Human Myeloid Leukemia Cell Line Research Articles

The Influence of Glutamic Pyruvate Transaminase 2 to Biological Characteristics of Acute Myeloid Leukemia Cell HL-60

To investigate the influence of GPT2（glutamic pyruvate transaminase 2）to biological characteristics of human acute myeloid leukemia cell line HL-60. The expression of GPT2 in hematological tumor and AML cell was detected. The lentvirus-mediated of short-hairpin RNA (shRNA) was constricted, and the knock-down efficiency of HL-60 in AML cell after infected by lentvirus-mediated was detected by Western blot and Q-PCR. CCK-8 assay and soft agar colony formation assay were used to detect the effect of GPT2 gene deletion to the cell proliferation potential. Fluorescence activated cell sorting(FACS) was used to analyze the effect of gene deletion to the cell cycle and Caspase 3/7 Activity Assay Kit was used to analyze the effect of GPT2 gene deletion to the cell apoptosis. GPT2 showed mRNA high expression in AML patients. CCK-8, soft agar assay, and Caspase 3/7 Activity Assay Kit results showed that compared with shCtrl group, the cells in shGPT2-1、shGPT2-2、shGPT2-3 group showed the slowing down on proliferation, decreasing on colony ability, and the apoptosis of the cells was increasing significantly. FACS showed that GPT2 gene was related to the cycle of HL-60 cell. GPT2 appears to involve the proliferation, cycle distribution and apoptosis of AML cell HL-60. The deletion of GPT gene can lead to the inhibitation of cells proliferation and increase apoptosis.

Mitochondria in human acute myeloid leukemia cell lines have ultrastructural alterations linked to deregulation of their respiratory profiles

Mitochondria not only are essential for cell metabolism and energy supply but are also engaged in calcium homeostasis and reactive oxygen species generation and play a key role in apoptosis. As a consequence, functional mitochondrial disorders are involved in many human cancers including acute myeloid leukemia (AML). However, very few data are available on the deregulation of their number and/or shape in leukemic cells, despite the evident link between ultrastructure and function. In this context, we analyzed the ultrastructural mitochondrial parameters (number per cell, mitochondria area, number of cristae/mitochondria, cristal thickness) in five leukemia cell lines (HEL, HL60, K562, KG1, and OCI-AML3) together with the functional assay of their respiratory profile. First, we describe significant differences in basal respiration, maximal respiration, ATP production, and spare respiratory capacity between our cell lines, confirming the various respiratory profiles among leukemia subtypes. Second, we highlight that these variations are obviously associated with significant interleukemia heterogeneity of the number and/or shape of mitochondria. For instance, KG1, characterized by the smallest number of mitochondria together with reduced cristal diameter, had a particularly deficient respiratory profile. In comparison, the HEL and K562 cell lines, both with high respiratory profiles, harbored the largest number of mitochondria/cells with high cristal diameters. Moreover, we report that K562, carrying the ASXL1 mutation, presents significant mitochondria-endoplasmic reticulum deficiency reflected by decreases in the numbers of matrix granules and mitochondria-associated endoplasmic reticulum membrane (MAM) and mitochondrial-derived vesicle (MDV) precursors, which are implicated in the regulatory pathways of cell mortality via the processes of mitophagy and calcium homeostasis. Contrarily, HL60 carried high levels of matrix granules and MAMs and had a higher sensitivity to drugs targeting mitochondria (rotenone/antimycin). We confirm the implication of ASXL1 mutation in this mitochondria dysregulation through the study of transcript expression (from 415 patients with public data) involved in three mitochondrial pathways: (1) endoplasmic reticulum-mitochondria contacts (MAMs), (2) matrix granule homeostasis, and (3) MDV precursor production. Our study offers new and original data on mitochondria structural alterations linked to deregulation of respiration profiles in AMLs and some genetic characteristics, suggesting that modifications of mitochondrial shape and/or number in leukemic cells participate in chemoresistance and could be a targeted mechanism to regulate their proliferative potential.

The Agglutinin of Common Nettle (Urtica dioica L.) Plant Effects on Gene Expression Related to Apoptosis of Human Acute Myeloid Leukemia Cell Line

Treatment of acute myeloid leukemia (AML) requires new drugs as result of a rise in new cases and high disease relapse. Plant lectins with the ability to bind carbohydrates on the cell surface have the potential to treat cancer. Urtica dioica L. agglutinin (UDA) is a low weight lectin with anti-benign prostatic hyperplasia (BPH) impact. Here, we examine the impact of UDA on HL-60 cell line. Cytotoxicity and cytostatic effects were assessed in HL-60 cells treated with UDA and vincristine (positive control). The effects of the lectin on cell cycle phases and cell death mechanism were surveyed by propidium iodide (PI) staining and annexin V/PI, respectively. The activation status of the apoptosis pathway was determined by western blotting. Finally, the expression levels of 84 genes were examined by the Human cancer drug target gene PCR array kit. The results indicated that the increase in UDA concentration inhibited the proliferation of HL-60 cells as well as apoptosis induction. Cell cycle analysis showed that the number of sub G1 cells increased essentially. Experimental observations showed that UDA can induce cell apoptosis through a caspase 9-dependent pathway. The expression changes of 21 genes confirmed the apoptotic events in HL-60 cells treated with UDA. In this, we have presented the first investigation on the cytotoxic and apoptotic effects of a lectin isolated from rhizomes and roots of Urtica dioica L. on human AML cells. Generally, the results suggest that UDA may have therapeutic value for leukemia and would be studied further as a new drug for AML later on.

Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents

We established the following two variants of the MOLM-13 human acute myeloid leukemia (AML) cell line: (i) MOLM-13/DAC cells are resistant to 5-aza-2′-deoxycytidine (DAC), and (ii) MOLM-13/AZA are resistant to 5-azacytidine (AZA). Both cell variants were obtained through a six-month selection/adaptation procedure with a stepwise increase in the concentration of either DAC or AZA. MOLM-13/DAC cells are resistant to DAC, and MOLM-13/AZA cells are resistant to AZA (approximately 50-fold and 20-fold, respectively), but cross-resistance of MOLM-13/DAC to AZA and of MOLM-13/AZA to DAC was not detected. By measuring the cell retention of fluorescein-linked annexin V and propidium iodide, we showed an apoptotic mode of death for MOLM-13 cells after treatment with either DAC or AZA, for MOLM-13/DAC cells after treatment with AZA, and for MOLM-13/AZA cells after treatment with DAC. When cells progressed to apoptosis, via JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) assay, we detected a reduction in the mitochondrial membrane potential. Furthermore, we characterized promoter methylation levels for some genes encoding proteins regulating apoptosis and the relation of this methylation to the expression of the respective genes. In addition, we focused on determining the expression levels and activity of intrinsic and extrinsic apoptosis pathway proteins.

Design, synthesis, and biological evaluation of novel bromo-pyrimidine analogues as tyrosine kinase inhibitors

In the present investigation we designed, synthesized and evaluated a novel series of bromo-pyrimidine analogues (6a-j,7a-e,9a-f,and10a-f) as anticancer agents. The compounds were characterized using spectroscopic studies and elemental analysis and screened for theirin vitrocytotoxic activity by MTT assay against four cancer cell lines including HCT116 (human colon cancer cell line), A549 (human lung cancer cell line), K562 (human chronic myeloid leukemia cell line), U937 (human acute monocytic myeloid leukemia cell line) as well as the normal human liver cell line, L02. Most of the compounds showed potent activity on K562 cells. Considering this, the compounds were evaluated for Bcr/Abl tyrosine kinase inhibitory activity by ADP-Glo assay. Dasatinib was used as standard drug for both cytotoxicity and tyrosine kinase inhibition studies. The compounds,6g,7d,9c,and10eemerged as potent Bcr/Abl kinase inhibitors. Hence, the potent compounds that arose out of this investigation are potential lead molecules to develop as an alternative to existing dasatinib therapy.

Induction of Apoptosis in the U937 Cell Line Co-cultured with Adipose-derived Stem Cells Secreting Bone Morphogenetic Protein-4.

Transforming growth factor-beta (TGF-β) plays a significant role in tumorigenesis. MiR-181b is a multifunctional miRNA involved in numerous cellular processes, such as cell fate and cell invasion. This study aimed to examine whether the co-culture of adipose-derived stem cells (ADSCs), highly expressing bone morphogenetic protein-4, with the U937 cell line, which is a human myeloid leukemia cell line, is able to induce cell death in this cancer cell line, considering the potential ability of ADSCs to migrate from tumor sites. Cell surface markers, namely CD73 and CD105, were analyzed to verify the identity of mesenchymal stem cells isolated from adipose tissue. Besides, the osteogenic and adipogenic differentiation potentials of ADSCs were evaluated. The induction of cell death and apoptosis in the U937 cell line was assessed using MTT and annexin V/ PI assays, respectively. The expression levels of miR-181 and TGF-β were determined in the co-culture system using real-time PCR. The results of MTT and annexin V/ PI assays showed that BMP4-expressing ADSCs could inhibit cell viability and induce apoptosis in U937 cells in the co-culture system. The co-culture of ADSCs, highly expressing BMP-4, with the U937 cell line led to the downregulation of miR-181 and TGF-β genes in the human cancer cell line. ADSCs may further be studied as a candidate for the treatment of hematological cancers.

The Synthesis and Biological Evaluation of Some C-9 and C-10 Substituted Derivatives of the RNA Polymerase I Transcription Inhibitor CX-5461

The regio-isomeric alkynyl-substituted derivatives, 2 and 3, of the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 (1) were prepared and the active one (compound 3) subjected to click reactions ([3 + 2]-cycloaddition reactions) with certain alkyl azides bearing biotin or fluorescent tags. Compounds 2 and 3, as well as four [3 + 2]-cycloadducts of the latter, were subjected to biological evaluation in a human acute myeloid leukemia cell line model. Among the six compounds tested only alkyne 3 remained active but this was less potent than parent 1.

Hsa-let-7b-5p Inhibits Proliferation of Human Leukemia THP-1 Cells via FTO/m6A/MYC Signaling Pathway

To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m6A/MYC signaling pathway. The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m6A enrichment level in transfected cells was analyzed by dot blot, the expression levels of let-7b-5p, FTO and c-MYC were assayed by RT-PCR, the expressions of FTO and c-MYC protein were verified by Western blot, and the proliferation level of cells after transfection was detected by MTT assay. Compared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m6A enrichment, and then up-regulated the expression of FTO in transfected PBMNC cells, the effects of which were significant (P<0.05). However, let-7b-5p mimic up-regulated c-MYC mRNA m6A enrichment level and down-regulated the expression of FTO in the transfected THP-1 cells, and the cell proliferation rate was significantly lower than that in the negative control group (blank vector) (P<0.05). Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m6A axis and promotes the proliferation of leukemia cell line THP-1.

Effects and Mechanism of PARP Inhibitor Olaparib on the Expression of NKG2D Ligands in HL-60 Cells

To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells. After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method. 10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells. Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.

An iPSC line derived from a human acute myeloid leukemia cell line (HL-60-iPSC) retains leukemic abnormalities and displays myeloid differentiation defects.

Cancer-derived iPSCs have provided valuable insight into oncogenesis, but human cancer cells can often be difficult to reprogram, especially in cases of complex genetic abnormalities. Here we report, to our knowledge, the first successful generation of an iPSC line from a human immortalized acute myeloid leukemia (AML) cell line, the cell line HL-60. This iPSC line retains a majority of the leukemic genotype and displays defects in myeloid differentiation, thus providing a tool for modeling and studying AML.

Transcriptional Control of HP1a By the RNA Binding Proteins Rbmx/L1 Maintain Chromatin State in Myeloid Leukemia

Mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to contribute to leukemia development (Prieto and Kharas, CSH, 2020). Previously we have identified the RBP RBMX (RNA binding motif protein, X-linked) in our shRNA in vivo screen using murine MLL-AF9 driven leukemia (Prieto et al, ASH abstract 2018). Here we uncover the role of RBMX and its retrogene RBMXL1 in normal hematopoiesis and leukemogenesis in mouse and human systems. To investigate RBMX function in normal hematopoiesis, we created an Rbmx Mx1-Cre conditional knockout model to specifically delete Rbmx in the hematopoietic system. We gender stratified our studies since Rbmx is sex-linked. Deletion of Rbmx in both female and male primary mice as well as in non-competitive transplant animals did not affect hematopoiesis. Additionally, Rbmx knockout (KO) leads to a mild reduction in multipotent progenitors (MPP2 and MPP4) in female competitive transplanted mice although no defects in long-term hematopoiesis was observed in male competitive transplanted mice. These data suggest that Rbmx is dispensable for normal hematopoiesis. To identify the role of RBMX in leukemogenesis we knockout Rbmx in MLL-AF9 murine leukemia cells and found it significantly reduced colony formation in vitro and delayed leukemogenesis in vivo, indicating that Rbmx is required for leukemia maintenance. We observed, however, that MLL-AF9 transformed cells from Rbmx KO donor mice showed no delay in leukemia initiation versus cells from wildtype. We then determined that while Rbmx deletion is effective with complete depletion of mRNA, the retrogene RBMXL1 expression is maintained in Rbmx deficient cells, which may compensate for Rbmx deletion in leukemia initiation. Indeed, depletion of RBMXL1 by shRNAs (KD) in Rbmx deficient leukemia cells results in a drastic reduction in colony formation, increased colony myeloid differentiation, and induced apoptosis in cells deficient for both RBMXL1 and RBMX compared to those only depleted of RBMX. Correspondingly, RBMXL1 KD in Rbmx deficient leukemia cells resulted in further delayed leukemogenesis in vivo, indicating that RBMXL1 is functionally redundant to RBMX and both genes are required for leukemia development and maintenance. We next investigated the role of RBMX/L1 in normal human hematopoietic and leukemia cells. We found that RBMX/L1 expression were higher in AML cell lines (n=10/11) and primary AML patient samples (n=2/4) compared to healthy individuals. RBMX/L1 KD by shRNAs led to a dramatic decrease in cell proliferation, induction of apoptosis and myeloid differentiation in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX/L1 depletion significantly delayed leukemogenesis in vivo of AML cell lines (median survival of 51.5 days in control vs. median 'not reached' in shRNA1 and shRNA2), and of primary leukemia cells derived from an AML patient (median 50 days in control vs. "not reached" in shRNA2). We next depleted RBMX/L1 in human CD34+ stem and progenitor cells and observed reduced colony formation but no increase in apoptosis. Taken together, our data suggest that RBMX/L1 are differentially required in leukemia cells versus normal cells in both human and mouse systems. To uncover the mechanism of RBMX/L1 function, we performed complex chromosomal karyotyping analysis of RBMX/L1 depleted MOLM13 cells and revealed increased metaphases with breaks and gaps. In addition, ATAC-seq analysis showed profound genome-wide changes in chromatin accessibility and compaction upon RBMX/L1 depletion. Analyses of RNA-seq and transcriptome-wide RBMX/L1 binding targets (PAR-CLIP; Liu et al. 2017) revealed that RBMX/L1 directly binds to mRNAs and affects transcription of multiple loci including the heterochromatin protein 1 alpha (HP1a) mRNA. Using single molecule RNA FISH, we uncovered that RBMX/L1 controls the nascent transcription of the HP1a locus. Forced HP1a expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through its downstream target HP1a. This study describes a novel genetic compensation phenomenon in leukemia and illuminate a mechanism for RBPs directly promoting transcription. Our results suggest RBMX/L1 as well as HP1a as potential novel therapeutic targets in myeloid malignancies. Disclosures Steidl: Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy. Kharas:Accent Therapeutics: Consultancy; 28-7: Research Funding.

Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

Screening of Twenty-one Ethiopian Medicinal Plants for their Antiproliferative Activity against Human Acute Myeloid Leukemia (MV4-11) Cell Line

In vitro antiproliferative activities of the 80% methanol extracts of 21 traditionally used Ethiopian medicinal plants were studied using human acute myeloid leukemia (MV4-11) cell line. Among the tested plants, Euphorbia schimperiana Scheele and Gnidia involucrata Steud. ex A. Rich. exhibited significant antiproliferative activity with GI50 values of 20.6 ± 10.3 μg/ml and 16.6 ± 12.3 μg/ml, respectively. The active extracts were further fractionated using n-hexane, chloroform, ethyl acetate, n-butanol and water, and the antiproliferative assay results indicated that the n-hexane (GI50 = 0.8 ± 0.2 μg/ml) and ethyl acetate (GI50 = 6.1 ± 0.6 μg/ml) fractions of E. schimperiana and G. involucrate, respectively, demonstrated better antiproliferative activity when compared with their respective hydroalcoholic extracts. The findings of this study suggest that further phytochemical studies on the active fractions may lead to the discovery of novel anticancer compounds.&#x0D; Keywords: antiproliferative activity, Ethiopian medicinal plants, acute myeloid leukemia cell line, Euphorbia schimperiana, Gnidia involucrata

An Evaluation of the Activity of Histidine-Rich Glycoprotein on Differentiated Neutrophil-Like Cells from Human Cell Lines.

Histidine-rich glycoprotein (HRG) treatment ameliorated the survival rate of septic mice by suppressing excess immunothrombus formation. Although such findings suggested that HRG may be one of the most useful drugs for sepsis, obtaining a stable experimental system to standardize the HRG drug product is difficult to achieve using neutrophils isolated from volunteers. This is due to the short survival time and individual differences of human neutrophils. In the present study, we determined whether the differentiated neutrophil-like cell lines exhibited similar responses to HRG compared with human purified neutrophils. All-trans retinoic acid (ATRA) was employed to induce the differentiation of the human myeloid leukemia cell lines HL-60 and NB-4. Thereafter, the cells were treated with Hank's balanced salt solution, human serum albumin, or HRG. The effects of HRG on these cells were evaluated according to cell shape, microcapillary passage, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETs) formation, the expression of activated CD11b, and cell viability. HRG maintained the round shape of differentiated neutrophil-like cells, decreased the time required by cells to pass through the microcapillaries, and inhibited ROS production, NETs formation, and the expression of activated CD11b on the cell surface. Moreover, the cells could survive longer in the presence of HRG than the control. The ATRA-induced differentiated cell lines could be used as alternatives to neutrophils to investigate the effects of HRG on neutrophils. This method can thus be used as an essential standardization test in pharmaceutical development. SIGNIFICANCE STATEMENT: Human neutrophils exhibit varying responses to histidine-rich glycoprotein (HRG); however, all-trans retinoic acid-induced differentiated neutrophil-like cell lines can be used as reliably proxies to investigate the effects of HRG on neutrophils. Additionally, these cell lines can be employed in the development of therapies for the treatment of sepsis.

Inhibitory Effect of S1PR2 Antagonist JTE-013 on Proliferation of Chronic Myeloid Leukemia Cells

To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562. K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression. JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G0/G1 phase. Further detection of the protein expressions of G1 phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased. JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G0/G1 phase.

The Transcriptional Network That Controls Growth Arrest and Macrophage Differentiation in the Human Myeloid Leukemia Cell Line THP-1.

The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely studied to test candidate leukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed Cap Analysis of Gene Expression (CAGE) to analyze a dense time course of transcriptional regulation in THP-1 cells treated with phorbol myristate acetate (PMA) over 96 h. PMA treatment greatly reduced the numbers of cells entering S phase and also blocked cells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increased progressively over the duration of the time course. Within 1–2 h PMA induced known targets of tumor protein p53 (TP53), notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 h, PMA induced immediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN, MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). The dense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of the time course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedback regulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGE also identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lack functional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBU platform allowing comparison to FANTOM4 and FANTOM5 data.

Production Enhancement of an Anticoagulant Trypsin Inhibitor from Oceanimonas sp. BPMS22 and Its Anti-cancer Activity

The significant role of proteases in the cancer progression exposed them as potential targets for drug discovery. Regulating protease activities using protease inhibitors is one of the major therapeutic strategies for the treatment of cancer. The proteolysis activities of serine proteases in different stages of metastasis can be regulated by serine protease inhibitors. In the present work, the application of a peptide trypsin inhibitor (TI) from Oceanimonas sp. BPMS22 in cancer treatment was exploited. Initially, the process parameters such as initial medium pH, glucose, yeast extract, and sodium chloride concentrations were optimized for the maximum inhibitor activity using Response surface methodology. The maximum specific trypsin inhibitor activity observed was 40.16 ± 1.33 IU/mg protein at predicted critical conditions, glucose, 19.51 g/L; yeast extract, 23.04 g/L; sodium chloride, 28.29 g/L; and pH 7.26. Further, the apoptotic cell death of purified trypsin inhibitor-treated human chronic myeloid leukemia (K562) cell lines was demonstrated by MTT assay, AO/EtBr staining, and DNA ladder assay. The cellular oxidative stress in K562 cells was confirmed by glutathione levels and fluorescence imaging of ROS generation.

A new cytotoxic isoflavane from Dalbergiae Odoriferae Lignum

Fourteen compounds were isolated from the ethanol extract of Dalbergiae Odoriferae Lignum by various chromatographic techniques, including column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. Their structures were determined by spectroscopic techniques as S-3'-hydroxy-7,2',4'-trimethoxyisoxane(1), 2-(2',4'-dimethoxyphenyl)-6-hydroxybenzofuran(2), 2-(2'-hydroxy-4'-methoxyphenyl)-6-methoxybenzofuran(3), 7,2',4'-trimethoxydihydroisoflavone(4), sativanone(5), 3,9-dimethoxy-6H-benzofuro[3,2-c]chromen-6-one(6),(6 aS,11 aS)-homopterocarpin(7),(6 aS,11 aS)-8-hydroxy-3,9-dimethoxypterocarpan(8),(6 aS,11 aS)-3,8,9-trimethoxypterocarpan(9), isodalbergin(10), isoliquiritigenin(11), butein(12), butin(13) and 3,7,4'-trihydroxyflavone(14). Among them, compound 1 was a new compound, while 2 and 3 were new natural products, 6, 8, 9 and 14 were isolated for the first time from Dalbergiae Odoriferae Lignum. Compounds 1-14 were tested for their cytotoxic activity against human hepatoma cell line BEL-7402, human gastric cancer cell line SCG-7901, human lung cancer cell line A549, human chronic myeloid leukemia cell line K562 and HeLa human cervical cancer cellline by MTT method. Compound 1 exhibited significant cytotoxicity with IC_(50) values ranging from 2.85 to 11.62 μg·mL~(-1). In addition, 2, 11 and 12 showed weak cytotoxic activities.

Costunolide Suppresses Proliferation of K562 Cells through JAK/STAT Signaling Pathway

To investigate the inhibitory effect of costunolide on the proliferation of human chronic myeloid leukemia cell line K562 cells and the underlying mechanisms. The effect of costunolide on the viability of K562 cells was detected by CCK-8 assay. The cell apoptosis and the level of ROS in K562 cells were detected by flow cytometry. The expression level of related proteins were analyzed using Western blot. The CCK-8 viability assay showed that costunolide at the gradient concentrations of 0-50 μmol/L significantly inhibited the proliferation of K562 cells after exposure for 24 h. The IC50 value of costunolide was approximately 15.70±2.13 μmol/L, with statistically significant difference (P＜0.05). Treatment with 15 μmol/L costunolide for 24 and 48 h induced apoptosis of K562 cells with the apoptotic rate significantly increasing from (1.77±0.59) % in the control group to (17.68±2.84) % and (30.65±4.54) % (P＜0.05) respectively. Furthermore, following treatment, the level of ROS was significantly increased, from (3.52±1.08) % to (23.56±3.52) % and (36.68±4.22) % (P＜0.05). The study results also revealed that costunolide treatment significantly inhibited the expression of p-JAK2, p-STAT3 and BCL-2, and increased the expression of BAX, cytochrome C, cleaved-caspase-3 and cleaved-PARP. Costunolide suppresses the proliferation of K562 cells through JAK/STAT signaling pathway.

Effect of B4GALT1 on Proliferation of Its Co-cultured Human Acute Myeloid Leukemia Cell Line in Bone Marrow Stromal Cells

To investigate the effect of bone marrow stromal cell glycosyltransferase B4GALT1 expression on hematopoietic cell proliferation and its upstream regulation mechanism. B4GALT1 was overexpressed in human bone marrow stromal cell line HS5, which was then co-cultured with acute myeloid leukemia cell line KG1a. And its effect on hematopoietic cell proliferation was detected by flow cytometry. Dual luciferase reporter assay, real-time PCR and Western blot were used to predict and validate upstream transcription factors that regulate stromal cell B4GALT1 expression. Overexpression of B4GALT1 in HS5 significantly promoted the proliferation of KG1a in the co-culture system. B4GALT1 expression in stromal cells positively correlated with upstream c-Jun expression, which was verified by JNK/c-Jun inhibitors. The differential expression of glycosyltransferases and their corresponding glycosylation in the hematopoietic microenvironment play an important role.