Human Leukemia Cell Line K562 Research Articles

Synthesis, antiproliferative activity and possible mechanism of action of novel 2-acetamidobenzamides bearing the 2-phenoxy functionality

Several new 2-(2-phenoxyacetamido)benzamides 17a–v, 21 and 22 were synthesized by stirring in pyridine the acid chlorides 16a–e and the appropriate5-R-4-R1-2-aminobenzamide 15a–e and initially evaluated in vitro for antiproliferative activity against the K562 (human chronic myelogenous leukemia) cell line. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell line panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). The most active compounds caused an arrest of K562 cells in the G0–G1 phase of cell cycle and induction of apoptosis, which was mediated by caspase activation.

Ruxolitinib induces autophagy in chronic myeloid leukemia cells.

Ruxolitinib is the first agent used in myelofibrosis treatment with its potent JAK2 inhibitory effect. In this novel study, we aimed to discover the anti-leukemic effect of ruxolitinib in K-562 human chronic myeloid leukemia cell line compared to NCI-BL 2171 human healthy B lymphocyte cell line. Cytotoxic effect of ruxolitinib was determined by using WST-1 assay. IC50 values for K-562 and NCI-BL 2171 cell lines were defined as 20 and 23.6μM at the 48th hour, respectively. Autophagic effects of ruxolitinib were detected by measuring LC3B-II protein formation. Ruxolitinib induced autophagic cell death in K-562 and NCI-BL 2171 cell lines 2.11- and 1.79-fold compared to control groups, respectively. To determine the autophagy-related gene expression changes, total RNA was isolated from K-562 and NCI-BL 2171 cells treated with ruxolitinib and untreated cells as control group. Reverse transcription procedure was performed for cDNA synthesis, and gene expressions were shown by RT-qPCR. Ruxolitinib treatment caused a notable decrease in expression of AKT, mTOR, and STAT autophagy inhibitor genes in K-562 cells, contrariwise control cell line. Ruxolitinib is a promising agent in chronic myeloid leukemia treatment by blocking JAK/STAT pathway known as downstream of BCR-ABL and triggering autophagy. This is the first study that reveals the relationship between ruxolitinib and autophagy induction.

Synergistic effect of HMGB1 knockdown and cordycepin in the K562 human chronic myeloid leukemia cell line.

The high-mobility group box 1 (HMGB1) protein is a DNA-binding nuclear protein, which is overexpressed in leukemia cells. Cordycepin is characterized by strong antileukemic properties and is regarded as an effective natural compound for leukemia therapy. The aim of the present study was to investigate the impact of HMGB1 knockdown and cordycepin treatment on proliferation, apoptosis, reactive oxygen species (ROS) levels and adhesion of K562 human chronic myeloid leukemia cells. The Cell Counting kit‑8 assay was used to determine the proliferation of K562 cells. The cell cycle and apoptosis of K562 cells was determined using flow cytometric analysis. In addition, a cell adhesion assay was performed. Western blotting was used to determine the protein expression of cyclooxygenase 2, Bax, receptor for advanced glycation end-products and Bcl‑2. The data collected demonstrated that HMGB1 knockdown combined with cordycepin treatment had significant anti‑proliferative and pro‑apoptotic effects. In addition, it increased the ROS levels and reduced the adhesion of K562 cells. It was also identified that HMGB1 knockdown had synergistic effects with cordycepin, which aided in accelerating apoptosis, and inhibiting proliferation and adhesion in chronic myeloid leukemia cells. These results indicated that HMGB1 may be used as a potential therapeutic target, with cordycepin having potential as an auxiliary drug. Therefore, it is suggested that HMGB1 knockdown and corycepin treatement may present a promising therapeutic strategy for leukemia.

Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase.

Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix-coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti-β1-integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, incubation with blocking anti-β1-integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia.

Evidence base of Mistletoe drug -interaction in Supportive- care.

e20746 Background: Mistletoe is often used as complimentary therapy in Europe . There is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Like other Herbal extracts etc. , Viscum Album extract [VAE] is used as supportive care during chemotherapy. The anti-tumor effects of VAE are well documented . However the clinical activity of VAE treatment still remains contraversal. The effect of VAE was investigated with common chemo-drugs usings different cell lines in vitro . Methods: Human leukemia cell lines K562 and KG1a were treated with Vincistrin, Mafosfamide, Idarubicine and Cisplatin. Breast cell carcinoma cell lines HCC1937 and HCC1143were treated with Doxorubicin HCl , prostate carcinoma cell line DU145, with Docetaxel and Mitoxantrone HCl and lung carcinoma cell line NCL-H460 was treated with Docetaxal and Cispaltin . Each dose of chemotherapeutic drug was combined with VAE in clinically relavent concentrations and proliferaton and apoptosis were measured. Results: VAE inhibited colony formation and did not exert any protective activity against induced inhibition of colongenicity .VAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of experimental settings . Conclusions: The study provides evidence that VAE is capable of enhancing cytotxicity of antcancer agents in vitro. Hence no risk of safety by herb drug- interactions is to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE at the same time. The study shows moderate to high evidence and further studies are warrented for clinical trial with optimized study design and treatment conditions.

Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways

MWCNT buckypaper (BP) shows physico-chemical and mechanical properties that make it potentially useful as a substrate in nano-bio interface research including in tissue engineering. When used as a scaffold material, BP comes into contact with host cells and surrounding tissues; therefore it is critical to determine its biocompatibility and interaction with living systems. The aim of this study was to investigate BP effects on cell growth, apoptosis and reactive oxygen species (ROS) production in three human leukemia cell lines HL-60, U-937 and K-562. BP was able to induce both the reduction of cell proliferation, associated with an arrest in G0/G1 phase of cell cycle and the increase of apoptosis in leukemic cell lines, thus exerting both cytostatic and cytotoxic effects. The growth inhibitory effect was likely mediated by the decrease of cyclins D, E, A, B1 levels and CDK4 expression; meanwhile, the apoptotic effect, not mediated by ROS production, was presumably due to the combined action of the survival and pro-apoptotic AKT and MAPK signal transduction pathways. These results raised the issue of biocompatibility of MWCNT BP for the creation of carbon nanotubes based scaffolds to utilize as prostheses in tissue engineering.

485. Targeted Epigenome Editing by CRISPR/Cas9-Based Repressors for Silencing of Distal Regulatory Elements

The CRISPR/Cas9 genome engineering platform has emerged as a powerful tool for gene regulation. Catalytically inactivated, “dead” Cas9 (dCas9) can be fused to a variety of transcriptional modulators to activate or silence gene expression. The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been used to repress oncogenes, inhibit viral replication, and treat dominant negative diseases. However, the specificity of silencing endogenous genes and the distance of heterochromatin formation catalyzed by dCas9-KRAB repressors have not been studied. Understanding the precision of dCas9-KRAB repressors is critical to their further therapeutic application. As a model system to investigate the epigenetic consequences of KRAB-based silencing, dCas9-KRAB was targeted to the HS2 enhancer of the globin locus in the K562 human erythroid leukemia cell line. Enhancers are regulatory elements characterized by open chromatin that can activate distal genes targets. Genetic variation in enhancer sequences has been linked to many disease states, including cancer and cardiovascular disease. The HS2 enhancer is a potent activator of the developmentally regulated globin genes, which are involved in sickle cell anemia and beta thalassemia. We demonstrated that dCas9-KRAB targeted to HS2 by a single guide RNA (sgRNA) can disrupt the expression of multiple downstream globin genes. These findings expand the capabilities of synthetic repressors by showing that dCas9-KRAB targeted to a distal regulatory element can silence expression of multiple genes located 10 to 50 kb away. Genome-wide analysis of DNA-binding and mRNA expression by ChIP-seq and RNA-seq, respectively, established that RNA-guided synthetic repressors were highly specific for targeting and silencing the globin genes. To further investigate the utility of dCas9-KRAB repressors as a targeted epigenetic modifier, we performed ChIP-seq for tri-methylation of H3K9, a mark of heterochromatin, and DNase I hypersensitivity-sequencing (DNase-seq), a genome-wide measure of chromatin accessibility. dCas9-KRAB repressors induced H3K9 methylation at the HS2 enhancer. Off-target H3K9 methylation events were minimal and generally depended on the sgRNA. Reduced DNase I hypersensitivity was also observed at the enhancer region and its promoter targets, indicating that dCas9-KRAB repressors can modulate the chromatin structure of active enhancers to affect the activity of downstream target genes. These studies establish that dCas9-KRAB repressors can interfere with distal enhancer activity by highly specific remodeling of the epigenetic state of targeted loci. Enhancers play critical roles in differentiation, development, and disease, and understanding the epigenetic implications of targeting engineered repressors to distal regulatory elements will inform further use of genome engineering technologies for therapeutic applications.

Therapeutic efficacy of phenolic compounds from Limonium in acute lymphoblastic leukemia

Bioassay-guided fractionation of Limonium myrianthum crude extract led the isolation of four known phenolic compounds including myricetin (1), myricitrin (2), gallic acid (3) and 6''-galloylmyricetin-3-O-β-D-glucopyranoside (4). Their structures were elucidated by spectroscopic analyses including HR-MS, 1D, and 2D NMR. The in vitro anti-leukemia activity of the phenolic compounds 1 – 4 against two human leukemia cell lines HL60 and K562 were evaluated after 48h exposure. The estimated IC50 values (nM) found as 5.8, 23.6, 13.8, 38.0 for HL60 cells and 15.0, 25.0, 32.0, 48 for K562 cells for compound 1 – 4, respectively. There were no significant differences in the sensitivity of the above two cell lines to the four compounds. To our knowledge, this is the first time to evaluate their anti-leukemia against two human leukemia cell lines HL60 and K562.

Preparation and Characterization of Nanoliposomal Beta- Cryptoxanthin and its Effect on Proliferation and Apoptosis in Human Leukemia Cell Line K562

Purpose: To prepare beta-cryptoxanthin-loaded nanoliposomes and evaluate their anti-proliferative activity in leukemia K562 cell line, compared to free beta-cryptoxanthin.Methods: Beta-cryptoxanthin-loaded nanoliposomes were prepared by extrusion method. Morphological characterization of the nanoliposomes was performed by cryo-transmission electron microscopy (cryo-TEM). The anti-proliferation effect of beta-cryptoxanthin (BC) in free and liposomal forms on K562 cell line was studied using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Apoptotic activity, following treatment with beta-cryptoxanthin in the free and liposomal forms, was detected using flow cytometry.Results: Entrapment efficiency of beta-cryptoxanthin was 86.3 % ± 1.0. Cryo-TEM analysis revealed that the nanoliposomes have spherical shapes. In all conditions, beta-cryptoxanthin-loaded nanoliposomes exhibited greater anti-proliferative activity than than the free beta-cryptoxanthin (p < 0.001). Furthermore, in the presence of beta-cryptoxanthin-loaded nanoliposomes, the proportion of apoptotic cells was higher for free beta-cryptoxanthin (p < 0.001). Conclusion: The data obtained indicate that beta-cryptoxanthin, especially in the liposomal form, inhibits the growth of K562 cells and may therefore provide a basis for the development of leukemia therapies.Keywords: Beta-cryptoxanthin, Nanoliposome, Anti-proliferative, Apoptosis, Flow cytometry, Leukemia

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation.

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5′-CACGTGT-3′) sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole–imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5′- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.

Anti-CD20 antibody induces the improvement of cytokine-induced killer cell activity via the STAT and MAPK/ERK signaling pathways.

There is a current requirement for novel therapeutic strategies for the treatment of hematopoietic tumors. Residual tumor cells are the main origin of tumor relapse. The aim of this study was to eliminate the residual tumor cells of hematopoietic tumors. Cytokine-induced killer (CIK) cells are used in immunotherapy to deplete the residual cells. However, it is necessary to increase the antitumor activity and clinical applicability of CIK cells. The present study investigated the antitumor activity of CIK cells to the SU-DHL2 human B-cell lymphoma and K562 human chronic myelogenous leukemia cell lines. CD3+CD56+ cells from healthy donors were expanded in culture with cytokines and anti-CD20 monoclonal antibody (mAb; rituximab) to generate CIK cells. A preliminary investigation of their mechanism was then performed. The increase in the cytotoxicity of the CIK cells induced by the anti-CD20 mAb was associated with an increase in the expression of cytotoxic factors. The expression of components of the signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways was found to increase. Upregulation of the expression of STAT1, STAT3 and STAT5 is important as these co-stimulatory molecules enhance T-cell proliferation. Activation of the MAPK signaling pathway is a possible mechanism for the anti-apoptosis effect on the proliferation of CIK cells. In conclusion, anti-CD20 mAb may play an important role in the improvement of CIK-mediated cytotoxicity to tumor cells. These observations may aid in the improvement of the effects of immunotherapy in depleting the residual cells of hematopoietic tumors. Thus, the use of CIK cells cultured with anti-CD20 mAb could be a novel therapeutic strategy for the depletion of chemotherapy-resistant or residual cells in anaplastic large and B-cell lymphoma.

Naringenin suppresses K562 human leukemia cell proliferation and ameliorates Adriamycin-induced oxidative damage in polymorphonuclear leukocytes.

Treatments for leukemia remain unsatisfactory. Conventional chemotherapy agents that aim to kill tumor cells may also damage normal cells and thus result in severe side-effects. Naringenin, a natural polyphenolic compound with antioxidant effects, has been revealed to have significant antitumor effects with low toxicity in preliminary studies. Thus, it is considered as one of the most promising flavonoids in the treatment of leukemia. In the present study, the effects of naringenin on the K562 human leukemia cell line and the underlying mechanisms were explored in vitro. In addition, human peripheral blood polymorphonuclear leukocytes (PMNs) were used as a normal control in order to evaluate the effects of naringenin on normal granulocytes and in the mediation of Adriamycin (ADM)-induced oxidative damage. The results revealed that K562 proliferation was significantly inhibited by naringenin in a time- and concentration-dependent manner; however, minimal cytotoxic effects were observed in PMNs when naringenin was used at concentrations <400 μmol/l. Morphological changes indicative of apoptosis were observed in naringenin-treated K562 cells. Flow cytometric analysis indicated that the K562 cells were arrested in the G0/G1 phase of the cell cycle with a significantly upregulated rate of apoptosis. Furthermore, in the naringenin-treated K562 cells, the labeling index of proliferating cell nuclear antigen was observed to be increased by immunochemical staining, the mRNA and protein expression levels of p21/WAF1 were strongly upregulated in reverse transcription-polymerase chain reaction and western blot analyses, whereas p53 gene expression was not significantly changed. In PMNs to which naringenin (50~80 μmol/l) was added 1 h subsequent to ADM, the cell damage induced by ADM was significantly reduced, coincident with reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increases in the activity of superoxide dismutase and glutathione peroxidase. However, the cytotoxic effect of ADM in K562 cells was not significantly altered by naringenin, and the oxidative stress indices in K562 cells remained stable. In conclusion, the present study revealed the promising value of naringenin in leukemia treatment. Naringenin demonstrated a significant inhibitory effect on the growth of K562 cells but not on normal PMNs. Furthermore, naringenin protected PMNs from ADM-induced oxidative damage at low concentrations. Cell cycle arrest and apoptosis-inducing effects, achieved through p53-independent p21/WAF1 upregulation, are likely to be the mechanism of the antileukemic effects of naringenin, and the protective effect against ADM chemotherapy-induced damage in PMNs may be due to the antioxidant capability of this agent at low concentrations.

Anticancer Effect and Apoptosis Induction of Gambogic Acid in Human Leukemia Cell Line K562 In Vitro.

BackgroundThe aim of this study was to investigate the anticancer effect and related mechanisms of gambogic acid (GA), a traditional Chinese medicine, on human leukemia cell line K562, together with the effect on bone marrow mononuclear cells (MNCs).Material/MethodsK562 cells and MNCs were treated with various concentrations and treatment times of GA. Inhibitory rate was detected by use of the Cell Counting Kit-8 (CCK-8) assay. Apoptosis was analyzed by morphological detection, Annexin-V/PI doubling staining, and TUNEL assays. The expression changes of pivotal proteins were evaluated by Western blotting.ResultsGA not only suppressed cell proliferation, but also induced apoptosis of K562 cells in a dose-dependent manner. While it did not significantly inhibit cell proliferation of MNCs, it did induce apoptosis in a dose-dependent manner. CCK-8 assay revealed that the proliferation of K562 cells was significantly inhibited when the concentration of GA was more than 0.5 μM. Morphological detection showed the nuclei became denser and more intense orange in K562 cells after GA treatment compared with the untreated group. The expression levels of BCL-2, nuclear factor-κB (NF-κB), c-myc, phosphatidylinositol3-kinase (PI3K), and phosphorylation of serine-threonine kinase (p-AKT) were down-regulated by GA.ConclusionsGA significantly suppressed the proliferation of K562 cells, but has less effect on MNCs. The inhibition of K562 cells proliferation and apoptosis induced by GA might be related to the down-regulation of BCL-2, NF-κB, c-myc, PI3K, and p-AKT.

Dimethyl sulfoxide inactivates the anticancer effect of cisplatin against human myelogenous leukemia cell lines in in vitro assays.

Objectives:To investigate the effect of DMSO on cisplatin induced cytotoxicity (invitro) against K562 (Human mylogenous leukemia) cell line and to study the cisplatin-DMSO adduct formation using UV-spectrophotometer.Materials and methods:Effect of DMSO on the cytotoxicity of cisplatin was studied in K562 (Chronic mylogenous leukemia) cell line by MTT assay. Cisplatin-DMSO adduct formation was studied by continuously monitoring the increase in absorption peaks for 30 minutes using UV-spectrophotometer.Results:0.1-0.3% DMSO markedly reduced the cytotoxic activity of cisplatin in K562 cells. Cisplatin-DMSO adduct formation was detected using UV-spectrophotometer. Continuous increase in UV absorbance between 250nm-290nm was observed when cisplatin (0.5mg/ml) and DMSO (10%) were mixed.Conclusion:Present study revealed that DMSO inactivates the cytotoxicity of cisplatin. Cisplatin-DMSO mixture showed increased absorbance at 250-290nm. Therefore, using DMSO in invitro assays might result in misinterpretation of actual efficacy of drugs.

Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells

Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

Mass spectrometric analysis of proteasomes affinity puirified from the human myelogenous leukemia cells K562

Proteasomes carry out regulated proteolysis of most proteins and thereby play a crucial role in the regulation of different cellular processes. Dissecting subunit composition and post-translational modifications of proteasome is one of the important milestones in understanding their functions and mechanisms of regulation in the cell. To this end a strategy we followed a strategy for affinity purification of proteasomes from human myeloid leukemia cells with subsequent mass spectrometric analysis. Proteasomes were purified from the stable cell line K562 expressing HTBH tag-labeled 20S proteasome subunit β7 (PSMB4) by non-covalent affinity purification on biotin-avidin beads, followed by elution with TEV protease. We identified all known subunits of the 26S proteasome, as well as PA200 and regulators PA28γ amongst the eluted proteins, using MALDI-ICR mass spectrometry. We have shown that the proteasomes are associated with heat shock proteins, components of the ubiquitin-proteasome system of some cytoskeleton proteins. A number of novel phosphorylation, ubiquitination and N-terminal modification of proteasome subunits were found for 16 proteasome subunits. Our results might be useful for further proteomic studies of proteasomes.

Abstract 375: A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells

Abstract Introduction The adenosine deaminase acting on RNA (ADAR) family of RNA editases has been linked to the pathogenesis of diverse malignancies, including leukemia, breast cancer and hepatocellular carcinoma. We previously showed that human leukemia stem cells (LSC) from blast crisis (BC) chronic myeloid leukemia (CML) patients harbor increased ADAR1 expression compared with normal and chronic phase (CP) progenitors. Whole transcriptome RNA sequencing (RNA-Seq) revealed increased adenosine to inosine (A-to-I) RNA editing during CML progression concentrated within primate specific Alu-containing transcripts. However, detection of RNA editing by RNA-Seq in rare cell populations can be technically challenging, costly and requires PCR validation. Thus, the objectives of this study were to validate RNA editing of a subset of these LSC-associated transcripts in the context of lentivirally enforced ADAR1 expression, and to develop an RNA editing reporter reporter assay in human leukemia cells and a qPCR-based diagnostic test to rapidly detect CSC-associated aberrant RNA editing. Methods The BCR-ABL+ human leukemia cell line K562 was stably transduced with lentiviral human ADAR1 or vector. FACS-purified K562-ADAR1 cells were transfected with a luciferase-based reporter vector to confirm RNA editing activity. Two genes, MDM2 and APOBEC3D, were selected from our previous RNA-Seq studies of BC progenitors (Jiang et al, 2013). Targeted sequencing was performed on high fidelity PCR products using primers flanking each of 2 editing sites in each gene. RNA editing-specific qPCR primers were designed for each editing site using an allele-specific strategy that detects cDNA containing either an A or G(I) representing an RNA editing event. Both targeted sequencing and qPCR were used to detect RNA editing in K562-ADAR1 and primary cord blood-derived hematopoietic stem cells (HSC) lentivirally transduced with ADAR1. Results Lentivirally enforced ADAR1 expression promoted RNA editing activity as measured by luciferase reporter activity. Increased A-to-I changes in MDM2 and APOBEC3D were confirmed by targeted sequencing. In independent experiments, RNA editing site-specific qRT-PCR accurately detected RNA editing in K562-ADAR1 cells (n=3) and in primary HSC overexpressing ADAR1 (n=4). Site-specific primers distinguished G(I) bases at RNA editing sites in cDNA and as predicted gave no signal in gDNA. Relative A-to-I RNA editing ratios were increased by 2 to 3 fold in ADAR1-expressing cells at all four sites. Conclusions These results set the stage for development of primate-specific RNA editing as a novel diagnostic strategy for clinical LSC detection and identify ADAR1 as a potential therapeutic target in LSC. These data shed new light on the mechanisms of ADAR1-mediated generation of malignant progenitors that drive therapeutic resistance, disease progression and relapse in CML and may be applicable to other CSC-driven malignancies. Citation Format: Leslie A. Crews, Qingfei Jiang, Maria A. Zipeto, Angela C. Court, Christian L. Barrett, Marco A. Marra, Kelly A. Frazer, Catriona H. M. Jamieson. A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 375. doi:10.1158/1538-7445.AM2014-375

Synthesis and anticancer effects of some novel 2-(4-phenoxyphenyl)-1H-benzimidazole derivatives on K562 cell line.

A series of novel 2-(4-phenoxyphenyl)-1H-benzimidazole derivatives was synthesized and tested in vitro on human chronic myelogenous leukemia (CML) cell line K562. Benzimidazoles containing 5-amidino (10), 5-N-isopropylamidino (11), 5-bromo (13), and 5,6-dimethyl (14) derivatives exhibited remarkable cytotoxic activity. The quantitative analysis of apoptosis by flow-cytometry demonstrated that the percentages of early and late apoptotic K562 cells treated with these compounds were significantly higher than cells without treatment. We also investigated the effects of these compounds on the expression of apoptosis-related genes BAX, BCL-2, BAD and BIM. Treatment of K562 cells wih compounds 10-14 significantly increased the expression levels of the proapoptotic genes BAX, BAD and BIM, whereas compound 20 increased BAX and BAD.

BCR/ABL mRNA targeting small interfering RNA effects on proliferation and apoptosis in chronic myeloid leukemia.

To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5 nmol/L~50 nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations (IC50) of siRNA1384, siRNA1276 and siRNA1786 for 24 hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50 nmol/L siRNA transfection. Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

The natural chemopreventive agent sulforaphane inhibits STAT5 activity.

Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.