Human Acute Leukemia Cells Research Articles

Design of colloidal drug carriers of celecoxib for use in treatment of breast cancer and leukemia

Inflammation develops initiation and pathological process of cancer. Nanoscale drug carriers are required to be investigated for delivery of actives at cellular level for treatment of various cancer types. Solid lipid nanoparticles (CLX-SLN), nanostructured lipid carriers (CLX-NLC) and a nanoemulsion (CLX-NE) of celecoxib (CLX), a selective cyclooxygenase-2 inhibitor, were formulated for use in remedy of breast cancer and acute promyelocytic leukemia. The hot high pressure homogenization technique was employed to product formulations. Scanning electron micrographs were utilized for morphological characterization of formulations. Laser diffraction (LD), photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC) were used for examination of their physical stability by storing them at various temperatures. Drug release profiles of formulations were obtained. Their activity on cancer cells was investigated in the cell culture experiments. Stable formulations having homogenous size distribution were obtained below 200 nm with high drug payloads between 93.76% and 96.66%. Nanoparticles were ascertained to contribute controlled drug release. CLX-SLN induced the highest decrease in numbers of human breast cancer and human acute promyelocytic leukemia cells through the activation of the cell death cascades especially apoptosis in comparison to CLX-NLC, CLX-NE and the pure CLX application (p < 0.05). Nanoformulations of CLX optimized in this study were found to have various advantages expected from sophisticated drug delivery systems in order to achieve higher CLX efficiency at cellular level. Thus, they are able to be administered efficaciously alone and in combination therapies in remedy of breast cancer and acute promyelocytic leukemia.

Anticancer potential of kebar grass (Biophytum petersianum), an Indonesian traditional medicine

Biophytum petersianum Klotzsch (kebar grass) has been used in Indonesia as traditional medicine. The objective of the current study was to evaluate the cytotoxic activity against several human cancer cells. The plants were collected from Purworejo, Central Java, Indonesia and extracted using methanol and dichlormethane. The extracts were analyzed for its antioxidant activity using DPPH (2,2 diphenyl-1-picrylhydrazyl) method. Cytotoxicity was examined against human acute lymphocytic leukemia cell (CCRF-CEM), multidrug resistance human acute leukemia cell (CEM/ADR5000), human cervical cancer cell (HeLa), human pancreatic carcinoma (Mia-PaCa2) and breast cancer cell (MCF-7) using colorimetric assay for assessing cell viability. The results indicated that methanol extract exhibited higher antioxidant activity as compared to dichlormethane extract. Both extracts exhibited moderate cytotoxicity against several human cancer cells, such as those of CCRF-CEM, CEM/ADR500, Mia-PaCa2 and MCF. This finding was the first report suggested that kebar grass from Purworejo, Central Java, Indonesia was potential as antioxidant and anticancer. Further comprehensive studies on the mechanism of actions are necessary to support this finding.

S836 AZACITIDINE AND ASCORBATE INHIBIT COMPETITIVE OUTGROWTH OF HUMAN TET2‐MUTANT HSPCS IN A XENOGRAFT MODEL OF PRE‐LEUKEMIA

Background:The TET2 gene is frequently mutated in pre‐leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5‐methylcytosine (5‐mC) to 5‐hydroxymethylcytosine (5‐hmC), a key intermediate for DNA demethylation. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5‐methylcytosine can be blocked with azacitidine.Aims:To understand the mechanisms of TET2 mutation‐driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ stem and progenitor cells.Methods:First, in “Hit &amp; Run”, we use Cas9 with two single‐guide RNAs to delete an internal region and disrupt TET2. Second, we use homology‐directed repair (HDR) of Cas9‐mediated double‐strand breaks to disrupt TET2 by inserting GFP and/or mCherry expression cassettes to generate in vivo traceable cells. Global 5‐hmC profiling revealed time‐dependent loss of hydroxymethylation after 6 weeks of in vitro culture at predominantly genes and promoters, validating loss of TET2 enzymatic function. Thus, we have developed a tractable and cell‐traceable model that recapitulates TET2‐mutated pre‐leukemia and clonal hematopoiesis.Results:First, we examined the biological effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both methods of TET2 disruption decreased CD71+CD235+ erythroblasts compared to control safe‐harbor disrupted cells. Notably, exposure to ascorbate (HDR, n = 6, p &lt; 0.02) or azacitidine (HDR, n = 4, p &lt; 0.05) partially rescued the erythroid differentiation block. TET2 disruption consistently abolished erythroid colonies in methylcellulose and increased the numbers of granulocyte‐macrophage colonies upon serial re‐plating (HDR, n = 4 independent experiments, p &lt; 0.0001).In vivo, transplantation of TET2‐disrupted Hit &amp; Run CD34+ cells into NSG mice showed gradual expansion of TET2‐disrupted cells in the CD33+ myeloid population. At 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells in keeping with a time‐dependent myeloid skewing induced by TET2 mutation (lymphoid = 22.1%, myeloid = 73.0%, Mann‐Whitney U, p = 0.0485).We therefore performed in vivo competition studies to determine if TET2‐disrupted HSPCs could be selectively targeted by azacitidine or ascorbate treatment. In PBS control treated mice, the percentage of TET2‐disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2‐disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p = 0.036), but did not eradicate established TET2 pre‐leukemia. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p = 0.14). Combination studies of both drugs in a larger cohort are ongoing coupled with global 5‐hmC profiling.Summary/Conclusion:Our results indicate that disruption of TET2 in human hematopoietic stem and progenitor cells is sufficient to induce global changes in 5‐hmC and suggest azacitidine or ascorbate treatment can slow the expansion of TET2‐mutant human pre‐leukemic clones suggesting an approach to preventing CHIP progression to de novo AML.

Evaluation of the combined effects of doxorubicin and bortezomib on the human acute lymphoblastic leukemia cell line

Abstract Increasing numbers of oncological patients and growing drug resistance ensure that new methods of cancer treatment are intensively sought. Combining drugs for a synergistic effect is one of several possible ways to mitigate this problem. This leads to reducing the effective drug dose and the occurrence of side effects. Doxorubicin (DOX) is an antineoplastic agent that has several mechanisms of action. DOX intercalates between base pairs of DNA helix, inhibits topoisomerase II and also forms reactive oxygen species. Bortezomib (BZT) is an antitumor agent belonging to the group of proteasome inhibitors. It has been observed that BZT triggers an oxidative stress response in vitro and in vivo. Accumulation of oxidatively damaged proteins and the simultaneously blocking of the proteasome can be very damaging to the tumour cell. For this reason, the aim of the study was to assess the potentially synergistic effect of DOX and BZT on human acute lymphoblastic leukemia (ALL). In the work, the cells were treated with both agents and their combinations and the effect was evaluated on the basis of morphological assessment, MTT assay and level of reduced glutathione measurement. The study has shown that on acute lymphoblastic leukemia cells, synergistic effects came about in the combination of 1nM BZT with a wide range of concentrations of DOX. Herein, the visible, coactive effect of DOX and BZT was observed on oxidative stress levels. This phenomenon can be essential in blunting the possibility of rapid manifestation of resistance seen in BZT monotherapy. In addition, the needed very low concentrations of DOX reduce the risk of therapy side effect.

Imaging Flow Cytometric Analysis of Stilbene-Dependent Apoptosis in Drug Resistant Human Leukemic Cell Lines.

Background: The natural compounds have been researched extensively as an alternative to the conventional chemotherapy and radiation. Stilbene derivatives appear as a group of therapeutics which deserves special attention. The present study was designed to analyze the effects of stilbene derivatives on drug resistant human leukemic cells. The aim of this work was to evaluate the apoptotic effect of stilbene derivatives in various concentrations on leukemic cells (LC) with and without resistant phenotype. Methods: Human acute promyelocytic leukemia (APL) cell lines (HL60, HL60/MX1, HL60/MX2) and acute lymphoblastic leukemia (ALL) cell lines (CEM/C1, CCRF-CEM) were studied. T-resveratrol, piceatannol, rhaponticin, deoxyrhaponticin, pterostilbene were used to stimulate apoptosis. Mitoxantrone (MIT) was applied to induce drug resistance. Results: t-Resveratrol (RES), deoxyrhaponticin (D-RHAP), rhaponticin (RHAP), pterostilbene (PTER), and piceatannol (PIC) influenced viability and induced apoptosis in all investigated cell lines. Conclusions: Our results confirmed that RES, PIC, RHAP, D-RHAP, and PTER are essential therapeutic compounds with anticancer activity exhibited by induction of apoptosis in leukemic cells with and without resistant phenotype. Stilbene-induced apoptosis in HL60/MX1, HL60/MX2, CEM/C1, and CCRF-CEM leukemia cell lines have been presented in very few studies so far and our research is an important contribution to the investigation of these substances.

Preliminary results indicate resveratrol affects proliferation and apoptosis of leukemia cells by regulating PTEN/PI3K/AKT pathway.

PTEN-PI3K/AKT signaling pathway is widely involved in the regulation of cell proliferation, cell cycle, apoptosis, and invasion. Resveratrol (Resv) is a natural botanical ingredient involved in several biological activities. It is still unclear in terms of whether Resv may exert anti-leukemia effects by regulating the PTEN-PI3K/AKT pathway. This study investigated the effect of Resv on leukemia cell proliferation and apoptosis by regulating PTEN-PI3K/AKT pathway. Human normal peripheral blood PBMC cells, and human acute promyelocytic leukemia (APL) cell line NB-4 and HL-60 cells were cultured in vitro. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect Phosphatase and tensin homolog (PTEN) mRNA expression. Western blot was adopted to test PTEN protein expression. HL-60 and NB-4 cells were treated with 0, 5, 10, and 20 μM Resv, respectively. Cell proliferation was analyzed by cell counting kit8 (CCK-8) assay. The level of caspase-3 was measured by Western blot. HL-60 cells were divided into control group, 20 μM Resv treatment group, and Resv+PTEN inhibitor SF1670 group. Cell apoptosis was determined by flow cytometry. Cell proliferation was assessed by EdU staining. Compared with peripheral blood mononuclear cell (PBMC), PTEN mRNA and protein levels were significantly decreased in NB-4 and HL-60 cells. Resv significantly inhibited the proliferation activity in HL-60 and NB-4 cells, and increased the activity of caspase-3. Resv treatment up-regulated the expression of PTEN and reduced the expression of p-AKT protein in HL-60 cells. However, Resv treatment markedly suppressed the proliferation of HL-60 and induced apoptosis. SF1670 treatment in the presence of Resv significantly antagonized the down-regulation of p-AKT protein expression induced by Resv, resulting in decreased apoptosis and enhanced cell proliferation. Resv can up-regulate PTEN expression and inhibit the activity of PI3K/AKT pathway to play an anti-leukemia effect through suppressing cell proliferation and inducing apoptosis.

Anticancer potential of metabolic compounds from marine actinomycetes isolated from Lagos Lagoon sediment

Thirty-two actinomycetes strains were isolated from sediment samples from 12 different sites at Lagos Lagoon and identified using standard physiological and biochemical procedures as well as 16S rDNA gene sequence analysis. Secondary metabolites were extracted from the strains and their anticancer activity on the K562 (Human acute myelocytic leukemia), HeLa (cervical carcinoma), AGS (Human gastric), MCF-7 (breast adenocarcinoma) and HL-60 (Human acute promyelocytic leukemia) cell lines was determined. The metabolic extracts exhibited cytotoxicity with IC50 values ranging from 0.030 mg/mL to 4.4 mg/mL. The Streptomyces bingchenggensis ULS14 extract was cytotoxic against all the cell lines tested. The bioactivity-guided extraction and purification of the metabolic extracts from this strain yielded two purified anticancer compounds: ULDF4 and ULDF5. The structures of the extracted compounds were determined using spectroscopic analyses, including electrospray ionization mass spectrophotometer and nuclear magnetic resonance (1 Dimensional and 2 Dimensional), and were shown to be structurally similar to staurosporine and kigamicin. The IC50 of ULDF4 and ULDF5 against the HeLa cell line was 0.034 μg/mL and 0.075 μg/mL, respectively. This study is the first to reveal the anticancer potential of actinomycetes from Lagos Lagoon, which could be exploited for therapeutic purposes.

Effect of microRNA-153 targeting phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway on proliferation in acute lymphoblastic leukemia cell

Objective To investigate the effect of microRNA (miRNA, miR)-153 on the proliferation and apoptosis of in human acute lymphoblastic leukemia (ALL) cell line Jurkat cells by phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (Akt) pathway. Methods After miR-153 mimics (miR-153 mimics group) and miR-153 inhibitor (miR-153 inhibitor group) were transfected into Jurkat cells, cell proliferation, cycle and apoptosis. was detected; the expression of miR-153, PI3K and Akt mRNA and protein was measured; whether PI3K was the target gene of miR-153. was confirmed. Results Compared with the negative control group, the expression of miR-153 in the miR-153 mimics group was significantly increased by approximately 10-fold (t=39.596, P<0.01), and the expression of miR-153 was significantly decreased in the miR-153 inhibitor group (t=14.651, P<0.01). The activity of the cells in the miR-153 mimics group was significantly decreased (t=10.931, P<0.01), and the activity of the miR-153 inhibitor group was significantly increased (t=7.640, P<0.01). The expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 mimics group was significantly decreased (t=7.161, P<0.01; t=6.684, P<0.01; t=10.769, P<0.01), and the expression of PI3K total protein, PI3K mRNA and p-Akt in the miR-153 inhibitor group was significantly increased (t=3.998, P<0.05; t=8.902, P<0.01; t=7.078, P<0.01). The proportion of G0/G1 phase cells increased significantly, while the S phase and G2/M phasecells decreased significantly (t=3.786, P<0.05; t=3.012, P<0.05; t=4.948, P<0.01) in the miR-153 mimics group; the G0/G1 phase cells decreased significantly, and the proportion of cells in S phase and G2/M phase increased significantly (t=5.356, P<0.01; t=3.055, P<0.05; t=3.893, P<0.05) in the miR-153 inhibitor group. The apoptosis rate of the miR-153 mimics group was significantly increased (t=4.673, P<0.05), while the apoptotic rate of the miR-153 inhibitor group was significant decline (t=5.618, P<0.01). Compared to the empty plasmid group, the relative luciferase activity of 3’untranslated regions (3’UTR) plasmid group was significantly reduced (t=7.925, P<0.01). Conclusion MiR-153 can directly target PI3K/Akt signaling pathway and regulate Jurkat cell proliferation and apoptosis. Key words: Acute lymphoblastic leukemia; MicroRNA-153; Phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway

The novel CD19-targeting antibody-drug conjugate huB4-DGN462 shows improved anti-tumor activity compared to SAR3419 in CD19-positive lymphoma and leukemia models.

Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies.

Preparation of immobilized trypsin micro monolithic column and its application to rapid digestion of trace proteins

A method for the preparation of a micro monolithic column with immobilized trypsin was developed for the rapid and efficient digestion of proteins. The micro monolithic column was prepared by photo-polymerization inside the tip of 20 μL pipette. The polymerization solution was composed of the functional monomers 4-pentenoic succinimide ester (PAS) and 2-hydroxyethyl methacrylate (HEMA), crosslinker pentaerythritol triacrylate (PETA) dissolved in a ternary porogenic system comprising dimethyl sulfoxide (DMSO), formyldimethylamine (DMF) and 1-dodecanol. Immobilization of trypsin was achieved by a chemical reaction between the amino group and succinimide. The effects of the active ester content in the polymerization mixture and the volumes of the monolithic bed on the column capacities of the immobilized trypsin were systematically investigated. The digestion efficiency as well as the stability and repeatability of the immobilized trypsin were systematically investigated by using standard proteins cytochrome C and bovine serum albumin. The experimental results indicated that high digestion efficiency with great reproducibility between batches and the digestion procedure could be obtained within 10 min. The trypsin immobilized columns were applied to the digestion of proteins extracted from 1×105 human acute promyelocytic leukemia (NB4) cells and human acute T-cell leukemia (Jurkat T) cells. A total of 2489 and 2572 proteins were readily identified by Nano-LC-MS/MS analysis. The quantity ratio of the identified proteins increased 2.2% and 6.1%, respectively, compared to the case of in-solution digestion, demonstrating the robustness of the trypsin immobilized micro column and its potential application to proteome studies.

The mechanism of apoptosis in human acute promyelocytic leukemia cells treated with sorafenib and lithium chloride

The mechanism of apoptosis in human acute promyelocytic leukemia cells treated with sorafenib and lithium chloride

Achieving Automated Organelle Biopsy on Small Single Cells Using a Cell Surgery Robotic System.

Single cell surgery such as manipulation or removal of subcellular components or/and organelles from single cells is increasingly used for the study of diseases and their causes in precision medicine. This paper presents a robotic surgery system to achieve automated organelle biopsy of single cells with dimensions of less than 20μm in diameter. The complexity of spatial detection of the organelle position is reduced by patterning the cells using a microfluidic chip device. A sliding mode nonlinear controller is developed to enable extraction of organelles, such as the mitochondria and the nucleus, from single cells with high precision. An image processing algorithm is also developed to automatically detect the position of the desired organelle. The effectiveness of the proposed robotic surgery system is demonstrated experimentally with automated extraction of mitochondria and nucleus from human acute promyelocytic leukemia cells and human fibroblast cells. Extraction is followed by biological tests to indicate the functionality of biopsied mitochondria as well as the cell viability after removal of mitochondria. The results presented here have revealed that the proposed approach of automated organelle biopsy on single small cells is feasible.

The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

BackgroundThere are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells.ResultsMPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model.ConclusionsCollectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.

Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia

The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine.To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in “Hit & Run,” we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis.First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p<0.02). This underscores the importance of TET2 in promoting erythroid differentiation and suggests TET2 mutations can exert a myeloid lineage skewing sensitive to ascorbate.Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p<0.0001; HDR, n=3 independent experiments, p<0.0001) and absence of erythroid BFU-E. Interestingly, analysis of indels in Hit & Run colonies showed that serial replating enriched for a 65 base pair deletion that results in a null allele, suggesting that TET2-disrupted cells outcompete normal HSPCs in vitro.Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease.Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14).Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. DisclosuresNo relevant conflicts of interest to declare.

Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models.We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D).We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+ cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/- clonal cell lines are distinct from their isogenic IKZF1-/- counterparts.Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/- cells and IKZF1IK6/+ cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. DisclosuresNo relevant conflicts of interest to declare.

Expression of the miR-150 tumor suppressor is restored by and synergizes with rapamycin in a human leukemia T-cell line

miR-150 functions as a tumor suppressor in malignancies of the lymphocyte lineage and its expression is significantly reduced in these cells. However, the mechanism of miR-150 repression is unknown and so are pharmacological interventions that can reverse it. Here, we report that reduced expression of miR-150 in human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells is mediated by constitutive mTOR signaling, a common characteristic of T-ALL cell lines and clinical isolates. Activating mTOR signaling in non-malignant T cells also resulted in a significant miR-150 down-regulation. Conversely, treatment with a pharmacological mTOR inhibitor, rapamycin, increased miR-150 expression in a dose-dependent manner in Jurkat cells, as well as in other leukemia cells. Interestingly, ectopic over-expression of miR-150 acted in a feed-forward loop and further sensitized Jurkat cells to a rapamycin-induced cell cycle arrest by targeting a large network of cell cycle genes. These findings suggest that miR-150 is normally expressed in quiescent T lymphocytes to reinforce an anti-proliferative state, and that mTOR signaling promotes cell proliferation in part by inhibiting miR-150 expression. Restoration of the miR-150-dependent anti-proliferative loop constitutes a novel mechanism underlying the efficacy of rapamycin in a T-ALL cell line. Further investigation of this mechanism in clinical isolates of T-ALL and other hematopoietic malignancies could help better guide development of targeted therapies.

Azadirachta indica based biosynthesis of silver nanoparticles and evaluation of their antibacterial and cytotoxic effects

Due to the growing threat to the environment by the climate change, the development of sustainable and environmental friendly methods for the preparation of metallic nanoparticles (NPs) is highly desirable. Herein, we demonstrate a simple and eco-friendly method for the preparation of silver nanoparticles (AgNPs) using Azadirachta indica leaf extract and aqueous solution of silver nitrate (AgNO3). The effect of concentration of leaf extract and temperature on the synthesis of AgNPs is studied. It was revealed that 1 mM of AgNO3, leaf extract concentration of 60 mg/mL, 30 min of reaction time and a temperature 85 °C were found to be optimum for the synthesis good quality AgNPs. The formation of AgNPs was confirmed by UV–Vis analysis, where the UV spectrum exhibited a highest absorption peak at ∼410 nm corresponding to the surface Plasmon Resonance (SPR) of the AgNPs. Furthermore, the antibacterial activity of biosynthesised AgNPs was investigated and the inhibitory concentrations of 0.5 µg/mL for Escherichia coli and 1.0 µg/mL for Staphylococcus aureus were found. Besides, the in vitro cytotoxicity investigation of biosynthesized AgNPs against human acute lymphoblastic leukemia cells via MTT assay showed that the minimum inhibitory concentration of 15.6 µg/mL was required. Therefore, the eco-friendly and cost effective synthesis of AgNPs has potential for biomedical applications and this method can be used for the large scale production of AgNPs.

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

Zinc-chitosan nanoparticles induced apoptosis in human acute T-lymphocyte leukemia through activation of tumor necrosis factor receptor CD95 and apoptosis-related genes

Chitosan (CS), a novel biomaterial is widely used as a drug nano-carrier for cancer treatments. Towards this aim, anticancer and antibacterial activities of CS-nanoparticles-linked zinc (Zn-CSNPs) were evaluated. The particle size of CSNPs was lowered (113.09 nm) compared to Zn-CSNPs (160.7 nm). Both nanoparticles (CSNPs and Zn-CSNPs) were spherical in shape, polydispersive and homogenous. Fourier transforms infrared spectrophotometer (FTIR) and energy dispersive X-ray spectroscopy (EDX) analysis confirmed the different molecular arrangement of NPs and the presence of Zn in Zn-CSNPs and CS in both NPs, respectively. Zn-CSNPs had higher inhibitory activity against tested pathogens with a minimal inhibitory concentration (MIC) of 9.25–13.5 μg·mL−1 and showed the complete inhibition of Staphylococcus aureus and Escherichia coli. Zn-CSNPs have triggered the apoptosis through activation of first apoptosis signal receptor/cluster of differentiation 95 (Fas/CD95), and apoptotic-regulatory genes and caused 65–70% of cellular damage in human acute T-lymphocyte leukemia (6T-CEM) cells. Overall, internalizing properties of Zn from CSNPs is a promising therapeutic approach to treat Zn-deficiency related diseases particularly human acute leukemia (HAL).

Analysis of Candidate Idarubicin Drug Resistance Genes in MOLT-3 Cells Using Exome Nuclear DNA.

Various gene alterations related to acute leukemia are reported to be involved in drug resistance. We investigated idarubicin (IDR) resistance using exome nuclear DNA analyses of the human acute leukemia cell line MOLT-3 and the derived IDR-resistant cell line MOLT-3/IDR. We detected mutations in MOLT-3/IDR and MOLT-3 using both Genome Analysis Toolkit (GATK) and SnpEff program. We found 8839 genes with specific mutations in MOLT-3/IDR and 1162 genes with accompanying amino acid mutations. The 1162 genes were identified by exome analysis of polymerase-related genes using Kyoto Encyclopedia of Genes and Genomes (KEGG) and, among these, we identified genes with amino acid changes. In resistant strains, LIG and helicase plurality genes showed amino-acid-related changes. An amino acid mutation was also confirmed in polymerase-associated genes. Gene ontology (GO) enrichment testing was performed, and lipid-related genes were selected from the results. Fluorescent activated cell sorting (FACS) was used to determine whether IDR permeability was significantly different in MOLT-3/IDR and MOLT-3. The results showed that an IDR concentration of 0.5 μg/mL resulted in slow permeability in MOLT-3/IDR. This slow IDR permeability may be due to the effects of amino acid changes in polymerase- and lipid-associated genes.