reduced PARP cleavage and caspase-3 activation. Taken together, the lines of evidence demonstrate that betulin triggers apoptosis of human cancer cells through the intrinsic apoptotic pathway. © 2010 Wiley-Liss, Inc. [source] The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562MOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 6 2010Sofie Lust Abstract We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G2/M phase and stimulated an accumulation of the cells in the sub-G0 phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-xL. Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78,kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib. [source] Photodynamic Action of Benzo[a]pyrene in K562 CellsPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2007Daza De Moraes Vaz Batista Filgueira Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA,protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA. [source] Cytotoxicity in vitro of naphthazarin derivatives from Onosma arenariaPHYTOTHERAPY RESEARCH, Issue 7 2006Tatjana Kundakovi Abstract The cytotoxicity of naphthazarin derivatives isolated from the roots of Onosma arenaria on human cervix adenocarcinoma cells (HeLa) and leukaemia K562 cells, as well on non-malignant peripheral blood mononuclear cells (PBMC) was studied. The results show that , -hydroxyisovalerylalkannin, acetylalkannin and the pigment fraction exhibited high cytotoxicity in vitro against the tested cell lines, as well the healthy PBMC before or after activation with phytohaemagglutinin. Copyright © 2006 John Wiley & Sons, Ltd. [source] In Vitro culture studies of FlorEssence® on human tumor cell linesPHYTOTHERAPY RESEARCH, Issue 2 2005Joseph Tai Abstract FlorEssence® (FE) is an herbal tea widely used by patients to treat chronic conditions in North America, particularly cancer patients during chemo- and radiation therapy. Although individual components of FE have antioxidant, antiestrogenic, immunostimulant and antitumor properties, in vitro evidence of anticancer activity for the herbal tea itself is still lacking. We studied the antiproliferative effect of FE on MCF7 and MDA-MB-468 human breast cancer, and Jurkat and K562 leukemia cell lines. We found that FE significantly inhibited the proliferation of both breast and leukemia cells in vitro only at high concentrations, with 50% inhibition of MDA-MB-468 cells at about 1[sol ]20 dilution, Jurkat cells at about 1[sol ]10 dilution and MCF7 and K562 cells at less than 1[sol ]10 dilution. Flow cytometry analysis showed that treatment with a high concentration of FE induced G2[sol ]M arrest in MCF7 and Jurkat cells, with also an increased SubG0[sol ]G1 fraction in MCF7 cells. MDA-MB-468 cells showed a significantly increased Sub G0[sol ]G1 fraction after treatment with 1[sol ]10 dilution of FE while the cell cycle of K562 was unaffected. When MCF7 and MDA-MB-468 breast cancer cells were treated with a combination of FE with either paclitaxel or cisplatin, results showed that only the combination of 1[sol ]20 dilution of FE with 0.5 µM cisplatin resulted in a small but significantly higher MCF7 cell survival than 0.5 µM cisplatin treatment alone. FE at 1[sol ]20 and 1[sol ]50 dilutions did not affect the antiproliferative properties of these two commonly used chemotherapeutic agents. The results suggest that FE at high concentrations show differential inhibitory effect on different human cancer cell lines. Further studies are needed to assess the biological activities of FE. Copyright © 2005 John Wiley & Sons, Ltd. [source] ORIGINAL ARTICLE: Suppression of Natural Killer Cell Cytotoxicity in Postpartum WomenAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2010Maureen Groer Citation Groer M, El-Badri N, Djeu J, Harrington M, Van Eepoel J. Suppression of natural killer cell cytotoxicity in postpartum women. Am J Reprod Immunol 2010; 63: 209,213 Problem, Natural Killer (NK) cell numbers and cytotoxicity are suppressed during pregnancy. Little is known about postpartum NK number and function. Method of study, Postpartum women (n = 39) were studied at one week and then monthly over the first six postpartum months. The standard natural killer cell cytotoxicity assay (NKCA) was performed. This is a Cr51 release assay from K562 cells cultured with peripheral blood mononuclear cells (PBMCs). Results, Data indicate suppression of NK cytotoxicity in postpartum women. Cytotoxicity at each effector:target (E:T) ratio showed a drop from 1 week postpartum, reaching a nadir at around 2 months, and a trend towards recovery of cytotoxicity from 3 to 6 months. Lytic units (LUs) from pre-incubated cells from postpartum women were lower than age-matched, non-pregnant, non-postpartum controls through the fifth postpartum month. Conclusion, These data suggest that the postpartum period, like pregnancy, is characterized by decreased NK cytotoxicity activity. This suppressed NK cytotoxic effect may result as a response to interaction with tolerized fetal microchimeric cells accumulated during pregnancy in maternal blood and tissues. [source] Inhibition of term decidual NK cell cytotoxicity by soluble HLA-G1AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5-6 2006Tobias G. Poehlmann Objectives, Soluble (s)HLA-G1 is produced by trophoblast cells. Aim was to analyze the capacities and mechanisms of sHLA-G1 to regulate interleukin (IL)-2-induced cytotoxicity of natural killer (NK) cells from human deciduas. Methods, Natural killer cells were isolated from decidual layers of term placentae, stimulated or not with IL-2 and supplemented with various concentrations of recombinant soluble HLA-G1 (sHLA-G1). For NK cell cytotoxicity assays, K562 cells were used as targets. Expression of signal transducer and activator of transcription 3 (STAT3) and perforin was analyzed by Western blotting. Apoptosis was examined by assessment of poly(ADP-ribose) polymerase cleavage. NK cells were analyzed by flow cytometry for IL-2receptor- , (IL-2R,; CD25) and transferrin receptor CD71 expression. Results, Interleukin-2 increases CD71, STAT3, perforin expression and cytotoxic potential of NK cells. Expression of CD71, STAT3 and perforin decreased simultaneously with cytotoxicity and dose-dependently when sHLA-G1 (1.6 ,g/mL,1.6 ng/mL) was added to IL-2 stimulated cultures. sHLA-G1 did not induce apoptosis and CD25 expression was not affected. Conclusion, Interleukin-2R, expression is not controlled by sHLA-G1, but its signal transducer STAT3 as well as several downstream effects, such as perforin expression, proliferation and cytotoxicity. The control of STAT3 bioavailability through sHLA-G1 may be a key regulator of the mentioned effects. [source] Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 6 2009Yong-Wook Park Objective To determine the cytotoxicity of natural killer (NK) cells and the level of differentiation of hematopoietic stem cells (HSCs) into NK cells in systemic lupus erythematosus (SLE). Methods Patients with SLE (n = 108), rheumatoid arthritis (RA; n = 90), Behēet's disease (n = 39), or ankylosing spondylitis (n = 41) and healthy control subjects (n = 173) were enrolled in the study. NK cell levels, NK cell cytotoxicities, and lymphokine-activated killer (LAK) activities against K562 cells were measured by flow cytometry. Gene expression was assessed by reverse transcription,polymerase chain reaction. NK cells were differentiated from peripheral blood and bone marrow HSCs in vitro. Results Percentages and absolute numbers of NK cells, cytotoxicities, and LAK activities were significantly lower in the peripheral blood of SLE and RA patients than in that of healthy controls. In particular, this NK cell deficiency was more prominent in patients with lupus nephritis and those with thrombocytopenia. Notably, purified NK cells derived from SLE patients, but not RA patients, were found to have lower cytotoxicities and LAK activities than those from healthy controls. This defect of NK cells in SLE patients was found to be related to lower numbers of NK precursors and to the down-regulation of perforin and granzyme in NK cells. The proliferative capacity of HSCs, the percentages of NK cells differentiated from HSCs, and NK cell cytotoxicities were significantly lower in SLE patients. Conclusion In SLE patients, circulating levels of NK cells were diminished and their cytotoxicities were impaired. Furthermore, the differentiation of HSCs into NK cells was found to be defective. These abnormalities possibly contribute to immune system dysregulation in SLE. [source] Mathematical model of the rate-limiting steps for retrovirus-mediated gene transfer into mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Venkata S. Tayi Abstract A quantitative understanding of the process of retrovirus-mediated gene transfer into mammalian cells should assist the design and optimization of transduction protocols. We present a mathematical model of the process that incorporates the essential rate-limiting transduction steps including diffusion, convection and decay of viral vectors, their binding at the cell surface and entry into the cell cytoplasm, reverse transcription of uncoated RNA to form DNA intermediates, transport of the latter through the cytosol to the cell nucleus and, finally, nuclear import and integration of the delivered DNA into the target cell genome. Cell and virus population balances are used to account for the kinetics of multiple vector infections which influence the transduction efficiency and govern the integrated copy number. The mathematical model is validated using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. Viral intermediate complexes derived from the internalized retroviral vectors are found to remain stable inside the K562 cells and the cytoplasmic trafficking time is consistent with the time scale for retrovirus uncoating, reverse transcription and transport to the cell nucleus. The model predictions of transduction efficiency and integrated copy number agree well with experimental data for both static (i.e., standard gravity) and centrifugation-based gene transfer protocols. The formulation of the model can also be applied to transduction protocols involving lenti- or foamy-viruses and so should prove to be useful for the optimization of several types of gene transfer processes. Biotechnol. Bioeng. 2010;105: 195,209. © 2009 Wiley Periodicals, Inc. [source] research paper: Role of the cold shock domain protein A in the transcriptional regulation of HBG expressionBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2010Raffaella Petruzzelli Summary Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate ,-thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the ,-globin gene (HBB) locus. To search for factors putatively involved in the expression of the ,-globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of ,-thalassaemia severity although they had the same ,BA - and ,,B cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans-acting repressor factor of HBG expression and modulates the HPFH phenotype. [source] Anti-miR-21 oligonucleotide sensitizes leukemic K562 cells to arsenic trioxide by inducing apoptosisCANCER SCIENCE, Issue 4 2010Yumin Li Arsenic trioxide (ATO), an ancient traditional Chinese medicine, has been successfully used as a therapeutic agent for leukemia. Drug resistance and toxicity are major concerns with the treatment. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that might modulate cellular sensitivity to anticancer drugs. miRNA-21 (miR-21) is one of the most prominent miRNAs involved in various aspects of human cancers. However, miR-21 has been rarely characterized in chronic myelogenous leukemia (CML). Here, we used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize K562 cells to ATO by degradation of miR-21. The results showed that both AMO-miR-21 and ATO caused growth inhibition, apoptosis, and G1-phase arrest in K562 cells. Meanwhile, AMO-miR-21 significantly promoted ATO-mediated growth inhibition and apotosis without affecting the G1 phase. Apoptotic cells were confirmed morphologically with Giemsa's staining. Furthermore, dual-luciferase reporter vector, containing two tandem miR-21 binding sites from PDCD4 3,UTR, validated that PDCD4 was directly regulated by miR-21. Therefore, AMO-miR-21 sensitized leukemic K562 cells to ATO by inducing apoptosis partially due to its up-regulation of PDCD4 protein level. The combination of ATO and AMO-miR-21 present therapeutic potential for CML. (Cancer Sci 2010; 101: 948,954) [source] LIM domain-containing adaptor, leupaxin, localizes in focal adhesion and suppresses the integrin-induced tyrosine phosphorylation of paxillinCANCER SCIENCE, Issue 2 2010Toshiyuki Tanaka Focal adhesion (FA) consists of multiple cellular proteins including paxillin and serves as a center for adhesion-mediated signaling. The assembly and disassembly of FAs is regulated by locally produced intracellular signals, and tyrosine phosphorylation of paxillin has been implicated in this process. A Lin-11 Isl-1 Mec-3 (LIM) domain-containing adaptor protein, leupaxin, a member of the paxillin family, is expressed in leukocytes as well as in certain cancer cells, and shares overall structural characteristics with paxillin. However, it remains unknown whether leupaxin and paxillin cooperate with or antagonize each other in integrin signaling. Here we show that leupaxin potently represses the tyrosine phosphorylation of paxillin. When expressed in mouse thymoma BW5147 cells bound to ICAM-1, leupaxin accumulated in FA-like patches in the cell periphery. When expressed in NIH3T3 and HEK293T cells, leupaxin localized to FAs upon cell adhesion to fibronectin and strongly suppressed the integrin-induced tyrosine phosphorylation of paxillin. In integrin-stimulated HEK293T cells, leupaxin's LIM3 domain appeared essential for selective FA localization and the suppression of paxillin tyrosine phosphorylation. Leupaxin's LD3 motif, which is critical for stable association with FAK, was dispensable for leupaxin's suppressive ability. In addition, leupaxin reduced the spreading of NIH3T3 cells on fibronectin, which required both the LD3 motif and LIM3 domain. When expressed in human leukocytic K562 cells, leupaxin significantly suppressed integrin ,5,1-mediated cell adhesion to fibronectin and the tyrosine phosphorylation of paxillin. These findings indicate that leupaxin functions as a paxillin counterpart that potently suppresses the tyrosine phosphorylation of paxillin during integrin signaling. (Cancer Sci 2009) [source] The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1CANCER SCIENCE, Issue 5 2009Junko H. Ohyashiki To evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the moleclular pathways responsible for antiproliferative effects on leukemia cells during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. The inhibitory concentration (IC50) of deferasirox was 17,50 µM in three human myeloid cell lines (K562, U937, and HL60), while those in fresh leukemia cells obtained from four patients it varied from 88 to 172 µM. Gene expression profiling using Affymerix GeneChips (U133 Plus 2.0) revealed up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) encoding p21CIP, genes regulating interferon (i.e. IFIT1). Pathways related to iron metabolism and hypoxia such as growth differentiation factor 15 (GDF-15) and Regulated in development and DNA damage response (REDD1) were also prominent. Based on the results obtained from gene expression profiling, we particularly focused on the REDD1/mTOR (mammalian target of rapamycin) pathway in deferasirox-treated K562 cells, and found an enhanced expression of REDD1 and its down-stream protein, tuberin (TSC2). Notably, S6 ribosomal protein as well as phosphorylated S6, which is known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells, and REDD1 small interfering RNA restored phosphorylation of S6. Although iron chelation may affect multiple signaling pathways related to cell survival, our data support the conclusion that REDD1 functions up-stream of tuberin to down-regulate the mTOR pathway in response to deferasirox. Deferasirox might not only have benefit for iron chelation but also may be an antiproliferative agent in some myeloid leukemias, especially patients who need both iron chelation and reduction of leukemia cells. (Cancer Sci 2009; 100: 970,977) [source] | ||||||||||||||||||||||||||||||||||