Glutathionylation of NF-Kb and Procaspase-3 Regulates Inducible Nitric Oxide Synthase Expression and Apoptosis of Chronic Myeloid Leukemia Cells

Abstract Chronic myeloid leukemia (CML), a myeloproliferative disorder, characterized by sustained neutrophilia and constitutive BCR-ABL tyrosine kinase activity. Constitutive expression of the BCR-ABL kinase and elevated reactive oxygen species (ROS) levels through mitochondria and NADPH oxidase 4 (NOX4) activation leads to genomic instability and enhanced cell survival. Nitric oxide (NO), a signaling molecule has been associated with hematopoesis and suppression of NOS activity may induce profound changes in hematopoietic stem cells/progenitor cells. NO addition or iNOS transfection in K562 cells (BCR-ABL+) altered genes expression involved in the iron metabolism, inhibited cell proliferation and enhanced apoptosis, which were reversed by addition of exogenous iron. Moreover, anti-cancer effect of farnesyltransferase inhibitor in these cells was also mediated by NO production/iNOS induction. The present study investigates status and regulation of NO/iNOS in neutrophils from CML patients.The present study was undertaken to explore NO generation/iNOS expression and its regulation in circulating neutrophils so as to access the role NO/iNOS in CML pathology. All CML patients (Drug/treatment naïve, n=70; imatinib responders, n=62; imatinib resistant, n=25) included in this study were diagnosed in chronic phase. The study protocol was approved by the ethical committees of CSIR-CDRI and KGMU, Lucknow and was conducted in accordance with the declaration of Helsinki. Total nitrite level in neutrophils (PMNs) was assessed by Griess reagent. Nitric oxide, Superoxide, ROS/RNS and mitochondrial ROS generation was assessed by DAF-2DA, DHE, DCF-DA and Mitosox Red respectively. H2O2was measured by Amplex red assay kit. Expression of iNOS gene was evaluated by a SYBR green real-time RT-PCR and Western blot. Binding of NF-kB (p50 and p65 subunit) to iNOS promoter was analysed by CHIP assay. NF-kB (p50 and p65 subunit) and procaspase-3 glutathionylation was assessed by Immunoprecipitation followed by Western blot. Findings in CML patients were further validated using in vitro experiments on K562 cells. Statistical analysis were performed by one way ANOVA test followed by Newman-Keul’s post hoc analysis using the Graph Pad prism software. PMNs total nitrite, NO level and iNOS expression in drug naïve and imatinib resistant patients were significantly less as compared to healthy subjects. However, significant recovery in all the parameters was observed in imatinib responsive patients. Superoxide, ROS/RNS, mitochondrial ROS generation as well as H2O2 level was significantly more in drug naïve and imatinib resistant patients and it was attenuated significantly in imatinib responsive patient’s PMNs. In vitro treatment of K562 cells with Imatinib (2µM) also showed augmented NO generation and iNOS expression, while superoxide, ROS/RNS and mitochondrial ROS generation was decreased. To decipher the molecular mechanisms underlying the modulation of iNOS in BCR-ABL+ cells, we examined binding of NF-κB to iNOS promoter/enhancer and protein S-glutathionylation. Binding of NF-κB (p50 and p65 subunits) to iNOS promoter/enhancer was less in BCR-ABL positive cells, while it was augmented following treatment with imatinib. Moreover, glutathionylation of p50, p65 and procaspase-3 was more in drug naïve as well as in imatinib resistant CML patients PMNs, while it was comparable to healthy subjects in imatinib responders CML patients PMNs. Glutathionylation of NF-κB (p50 and p65 subunit) and procaspase-3 was also attenuated in imatinib treated K562 cells. The results obtained suggest that reduced NO generation/iNOS expression in BCR-ABL positive cells was due to the S-glutathionylation of NF-κB, which decrease it’s binding to iNOS promoter. S-glutathionylation of procaspase-3 in CML however, inhibited apoptosis of BCR-ABL positive cells. The study thus highlights importance of S-glutathionylation as key regulators involved in the proliferation and apoptosis of BCR-ABL positive cells. Disclosures No relevant conflicts of interest to declare.

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Abstract 11852: Role of Lox-1 in Mitochondrial Dna Damage and NLRP3 Inflammasome Activation

Circulation ◽

10.1161/circ.130.suppl_2.11852 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Zufeng Ding ◽

Sadip Pant ◽

Abhishek Deshmukh ◽

Jawahar L Mehta

Keyword(s):

Dna Damage ◽

Mitochondrial Dna ◽

Nlrp3 Inflammasome ◽

Ros Generation ◽

Inflammasome Activation ◽

Mtdna Damage ◽

Mitochondrial Dna Damage ◽

Mitochondrial Ros ◽

Nlrp3 Inflammasome Activation

Objective: This study tested the hypothesis that mitochondrial DNA damage could trigger NLRP3 inflammasome activation during inflammation, and LOX-1 may play a critical role in this process. Methods and Results: We performed studies in cultured human THP1 macrophages exposed to ox-LDL or LPS,which are often used as inflammation stimuli in vitro . We examined and confirmed the increase in LOX-1 expression when cells were treated with ox-LDL or LPS. Parallel groups of cells were treated with LOX-1 Ab to bind LOX-1. In accordance with our previous studies in endothelial cells and smooth muscle cells, LOX-1 Ab markedly reduced ox-LDL- as well as LPS-stimulated LOX-1 expression. To assess mitochondrial ROS generation, MitoSOX™ Red mitochondrial superoxide indicator was used. Both fluorescence staining and flow cytometry analysis showed that LPS induced (more than ox-LDL) mitochondrial ROS generation. Pretreatment with LOX-1 Ab significantly attenuated mitochondrial ROS generation in response to ox-LDL or LPS. Then we observed mtDNA damage in THP1 cells exposed to ox-LDL or LPS. Importantly, pretreatment with LOX-1 Ab protected mtDNA from damage in response to both stimuli. This was also confirmed by q-PCR (mtDNA/nDNA ratio) analysis. Further, ox-LDL or LPS induced the expression of phos-NF-kB p65, caspase-1 p10 and p20, and cleaved proteins IL-1β and IL-18. Of note, NLRP3 inflammasome was activated in response to ox-LDL or LPS in a similar manner. Pretreatment of cells with LOX-1 Ab treatment blocked or significantly attenuated these inflammatory responses. Conclusions: These observations based on in vitro observations indicate that LOX-1 via ROS generation plays a key role in mtDNA damage which then leads to NLRP3 inflammasome activation during inflammation.

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In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater green alga, Spirogyra sp.

Fisheries and Aquatic Sciences ◽

10.1186/s41240-020-00172-9 ◽

2020 ◽

Vol 23 (1) ◽

Author(s):

Lei Wang ◽

You-Jin Jeon ◽

Jae-Il Kim

Keyword(s):

Nitric Oxide ◽

Green Alga ◽

Raw 264.7 Cells ◽

Ros Generation ◽

Prostaglandin E ◽

No Production ◽

Raw 264.7 ◽

Anti Inflammatory

Abstract Background Inflammation plays a crucial role in the pathogenesis of many diseases such as arthritis and atherosclerosis. In the present study, we evaluated anti-inflammatory activity of sterol-rich fraction prepared from Spirogyra sp., a freshwater green alga, in an effort to find bioactive extracts derived from natural sources. Methods The sterol content of ethanol extract of Spirogyra sp. (SPE) was enriched by fractionation with hexane (SPEH), resulting 6.7 times higher than SPE. Using this fraction, the in vitro and in vivo anti-inflammatory activities were evaluated in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells and zebrafish. Results SPEH effectively and dose-dependently decreased the production of nitric oxide (NO) and prostaglandin E2 (PGE2). SPEH suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β through downregulating nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells without cytotoxicity. The in vivo test results indicated that SPEH significantly and dose-dependently reduced reactive oxygen species (ROS) generation, cell death, and NO production in LPS-stimulated zebrafish. Conclusions These results demonstrate that SPEH possesses strong in vitro and in vivo anti-inflammatory activities and has the potential to be used as healthcare or pharmaceutical material for the treatment of inflammatory diseases.

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Impaired mitochondria-dependent vasodilation in cerebral arteries of Zucker obese rats with insulin resistance

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.90656.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. R289-R298 ◽

Cited By ~ 31

Author(s):

Prasad V. G. Katakam ◽

Ferenc Domoki ◽

James A. Snipes ◽

Anna R. Busija ◽

Yagna P. R. Jarajapu ◽

...

Keyword(s):

Nitric Oxide ◽

Insulin Resistance ◽

Cerebral Arteries ◽

Ros Generation ◽

Ros Scavenging ◽

Mitochondrial Depolarization ◽

Mitochondrial Ros ◽

Endothelial Denudation ◽

Obese Rats ◽

Oxide Synthase

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (KATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial KATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 ± 3% in ZL vs. 21 ± 4% in ZO rats ( P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial KATP channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity.

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Dasatinib (BMS-354825) Overcomes Multiple Mechanisms of Imatinib Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽

10.1182/blood.v106.11.1994.1994 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1994-1994 ◽

Cited By ~ 10

Author(s):

Francis Y. Lee ◽

Mei-Li Wen ◽

Rajeev Bhide ◽

Amy Camuso ◽

Stephen Castenada ◽

...

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Leukemic Cell ◽

K562 Cells ◽

Src Family Kinases ◽

Imatinib Resistance ◽

Over Expression ◽

Imatinib Resistant

Abstract Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science305:399, 2004; O’Hare et al, Cancer Res65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol23:565s, 2005; Talpaz et al, J Clin Oncol23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START’ program, and in Phase I evaluation in solid tumors.

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Interleukin 21 Enhances the Expansion and Anti-Tumor Cytotoxic Activity of Cytokine-Induced Killer Cells Derived from Both Peripheral Blood and Cord Blood In Vitro.

Blood ◽

10.1182/blood.v110.11.4887.4887 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4887-4887

Author(s):

Mingfeng Zhao ◽

Qi Deng ◽

Yuming Li ◽

Xuemei Lin ◽

Li Geng ◽

...

Keyword(s):

Cytotoxic Activity ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Culture Supernatant ◽

K562 Cells ◽

Target Cells ◽

Cik Cells ◽

Interleukin 21 ◽

Anti Tumor Activity

Abstract Interleukin 21(IL-21) is a new member of interleukin 2 cytokine families which was discovered in 2000. IL-21 is produced by activated CD4 positive cells, and is known to influence T, B, NK cells and DC, and has potent anti-tumor effects. For example, IL-21 can improve the proliferation of B lymphocytes, enhance the production of IgG1; improve the proliferation and enhance the anti-tumor activity of both NK and T cells. The cytokine-induced killer (CIK) cells, which are characterized with the phenotype of CD3+CD56+, are the effective cells on adoptive cellular immunotherapy against tumors. We hypothesize that IL-21 could also affect the proliferation and function of CIK cells, thus play a certain role in the anti-tumor immunotherapy by CIK cells. The peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated with anti-CD3 (OKT3) monoclonal antibody and IFNgamma and then expanded with IL-2 and with/without IL-21(200ng/ml). CD3+CD56+ CIK cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cells cytotoxicity against chronic myeloid leukemia cell line K562 and a variety of tumor target cells from patients. The concentration of the IFNgamma in the culture supernatant was measured by enzyme-linked-immunoassay, the quantity of IFNgamma RNA was measured by RT-PCR assay, and the cytotoxic activity against K562 cells by the culture supernatant was also detected. Cultured with IL-21, at day 14, the quantity of CIK cells was increased from a median of 17.5% to 26.5% (PBMC original) and from 33.8% to 55.9% (CBMC original); The cytotoxic activity rates against K562 cells by CIK cells were increased from 24.0% to 52.2% (PBMC original) and from 35.1% to 79.7% (CBMC original); The concentration of IFNgamma in the culture supernatant was increased for 1.9-fold (PBMC original), and for 3.2-fold (CBMC original); The cytotoxic activity against K562 cells by the culture supernatant was increased for 1.8-fold (PBMC original) and for 2.7-fold (CBMC original); The expression of IFNgamma RNA in CIK cells was also markedly increased derived from both PBMC and CBMC when cultured with IL-21. Moreover, the cytotoxic activity against leukemia cells from 11 patients (6 with acute lymphoblastic leukemia, 5 with acute myeloid leukemia) by CIK cells derived from CBMC were also detected. The cytotoxic activity rates were at a median of 68.3% (range, 34.7%–86.4%) when CIK cells were cultured with IL-21, rates that contrasted drastically to the cytotoxic activity rates when CIK cells were cultured without IL-21, which were only at a median of 37.4% (range, 16.1%–60.0%). In conclusion, our data indicated that IL-21 could enhance the expansion of CIK cells and their anti-tumor activity derived from both PBMC and CBMC in vitro, IFNgamma was evolved in this course although the mechanism still need to be explored. These observations open up the possibility of imagining a future clinical application of IL-21 in the anti-tumor immunotherapy by CIK cells.

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Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C

Blood ◽

10.1182/blood.v122.21.5158.5158 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5158-5158

Author(s):

Qi Shen ◽

Sichu Liu ◽

Yu Chen ◽

Lijian Yang ◽

Shaohua Chen ◽

...

Keyword(s):

Gene Expression ◽

Chronic Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Primary Cells ◽

Healthy Individuals ◽

Hoechst 33258 ◽

Imatinib Resistance ◽

Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML. Disclosures: Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

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Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-xL

Blood ◽

10.1182/blood.v95.3.1014.003k04_1014_1022 ◽

2000 ◽

Vol 95 (3) ◽

pp. 1014-1022 ◽

Cited By ~ 162

Author(s):

Charles Perkins ◽

Caryn N. Kim ◽

Guofu Fang ◽

Kapil N. Bhalla

Keyword(s):

Myeloid Leukemia ◽

Blast Crisis ◽

K562 Cells ◽

Cell Types ◽

Multidrug Resistant ◽

Leukemia Cells ◽

Protein Levels ◽

Growth Inhibitory ◽

Induced Apoptosis

We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As2O3 (0.5 to 2.0 μmol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-xL(HL-60/Bcl-xL), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC50 values for As2O3 treatment for 7 days against all these cell types ranged from 0.8 to 1.5 μmol/L. Exposure to 2 μmol/L As2O3 for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (▵Ψm) and the increase in reactive oxygen species (ROS). Treatment with As2O3 (2 μmol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As2O3-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-xL, Bax, Apaf-1, Fas, and FasL. Although As2O3 treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As2O3 potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As2O3 as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.

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Alternatively spliced truncated BCR-ABL1 protein in CML patients with resistance to kinase inhibitors

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.7026 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 7026-7026

Author(s):

J. Bruey ◽

H. Kantarjian ◽

W. Ma ◽

C. Yeh ◽

R. Peralta ◽

...

Keyword(s):

Structural Changes ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Residual Disease ◽

Chronic Phase ◽

K562 Cells ◽

Aurora Kinase ◽

Imatinib Resistant ◽

Alternatively Spliced

7026 Background: We have reported that some patients with imatinib-resistant chronic myeloid leukemia (CML) express an alternatively spliced BCR-ABL mRNA with a 35-bp insertion (BCR-ABL135INS), resulting in the addition of 10 residues and truncation of 653 residues. Molecular dynamic simulation suggested that this truncation and insertion of new 10 AA results in structural changes similar to those seen in BCR-ABL with T315I mutation. Here we evaluate the prevalence of BCR-ABL135INS in imatinib-resistant CML, examine the effect of this mutation on resistance to compared the efficiency of various kinase inhibitors in vitro, and suggest a model for persistent CML and a possible strategy to eradicate residual disease. Methods: Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in 288 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot. We then tested the effectiveness of various kinase inhibitors on human K562 CML cells expressing different levels of BCR-ABL135INS along with wild-type BCR-ABL1. Results: BCR-ABL135INS mRNA was detected in 210 (73%) of the 288 patients. Only 25% of BCR-ABL135INS positive cases showed coexistance of ABL1 kinase point mutation. Immunoprecipitation studies demonstrated that expression of the predicted 143-kD BCR-ABL135INS protein at levels proportional to those predicted by mRNA. Expression of BCR-ABL135INS in K562 cells was sufficient to conferred resistance to imatinib, dasatinib, and nilotinib in a dose-dependant fashion. However, no resistance was detected using aurora kinase inhibitor (MK 0457) or homoharringtonine (HHT). BCR-ABL135INS suppressed imatinib, nilotinib, and dasatinib-mediated dephosphorylation of CRKL, LYN, SRC, and STAT5, but had no effect on MK 0457-mediated dephosphorylation. The combination of imatinib with nilotinib or HHT showed strong synergy, overcoming BCR-ABL135INS-induced resistance in vitro. Conclusions: These findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS protein and may provide a strategy to treat resistant disease and eradicate residual CML. No significant financial relationships to disclose.

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Inhibitory Effect of Hydroxyapatite Nanoparticles on K562 Cells

Materials Science Forum ◽

10.4028/www.scientific.net/msf.685.352 ◽

2011 ◽

Vol 685 ◽

pp. 352-356 ◽

Cited By ~ 2

Author(s):

Hong Lian Dai ◽

Pei Chen ◽

Yin Chao Han ◽

Xin Yu Wang ◽

Shi Pu Li

Keyword(s):

Myeloid Leukemia ◽

K562 Cells ◽

Inhibitory Effect ◽

Inhibition Mechanism ◽

Hydroxyapatite Nanoparticles ◽

X Ray Diffraction ◽

Transmission Electron ◽

Cell Changes ◽

Crystallization Degree

HAP Nanoparticles Was Synthesized by Homogeneous Precipitation. the Size Distribution, Crystallization Degree and Morphology of the Precipitation Were Characterized by Laser Granularity Instrument, X-Ray Diffraction (XRD), and Transmission Electron Microscope (TEM) Respectively. the Prepared HAP Nanoparticles Were Used for the Treatment of Human Chronic Myeloid Leukemia K562 Cells. the Inhibition Effect of the Nanoparticles on the Proliferation of K562 Cells Was Measured by MTT Assay and Growth Curve Test. the Results Showed that the HAP Nanoparticles Inhibit the Proliferation of K562 Cells Dramatically in Vitro. the Likely Inhibition Mechanism of HAP Nanoparticles on the K562 Cells Is that the Nanoparticles Entered into the Dells, Induced a Series of Cell Changes, through Cell Death of Apoptosis, Oncosis and Autoschizis, Thus Led to the Death of K562 Cells.

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Vitamin E in Chronic Myeloid Leukemia (CML) Prevention

10.5772/intechopen.96452 ◽

2021 ◽

Author(s):

Lyudmyla Shvachko ◽

Michael Zavelevich ◽

Daniil Gluzman ◽

Gennadii Telegeev

Keyword(s):

Alkaline Phosphatase ◽

Vitamin E ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Blast Crisis ◽

K562 Cells ◽

Leukemic Cells ◽

Blast Cells ◽

Differentiation Block

The resistance to inhibitors of tyrosine kinase necessitates novel approaches to the therapy of chronic myeloid leukemia (CML). The progression of CML to blast crisis is associated with down-regulation of C/EBP-alpha being involved in the differentiation block in leukemic blast cells. Moreover, lowered C/EBP-alpha expression correlates with resistance to imatinib in CML. We have demonstrated that vitamin E up-regulates expression of C/EBP-alpha and down-regulates expression of Snail transcription factor in K562 cells in vitro contributing to the putative recovery of myeloid differentiation potential. In parallel with increased CEBP alpha expression, Vitamin E treatment results in the decreasing expression of placental-like alkaline phosphatase and increasing expression of tissue non-specific alkaline phosphatase. We suggest that vitamin E could be used as the plausible biological modulator to prevent the progression to blast crisis and to overcome drug resistance of leukemic cells in CML.

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