Myeloid Leukemia Cell Line HL-60 Research Articles

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

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

Gene expression profiling of HL-60 cells following knockdown of nucleostemin using DNA microarrays.

Nucleostemin (NS) plays an important role in tumorigenesis and progression. Most studies consider that NS plays its role through combining with p53 and inhibiting it, however our previous studies revealed that NS could also function without the existence of p53. To date, few studies have focused on the p53-independent pathway of NS, and its molecular mechanism remains unknown. The aim of the present study was to investigate the p53-independent pathway of NS in the human acute myeloid leukemia cell line HL-60 which was p53-null by using the DNA microarray technique. Lentivirus-mediated RNA interference technique was used to knock down NS expression in HL-60 cells, and then DNA microarray and bioinformatics were used to analyze the gene expression profiling changes. The microarray data showed that after knocking down NS in HL-60 cells, 2,628 differentially expressed genes were identified through ≥ 2 or ≤ 0.5-fold-change, in which 818 genes were upregulated and 1,810 genes were downregulated. Real-time quantitative polymerase chain reaction (qPCR) validated the reliability of DNA microarray data. Pathway analysis showed extensive signal pathways in HL-60 cells were influenced by inhibiting NS expression. In particular, the inhibition of PI3K-AKT pathway, JAK-STAT pathway, RAS-RAF-MEK-ERK1/2 pathway and activation of JNK pathway, p38 MAPK pathway may associate with the apoptosis of HL-60 cells after knocking down NS. The findings of this study provide insight to further explore the specific molecular mechanism of NS function in p53-null leukemia and they also lay the foundations for exploring new therapeutic targets for p53-null leukemia and even p53-null tumors.

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

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

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

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

Down-Regulation of Mcl-1 by Small Interference RNA Induces Apoptosis and Sensitizes HL-60 Leukemia Cells to Etoposide

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

Inhibiting effect of CaMKIIN up-regulation on leukemia cells growth and its mechanism

To investigate the inhibitory effects of CaMKIIN on acute myeloid leukemia cell line HL-60 to explore a novel therapeutic target of leukemia. Human CaMK II N gene expression vector pcDNA3.1/hCaMKIIN or empty vector pcDNA3.1/myc-His (-) B was transfected into HL-60 cells by Lipofectamine 2000. Human CaMK II N proteins of transfected cells were detected by Western blot. Cell proliferation affected by human CaMKIIN was determined by MTT. Colony-forming assay was performed by soft agar growth system. The cells transfected with CaMKIIN were stained with Hoechst 33342 to detect the apoptotic proportion under fluorescence microscopy. Cell cycle was analyzed by flow cytometry. Human CaMKIIN was stably transfected into HL-60 cells, and overexpression of human CaMKIIN inhibited the proliferation of HL-60/CaMKIIN cells compared to HL-60/mock cells and HL-60 cells [(0.44 ± 0.03) vs (0.94 ± 0.05) vs (0.94 ± 0.04), P<0.01]. The colony formation of HL-60/CaMKIIN was also markedly smaller[(21.00 ± 3.05)/500] than that of mock-transfected [(111.00±4.58)/500]] and control cells [(119.00±6.09)/500] (P<0.01). After 72 hrs-culture, the apoptotic proportion in cells transfected with CaMK II N was obviously higher than of cells transfected with mock DNA or control [(22.49 ± 2.15)% vs (7.17 ± 0.72)% vs (6.40 ± 0.55)%, P<0.01]. Up to (82.97 ± 2.90)% human CaMKIIN/HL-60 cells were arrested at G0/G1 phase, which was more than mock-transfected [(40.53 ± 2.38)%] and control cells [(41.63 ± 2.27)%] (P<0.05). Human CaMKIIN could down-regulate expression of Bcl-2 in transfected cells. CaMK IIN up-regulation could inhibit proliferation and induce apoptosis of human acute myeloid leukemia cell HL-60.

Novel Small-Molecule Human G-CSF Receptor Agonist Stimulates Neutrophil Counts In Monkeys and Displays Anti-Proliferative Effects On Tumor Cells Mediated By a Reduction Of Intracellular Iron

Previously we reported on the discovery of a series of novel, non-peptidyl, small molecules that selectively activate the human granulocyte colony stimulating factor receptor (hG-CSFR) in a manner distinct from G-CSF and similar to the mechanism of small-molecule human thrombopoietin receptor (hTPOR) agonists [Marschke KB, et al. ASH 53rd Annual Meeting Abstracts, Blood 2011 118(21):1453, Abstract 3391]. The lead compound, LG7455, activates STAT5 and STAT3 signal transduction and induces G-CSF-dependent hematopoietic cell growth, but not TPO-dependent or erythropoietin-dependent cell growth. LG7455 promotes the differentiation of human bone marrow cells into granulocytes, significantly increasing expression of the granulocyte-specific marker CD15 (FUT4). The G-CSF-like effect of LG7455 requires the expression of human or monkey G-CSFR, and a histidine in the transmembrane domain is needed for activity, similar to what has been found for small-molecule hTPOR agonists, such as eltrombopag. LG7455 increases the binding of GCSF in a manner consistent with allosteric receptor modulation. To test the ability of LG7455 to stimulate neutrophil counts in vivo, cynomolgus monkeys were dosed intravenously (IV) 3 times per day (TID) to increase LG7455 exposure. Increases in peripheral blood neutrophils were observed in male cynomolgus monkeys with LG7455 (2 mg/kg/dose, TID). Because of the potential for LG7455 to interact with metal ions, their role in the activity of LG7455 was investigated. The ability of LG7455 to activate luciferase expression in cells transiently transfected with an hG-CSFR expression vector and a STAT5-responsive luciferase reporter was dependent on the addition of zinc (II) ion (up to 30 µM) to the cell culture media. The addition of Zn(II) also significantly enhanced the efficacy (63% vs. 23% relative to rhG-CSF) and potency ( EC50 of 28 nM vs. 300 nM) by which LG7455 stimulated the growth of UT-7 cells made dependent on G-CSF by stable transfection of hGCSFR (UTP-hGCSFR). LG7455 did not activate luciferase expression or stimulate growth of cells incubated in the presence of additional iron (III), copper (II), magnesium (II) or calcium (II) ions. Similar to the antitumor effect of the well-described iron chelator salicylaldehyde isonicotinyl hydrazine (SIH), LG7455 inhibited the growth of a selection of human tumor cell lines, including the myeloid leukemia cell lines HL-60 and Kasumi-1, the lung carcinoma cell line A549, the embryonic kidney cell line HEK293, and the hepatocellular carcinoma cell line HepG2 (IC50's of 5.0, 12.1, 6.1, 1.4 and 1.0 µM, respectively). SIH did not stimulate but instead inhibited the growth of UTP-hGCSFR cells. The anti-proliferative effect of LG7455 on HL-60 cells was accompanied by an increase in apoptosis as determined by Annexin V/propidium iodide staining and FACS analysis. The addition of excess Fe(III) to the tumor cells was able to overcome the anti-proliferative effect of LG7455 in a concentration-dependent manner, indicating that a reduction in intracellular iron plays a role in the effect. The addition of Zn(II) and Cu(II) were less efficient at blocking the anti-proliferative effect of LG7455 than Fe(III). Addition of Mg(II) or Ca(II) had no effect. LG7455 increased the fluorescence of HL-60 cells loaded with calcein-AM, a fluorescent dye quenched when bound to iron, further supporting that a reduction of free intracellular iron occurs in cells treated with LG7455. Lastly, as has been shown with other iron chelators with anti-proliferative activity, LG7455 increased intracellular reactive oxygen species (ROS) formation in HL-60 cells loaded with the cell-permeant ROS indicator, CM-H2-DCFDA, and evaluated by flow cytometry. Further optimization of the LG7455 series may provide a novel, orally-available adjuvant anticancer therapy that is also a supportive care agent to treat neutropenia in patients receiving bone-marrow suppressive treatments. Disclosures:Marschke:Ligand Pharmaceuticals Incorporated: Employment. Vajda:Ligand Pharmaceuticals Incorporated: Employment. Zhi:Ligand Pharmaceuticals Incorporated: Employment.

Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells

Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.

A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion

We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C - MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50 μM. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50 μM. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization.

Effects of Rheb overexpression in HL-60 and K562 leukemia cell lines

mTOR (mammalian target of rapamycin) is the center for cellular activities. It controls many cell activities via inhibiting apoptosis and promoting cell growth. Rheb can activate mTOR signaling pathway and participate in genesis and development of multiple cancers. This study was purposed to explore the underlying role of Rheb in human myeloid leukemia by using the myeloid leukemia cell lines. Two myeloid leukemia cell lines HL-60 and K562 overexpressing Rheb were established with retrovirus containing Rheb. The mRNA and protein expressions of Rheb were determined by Real-Time PCR and Western blot respectively. Cell proliferation rate was examined by CCK-8 assay and apoptosis rate was analyzed using Annexin V and 7-AAD double-staining. The results showed that Rheb was overexpressed in both HL-60 and K562 cell lines. The Rheb overexpression cell lines were successfully established. It is found that overexpression of Rheb could promote cell growth. Furthermore, the overexpression of Rheb could accelerate cells entering into G2/M phase (P < 0.01), while did not affect the apoptosis. It is concluded that Rheb overexpression promotes myeloid leukemia cell proliferation through accelerating cell cycle progression.

Myeloid leukemia cells with a B7‐2+ subpopulation provoke Th‐cell responses and become immuno‐suppressive through the modulation of B7 ligands

Expression of the B7 family molecules in acute myeloid leukemia (AML) has been demonstrated by independent clinical studies. Intriguingly, the expression of the most potent costimulatory molecules B7-2 (CD86) and B7-H2 (ICOS Ligand) on AML cells has been associated with poor prognosis and disease severity. Here, this phenomenon was modeled in vitro with the myeloid leukemia cell line HL-60, which is capable of differentiating through the FAB M2/M3 and M4/M5 immunophenotypes. These derivatives of HL-60 harbored a B7-2(+) subpopulation and recapitulated the distribution of B7 ligands previously reported in primary AML cases. B7-2(+) AML cells significantly contributed to T-cell responses. This costimulatory activity enabled helper (Th)-cell activation, proliferation, and production of Th1-associated cytokines. Conversely, even a short-term incubation with stimulated T cells resulted in upregulation of inhibitory B7-H1 (PD-L1) and B7-DC (PD-L2), and downregulation of stimulatory B7-H2 molecules on leukemia cells. Purified from iHL-60-T-cell co-cultures, these myeloid leukemia cells severely suppressed Th-cell responses specifically through the PD-1 pathway. In conclusion, Th-cell responses can be directly supported by B7-2(+) leukemia subpopulations. However, this interaction can facilitate the acquisition of a suppressive character that may contribute to immune evasion in myeloid leukemia.

Euphohelioscopin A Is a PKC Activator Capable of Inducing Macrophage Differentiation

To identify small molecules that selectively control hematopoietic stem cell differentiation, we performed an unbiased screen using primary human CD34(+) cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34(+) cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the promonocytic effects of this natural product are mediated by PKC activation. In addition to shedding insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.

MiR-125b, a Target of CDX2, Regulates Cell Differentiation through Repression of the Core Binding Factor in Hematopoietic Malignancies

MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor β (CBFβ) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFβ was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFβ in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.

Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663.

BackgroundDifferentiation of the acute myeloid leukemia (AML) cell line HL-60 can be induced by all trans-retinoic acid (ATRA); however, the mechanism regulating this process has not been fully characterized.MethodsUsing bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation.ResultsSix microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro.ConclusionsOur results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies

Abstract 277: Genetic re-programming of the acute myeloid leukemia cell line HL-60

Abstract HL-60 cells represent an established line of human acute myeloid leukemia cells that can be induced to differentiate along several distinct myeloid lineages. Phorbol esters activate a signaling network, which includes the protein kinase C (PKC) signaling pathway, causing the leukemia cells to differentiate into macrophage-like cells. As the leukemia genetic program is converted to the macrophage program, the cells exit the cell cycle, undergo morphological changes, become adherent, display other macrophage properties, and ultimately initiate apoptosis. This study provides a detailed DNA microarray analysis of the genetic network of the leukemia cell during first 24 hours post-induction of differentiation. Approximately 1,000 genes were identified that exhibit significant changes in mRNA levels from the leukemia expression profile. A cluster analysis was performed to group these genes by their pattern of expression resulting in a collection of distinct expression profiles. Expression levels of selected genes within each cluster were validated by real time PCR. Among the most significant changes observed were dramatic increases in the expression of CCL chemokine family genes and a variety of transcription factor genes including members of the AP-1 and NF-κB families. Expression level changes in genes involved in cell cycle progression (ex. MYC and p21) were also observed along with genes involved in the regulation of apoptosis (ex. BCL2A1, TNF, IER2 and OLR1). Functional analysis at the protein level using the electrophoretic mobility shift assay (EMSA), western blotting and immunofluorescence imaging will also be presented. This study provides a comprehensive view of the re-programming events that occur in phorbol ester induced macrophage differentiation of HL-60 cells with regard to the inflammatory response, cell cycle exit, differentiation and initiation of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 277. doi:10.1158/1538-7445.AM2011-277

Mangiferin induces apoptosis by suppressing Bcl-xL and XIAP expressions and nuclear entry of NF-κB in HL-60 cells

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-D: -glucoside (C-glucosylxanthone), is a xanthone derivative that is widely distributed in higher plants. Recently, mangiferin was found to exhibit potential antitumor effects. However, the molecular mechanisms of this effect have not been elucidated. In the present study, we attempt to clarify the mechanism of mangiferin-induced apoptosis in the human acute myeloid leukemia cell line HL-60; mangiferin was found to induce apoptosis. We also observed a concurrent increase in caspase-3 activity and DNA fragmentation. Furthermore, on examining the survival signals expressed during apoptotic induction, we observed that mangiferin caused a remarkable decrease in the nuclear entry of NF-κB p65. However, there were no changes in the expression of other survival signals, such as extracellular signal-regulated kinase 1/2, protein kinase B, and p38 mitogenactivated protein kinase. In addition, mangiferin suppressed the expressions of Bcl-xL and XIAP; however, we did not note any changes in the levels of Bcl-2, Bax, and Bim. These results indicate that mangiferin induces apoptosis by suppressing NF-κB activation and expressions of Bcl-xL and XAIP. These findings suggest that mangiferin may be useful as an anticancer agent and can be used in combination therapy with other anticancer drugs for the treatment of acute myeloid leukemia.

Seed Oil of Brucea javanica Induces Apoptotic Death of Acute Myeloid Leukemia Cells via Both the Death Receptors and the Mitochondrial‐Related Pathways

Seed oil of Brucea javanica (BJO) is extracted from the seeds of herb medicine Brucea javanica (L.), and its emulsion formulation (BJOE) has been used clinically to treat carcinomas for many years in China. The antileukemia potential of BJO was investigated in human acute myeloid leukemia cell lines (AML) U937 and HL-60 in vitro and in a mouse U937 xenograft tumor model. BJO induced AML cell apoptosis through activation of caspase-8 and modulation of apoptosis-related proteins. Meanwhile, the inhibition of survivin and XIAP increased the cytotoxicity of BJO. Consistent with these findings, BJO also increased subG1 phase cells and cause PARP cleavage in AML patients' leukemia cells. In contrast, only weak cytotoxicity of BJO was found in peripheral blood lymphocytes (PBLs) of healthy volunteers. Moreover, oleic acid and linoleic acid were found to be the active components of BJO. Our study provided strong evidence for the first time that BJO induced apoptosis of both cultured and primary AML cells. Furthermore, intravenous injection of BJO significantly inhibited U937 tumor growth in the xenograft mouse model. These results suggest that BJO may have a therapeutic role in the treatment of human leukemia.

DIGE-Based Proteomic Analysis Identifies NPM/B23 and Nucleolin C23 as Overexpressed Proteins In Relapsed /Refractory Acute Leukemia

AbstractAbstract 1711Resistance to chemotherapy is a challenge in treatment of acute leukemia. Although the classic multidrug resistance (MDR) phenotype is often characterized by expression of drug efflux pump P-glycoprotein or by multidrug resistance-associated proteins, precise molecular mechanisms are largely unknown. To investigate novel protein changes involved in resistance mechanism, protein expression profiles between human myeloid leukemia HL-60 cell lines and adriamycin- resistant HL-60 cell lines (HL-60/ADR) was compared, which was based on a differential proteomic approach — 2 dimensional difference in gel Electrophoresis(2D-DIGE) followed by mass spectrometry (MALDI-TOF-MS) and complemented by western blot validation. 16 protein spots were identified as being differentially expressed (> 1.2 fold change and p≤ 0.05) between above two cell lines, among which 13 protein spots were identified as up-regulated and 3 as down-regulated in the HL-60/ ADR cell line. Proteins found to have higher abundance levels in the resistant HL-60/ADR cells included enzymes, proteins and oncogenes related to signal transduction, protein synthesis, cell growth regulation and metabolism. 3 lower abundance proteins are related to transcription. From 16 proteins, 2 proteins, nucleophosmin B23 (NPM B23)and nucleolin C23, were selected and verified in leukemia cell lines and primary leukemia samples by western blot. Compared to healthy control samples, which showed no expressions of these 2 proteins, leukemic cell lines revealed an obvious up-regulation of B23 and C23. Moreover, significantly higher expressions of B23 and C23 were found in 3 resistant leukemic cell lines, HL-60/ADR, K562/ADR and KG01 cells, compared to the parent HL-60 and K562 cells, and other leukemic cell lines. In de novo leukemia samples, 43.8%(35/80) expressed B23 and C23 proteins, 37.9% (22/58) AML and 59.1% (13/22) ALL respectively. Meanwhile, concomitant expression of B23 and C23, both positive or negative, was noted in 97%(79/80)patients. Over-expressions of B23 and C23 were observed in 68.8% relapased/refractory leukemia patients. With regard to treatment outcome,among those patients who achieved ongoing CR, fewer patients expressed 2 proteins, only 13.35% (7/52) AML and 46%(7/15) ALL respectively. It implicated that B23 and C23 may be involved in drug resistance and be useful in assessing treatment outcome and prognosis of leukemia. To a conclusion, these results provide a novel clue for the molecular mechanism of MDR and suggest that B23 and C23 are prognostic indicators for leukemia. Disclosures:No relevant conflicts of interest to declare.

Nampt/SIRT2 Hyperactivation Lead to Activation of ß-Catenin: a Possible Mechanism of Leukomogenic Transformation In CN Patients.

AbstractAbstract 1486Recently we demonstrated that nicotinamide phosphoribosyltransferase (NAMPT) is an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis via activation of NAD+/sirtuins/C/EBPs signaling cascade. Nampt levels were significantly elevated in plasma and in myeloid cells of patients with severe congenital neutropenia (CN). CN is characterized by a “maturation arrest” of granulopoiesis on the promyelocytic stage of differentiation and by leukemogenic tansformation of hematopoiesis in ca. 20 % of patients. The mechanism of the leukemic transformation is still unclear. Previously, we reported elevated levels of activated oncogene ß-catenin in nuclei of myeloid progenitor cells of CN patients. The activity and nuclear translocation of ß-catenin is regulated by glycogen synthase kinase-3 ß (GSK3ß), which activates ß-catenin degradation complex. In the present study we found that in myeloid cells of CN patients GSK3ß was inhibited by phosphorylation on Ser9, as compared to healthy individuals. Therefore, we assume that GSK3ß-ß-catenin pathway could be involved in the leukemogenic transformation of hematopoiesis. Since, Nampt was also elevated in CN patients, we aimed to investigate the connection between hyperactivated Nampt and ß-catenin in leukemogenesis. The Nampt functions in hematopoiesis are dependent on the dose of Nampt and NAD+. Thus, in vitro stimulation of CD34+ cells with Nampt led to granulocytic differentiation via activation of sirtuin/C/EBP-dependent pathway. At the same time, inhibitors of NAMPT have been identified as therapeutical targets for some cancers including leukemia. This suggested that different mechanisms are operating downstream of NAMPT in the “normal” and leukemogenic myeloid cells. Screening of the different sirtuins in primary acute myeloid leukemia (AML) blasts revealed significant upregulation of SIRT2 mRNA and protein levels, as compared to CD34+ and CD33+ hematopoietic cells of healthy individuals. SIRT2 levels were also elevated in myeloid cells of CN patients treated with G-CSF. Specific inhibition of NAMPT (using 10 nMol of FK866) or SIRT2 (using 100nMol of AC93253) significantly reduced proliferation and induced apoptosis in human myeloid leukemia cell lines (NB4, HL60 and U937). We further tested if inhibition of Nampt or SIRT2 has an effect on GSK3ß/ß-catenin pathway. GSK3ß is known to be inhibited by Akt and treatment of the acute myeloid leukemia cell lines NB4 and HL60 with FK866 or AC93253 resulted in the activation of Akt via phosphorylation on Thr308 and Ser473 and inactivation of GSK-3ß via inhibition of phosphorylation on Ser9. Moreover, activated ß-catenin protein was almost completely disappeared from the nucleus of cells treated with FK866. Taken together, our results provide strong evidence that NAMPT and SIRT2 participate in leukemogenic transformation via inactivation of GSK3ß leading to nuclear accumulation of oncogenic ß-catenin. Disclosures:No relevant conflicts of interest to declare.

Screening for methylation-silenced genes in acute myeloid leukemia HL-60 cell line by a quantitative proteomic approach.

To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment. Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.