Human Erythroleukemia Cell Line K562 Research Articles

Abstract 4040: Asciminib activates erythroid differentiation in K562 cell line

Abstract Background: Asciminib an allosteric tyrosine kinase inhibitor (TKI) was recently approved by the FDA for managing Philadelphia positive chronic myeloid leukemia (CML) patients in chronic phase who had failed to respond to previous TKIs. In this study, we used human erythroleukemic K562 cell line to investigate the role of asciminib as an inducer of erythroid differentiation. Additionally, imatinib, which has previously been reported as a potential inducer of erythroid differentiation, was also tested on these cells, both independently as well as in combination, in order to investigate the degree of erythroid differentiation in a K562 cell line. Methodology: In this study, we investigated the effect of asciminib (5-20nM) with and without imatinib (50-200nM) in differentiating the erythroleukemic cell line K562 towards erythroid lineage. K562 cells were cultured over a 12-day period in basic (IMDM) media and an EPO-based differentiation media to select optimal medium conditions. In addition, an in-vitro drug cytotoxicity experiment (XTT) was used to determine the preliminary toxicity of asciminib with IMDM media and EPO-based differentiation media CD235a and CD71 surface marker expressions were analyzed by flow cytometry at all time-points to determine the degree of differentiation achieved by each of the conditions Moreover, to analyze and identify hemoglobin producing cells ensuing differentiation, benzidine staining was performed at indicated time points. Results: Our results suggest that asciminib, imatinib or a combination of both drugs in EPO-based differentiation media results in efficient erythroid differentiation with high levels of GPA expression (approx. 90%) and significant upregulation of globin genes (p value = 0.0001). These results were further confirmed by benzidine staining, where drug addition and dose escalation significantly increased benzidine-positive cells. Our results also demonstrated that asciminib was able to achieve maximum differentiation at a much earlier time point as compared to imatinib. Moreover, asciminib, when combined with another TKI such as imatinib, can also aid in lowering the drug concentrations being used, hence resulting in lessening the toxicity of the drug when used alone required at high doses. Conclusion: The results from this study highlighted the novel role of asciminib in erythroid differentiation using an in-vitro K562 model that can give high-levels of surface markers and globin expression. Potential clinical application of asciminib in hematological diseases that are associated specifically with a failure in the expression of globin genes should be evaluated. Keywords:K562, erythroid differentiation, asciminib, imatinib, glycophorin A, transferrin receptor, hemoglobin Citation Format: Hammad Hassan, Sarah Yousuf, Syed Muhammad Ahmed, Karim Ruknuddin, Hadiqa Raees, Sadia Habib, Fizza Iftikhar, Afsar Ali Mian, Elnasir Lalani. Asciminib activates erythroid differentiation in K562 cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4040.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for β-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF’s potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the β-type globin’s expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of β-type globin’s locus, triggering the transcriptional events from γ- to β-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an “open” chromatin conformation across the γ-δ intergenic region and accomplish β-globin expression and hemoglobin switch.

Cytotoxicity of the gold complex based on mercaptotriazole – A comparison with the conventional cyanide electrolyte

The study presents the 3-(4,5-dımethylthıazol-2-yl)-2,5-dıphenyltetrazolıum bromıde (MTT) assay results on human erythroleukemia K562 ​cell line to compare the toxicity of electrolytes based on the mercaptotriazole gold complex (Au-MT) and gold potassium cyanide. Solutions immediately after mixing and after three months at different concentrations were compared, with a prolonged aging period, to a year, for the electrolytes at pH ​= ​9. Lower toxicity of new organic gold complex at pH ​= ​4, 7, and 12 than of alkaline cyanide electrolyte, but higher at pH ​= ​2, was determined. The solution of the Au-MT at pH ​= ​9 had comparable, but lower cell toxicity in the whole investigated period.

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast.

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a–c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a–c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82–97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7–30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6–18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.

Use of trans‐3′5'4‐trihydroxystilbene (Resveratrol) as an adjunct in the treatment of Sickle Cell Disease

Sickle Cell Disease (SCD) and □-thalassemia are among the most common forms of hemoglobinopathy and life-threatening genetic diseases worldwide. So far, the only approved drug for these disorders is the ribonucleotide reductase inhibitor hydroxyurea (HU) falls short of being curative. The efficacy of HU in the management of thalassemia and SCD is generally attributed to its limited ability to boost the levels of fetal hemoglobin (Hb F,α2γ2), which decreases sickling by interfering with the polymerization of hemoglobin in patients with SCD and can partially correct the globin chain imbalance in □-thalassemia patients, while at the same time facilitating O2 transport to myoglobin in peripheral tissues. The naturally occurring dietary polyphenol resveratrol (trans-3′5'4-trihydroxystilbene, RV), has also been postulated to induce Hb F production via a biochemical mechanism like HU. Preclinical studies using the human erythroleukemic K562 cell line suggested RV can induce Hb F production in a more robust manner than HU. However, clinical studies on the efficacy and effectiveness of RV are limited and conflicting. This study sought to evaluate the clinical efficacy and safety of RV in managing SCD compared to HU. A systematic literature review was conducted in peer-reviewed, published full-text articles investigating RV in the management of SCD. Eligible studies were required to report explicitly, including participants diagnosed with β-thalassemia or SCD anemia or cultured cells with the SCD or thalassemia disease and where HU was included as a control. The analysis included thalassemia, RV type, RV dosage, duration of treatment, and outcome, Hb F induction and antioxidant activity. Additionally, a pilot study of RV in the management of adults with SCD who received treatment at National Hospital Abuja, Nigeria, was attempted but discontinued due to the COVID 19 pandemic. A case study of a single 23-year-old female SCD patient who had been taking RV [1000mg daily p.o.] for more than 6 years was included. Outcome variables evaluated include number of hospitalization episodes of pain crisis, need for blood transfusion, possible side effects and RBC indices. The early results of this metanalysis show that RV was as effective as HU in inducing production of Hb F in adult SCD patients and reducing hospital treatment days post RV treatment. Four primary treatment and curative therapies that have shown great potential and are currently used to prevent and treat acute and chronic complications in SCD, HU and blood transfusions, are the most well-established disease-modifying therapies used to prevent and treat complications associated with SCD. Other lesser used therapies include stem cell transplantation or the allogeneic hematopoietic stem cell transplant (HSCT), and the investigational gene therapy is the only known curative therapy that exists regarding SCD. The results of this study indicate that RV is an effective inducer of Hb F production in adults and shows promise as a cost effective and useful adjunct for the treatment of hemoglobinopathies including SCD and thalassemia.

Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Synthesis and Erythroid Induction Activity of New Thiourea Derivatives.

The use of medicinal agents to augment the fetal hemoglobin (HbF) accretion is an important approach for the treatment of sickle-cell anemia and β-thalassemia. HbF inducers have the potential to reduce the clinical symptoms and blood transfusion dependence in the patients of β- hemoglobinopathies. The current study was aimed to examine the erythroid induction potential of newly synthesized thiourea derivatives. Thiourea derivatives 1-27 were synthesized by using environmentally friendly methods. Compounds 3, 10 and 22 were found to be new. The structures of synthesized derivatives were deduced by using various spectroscopic techniques. These derivatives were then evaluated for their erythroid induction using the human erythroleukemic K562 cell line, as a model. The benzidine-H2O2 assay was used to evaluate erythroid induction, while HbF expression was studied through immunocytochemistry using the Anti-HbF antibody. Cytotoxicity of compounds 1-27 was also evaluated on mouse fibroblast 3T3 cell line and cancer Hela cell line using MTT assay. All the compounds (1-27) have not been reported for their erythroid induction activity previously. Compounds 1, 2, and 3 were found to be the potent erythroid inducing agents with % induction of 45± 6.9, 44± 5.9, and 41± 6.1, at 1.56, 0.78, and 0.78 μM concentrations, respectively, as compared to untreated control (12 ± 1 % induction). Furthermore, compound 1, 2, and 3 significantly induced fetal hemoglobin the expression up to 4.2-fold, 4.06-fold, and 3.52-fold, respectively, as compared to untreated control. Moreover, the compounds 1-4, 6-9, 11, 12, 15, 17, 19, 22, 23, and 25 were found to be non-cytotoxic against the 3T3 cell line. This study signifies that the compounds reported here may serve as the starting point for the designing and development of new fetal hemoglobin inducers for the treatment of β- hemoglobinopathies.

Facile One-Pot Synthesis of Benzo[h]isoquinolines via Strong Acid Triggered [1,2]-Sigmatropic Rearrangement: A Theoretical and Experimental Studies

Benzo[h]isoquinoline scaffold is of interest as a rigid subunit that can be useful for constructing biologically active products. However, no good-yielded synthetic pathway to this ring system has been reported yet. Herein, a facile one-pot synthesis from N-aryl itaconimides and 1,3-diarylisobenzofuran via strong acid triggered skeletal rearrangement reaction is described. Theoretical study for this rearrangement is provided at M11/cc-pVDZ level of theory. Antitumor activity of obtained benzo[h]isoquinoline derivatives against human erythroleukemia K562 cell line was evaluated in vitro by MTS-assay.

Thiourea derivatives induce fetal hemoglobin production in-vitro: A new class of potential therapeutic agents for β-thalassemia

Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of β-hemoglobinopathies like β-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562 cell line. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562 cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562 cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including β-thalassemia and sickle cell anemia.

Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course.

This sequence of labs was developed for an upper level undergraduate cell biology course at Fairfield University. The labs are based on the use of the K562 human erythroleukemia cell line, a model system that is exceptionally amenable to an undergraduate cell biology lab course due to its ease of maintenance and propagation and usefulness for studies of growth, death, and differentiation. The sequence of labs is conducted over a 6-week period, following a series of weekly cell biology labs covering basic cell and molecular biology techniques. Together, the lab series has four primary objectives 1) to teach students how to culture and maintain mammalian cells; 2) to build student competency in standard cell biology techniques; 3) to demonstrate the role of growth factors on cell proliferation and viability; and 4) to provide students with an opportunity to use these cells in an independent investigation on cell differentiation. We provide examples of student data and offer a range of experimental measurements depending on institutional capacity and facilities. Our assessment data suggest that students find great value in this lab series, enhancing their comprehension of key concepts, acquisition of important lab skills, and depth of understanding of the research process. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):263-271, 2019.

Genomic landscape of human erythroleukemia K562 cell line, as determined by next-generation sequencing and cytogenetics

We have performed detailed analysis of the genomic landscape of commercially available K562 cells, employing targeted enrichment of nearly 1300 cancer-related genes followed by next-generation sequencing (NGS) and also classical cytogenetics. Deep sequencing revealed 88 variants of potentially biological significance. Among them we have detected alterations in genes already known to be mutated in K562, such as TP53 but also in several other genes, which are implicated in tumorigenesis and drug resistance, such as MLH1, ASXL1 and BRCA1 as the most prominent examples. Fluorescence in situ hybridization (FISH) of interphases of K562 cells revealed multiplication of the BCR and ABL1 gene copies, as well as the amplification of the BCR-ABL1 fusion gene. Our results may help to better understand genomic instability of the blastic phase of CML represented by the K562 cell line and can help researchers who want to employ this cell line in various experimental settings.

Identification of physical interactions between genomic regions by enChIP-Seq.

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. Here, we show the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 and a single guide RNA. Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5'HS5 locus, which is involved in the regulation of globin genes expression at the β-globin locus, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5'HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism mediated by physical interactions between these loci. Thus, enChIP-Seq might be a potentially useful tool for detecting physical interactions between genomic regions in a nonbiased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.

Liver X Receptor (LXR) Activation Decreases Chronic Myelogenous Leukemia Cell Viability and Alters the Expression of Antiapoptotic and Cholesterol Genes

Liver X receptors (LXRs) are oxysterol‐activated nuclear receptors involved in the regulation of cholesterol transport, glucose metabolism, and inflammatory responses. Recently, LXRs have been shown to possess anti‐cancer effects in a variety of cancer types, in part due its capacity to deplete essential lipids required for cell proliferation. However, the role of LXRs in chronic myelogenous leukemia (CML) has yet to be elucidated. In this study, we investigated the effects of LXR activation on the human erythroleukemia K562 cell line – a model for CML. K562 cells were treated with the LXR agonist TO901317 (0μM, 1μM, 5μM, 10μM) to determine dose‐ and time‐dependent effects on cell viability, cell number and size, and the mRNA expression of genes related to cholesterol metabolism (ATP‐binding cassette transporter A1 (ABCA1), low‐density lipoprotein receptor (LDLR), and HMG‐CoA reductase (HMGCR)) and anti‐apoptosis (B cell leukemia‐xL (Bcl‐xL)). TO901317 treatment significantly increased ABCA1 mRNA expression at 24hr and 72hr, with greater expression at the lowest treatment concentration (1μM). HMGCR and Bcl‐xL mRNA expression was dose‐dependently reduced by LXR activation after 72hr, as were decreases in total cell number. Average cell size similarly decreased from 24hr to 72hr with 10μM TO901317 treatment. Further, K562 viability was decreased by TO901317 treatment at all concentrations at 24hr, 48hr, and 72hr, with dose‐dependent effects observed at 48hr only. Together, these findings suggest that LXR agonism decreases CML cell viability and number, and that these changes may be related to modulation of cholesterol metabolism and apoptotic pathways. Current studies are underway to further investigate these pathways, in addition to the capacity of TO901317‐induced LXR activation to modulate cellular differentiation.Support or Funding InformationThe Science Institute of the College of Arts and Sciences and Faculty Research Committee, Fairfield University

Nuclear DGKα regulates cell cycle progression in K562 cells

The existence of an independent nuclear inositide pathway distinct from the cytoplasmic one has been demonstrated in different physiological systems and in diseases (1). Phosphatidylinositols (PIs) play an important role in nuclear function regulation and behave differently from their counterparts in the cytoplasm. The autonomous nuclear PI cycle in eukaryotic cells is involved in different regulation processes, from cell proliferation to differentiation and many others (2). At nuclear level an array of kinases and phosphatases can modulate PIs. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol (DAG) and induce the synthesis of phosphatidic acid. We Investigated DGKα localization and function in human erythroleukemia cell line K562. Synchronization experiments at different cell cycle checkpoints showed an important expression of DGKα in the nuclear fraction of this cell model, slightly peaking at G2/M. This suggested that DGKα might have a function in nuclear signaling. In particular, nuclear DGKα expression can modulate cell cycle progression, leading to changes in the phosphorylated status of the Retinoblastoma protein (pRb), thus, regulating G1/S transition: DGKα silencing or downregulation leads to impaired G1/S transition and its overexpression leads to S phase progression. The molecular mechanism by which nuclear DGKα controls pRb phosphorylation and therefore cell cycle regulation in K562 cell line are still unclear. Further studies are needed to better understand the role of DGKα in relation to other pivotal PIs involved in cell cycle regulation in the hematopoietic system.

Topsensterols A-C, Cytotoxic Polyhydroxylated Sterol Derivatives from a Marine Sponge Topsentia sp.

Three new polyhydroxylated sterol derivatives topsensterols A–C (1–3) have been isolated from a marine sponge Topsentia sp. collected from the South China Sea. Their structures were elucidated by detailed analysis of the spectroscopic data, especially the NOESY spectra. Topsensterols A–C (l–3) possess novel 2β,3α,4β,6α-tetrahydroxy-14α-methyl Δ9(11) steroidal nuclei with unusual side chains. Compound 2 exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC50 value of 8.0 μM. Compound 3 displayed cytotoxicity against human erythroleukemia cell line K562 with an IC50 value of 6.0 μM.

Polyphyllin D induces apoptosis and differentiation in K562 human leukemia cells

Polyphyllin D, a compound derived from Paris polyphylla rhizoma, demonstrated strong anticancer activities in a previous study. Our results demonstrated that polyphyllin D exerts a growth inhibitory effect by inducing apoptosis and differentiation in the human erythroleukemia cell line K562. Polyphyllin D induced apoptosis via the mitochondrial apoptotic pathway, as evidenced by the decreased Bcl-2 and Bcr/Abl expression levels, the disruption of MMP and increased Bax, cytochrome c and cleaved-caspase-3 levels. At a low dose, polyphyllin D increased CD14 expression on the surface of K562 cells and induced cells to differentiate into monocytes or mature macrophages. These data suggest that polyphyllin D has the potential to be a potent therapeutic agent for treating human chronic myelogenous leukemia.

Non-cytotoxic copper overload boosts mitochondrial energy metabolism to modulate cell proliferation and differentiation in the human erythroleukemic cell line K562

Copper is integral to the mitochondrial respiratory complex IV and contributes to proliferation and differentiation, metabolic reprogramming and mitochondrial function. The K562 cell line was exposed to a non-cytotoxic copper overload to evaluate mitochondrial dynamics, function and cell fate. This induced higher rates of mitochondrial turnover given by an increase in mitochondrial fusion and fission events and in the autophagic flux. The appearance of smaller and condensed mitochondria was also observed. Bioenergetics activity included more respiratory complexes, higher oxygen consumption rate, superoxide production and ATP synthesis, with no decrease in membrane potential. Increased cell proliferation and inhibited differentiation also occurred. Non-cytotoxic copper levels can modify mitochondrial metabolism and cell fate, which could be used in cancer biology and regenerative medicine.

Cyclic RGD peptidomimetics containing 4- and 5-amino-cyclopropane pipecolic acid (CPA) templates as dual αVβ3 and α5β1 integrin ligands

4-Amino- and 5-amino-cyclopropane pipecolic acids (CPAs) with cis relative stereochemistry between the carboxylic and amino groups were used as templates to prepare cyclic peptidomimetics containing the RGD sequence as possible integrin binders. The peptidomimetic c(RGD8) built on the 5-amino-CPA displayed an inhibition activity (IC50=2.4nM) toward the αvβ3 integrin receptor (expressed in M21 human melanoma cell line) comparable to that of the most potent antagonists reported so far and it was ten times more active than the corresponding antagonist c(RGD7) derived from the isomeric 4-amino-CPA. Both compounds were also nanomolar ligands of the α5β1 integrin (expressed in human erythroleukemia cell line K562). These results suggest that the CPA-derived templates are suitable for the preparation of dual αvβ3 and α5β1 ligands to suppress integrin-mediated events as well as for targeted drug delivery in cancer therapy.

Anti-Helicobacter pylori activity of bioactive components isolated from Hericium erinaceus

Ethnopharmacological relevanceThe fungus Hericium erinaceus (Bull.) Pers is used in Chinese traditional medicine to treat symptoms related to gastric ulcers. Different extracts from the fungus were assessed for anti-Helicobacter pylori activity to investigate the antibacterial activity of the ethanol extracts from H. erinaceus and verify the traditional indication of use. Materials and methodsThe fruiting bodies of H. erinaceus were concentrated with ethanol by HPD-100 macroporous resin and the whole extract was partitioned by petroleum ether and chloroform to afford fractions with using a silica gel column. Several pure compounds of petroleum ether extracts were obtained and analyzed using nuclear magnetic resonance (NMR). The activity of the extracts and fractions towards H. pylori was assessed by the microdilution assay and by the disk diffusion assay in vitro. From the most active fraction, two pure compounds were isolated and identified as the main components with anti-H. pylori activity from the fungus H. erinaceus. The cytotoxicity of these two compounds against the human erythroleu-kemia cell line K562 was also evaluated. ResultsThe crude ethanol extracts from the fungus H. erinaceus were inhibitory to H. pylori. The petroleum ether extracts (PE1s, PE2s) and the chloroform extracts (TEs) demonstrated strong inhibition to H. pylori. The inhibition of H. pylori was observed through an agar dilution test with minimal inhibition concentration (MIC) values from 400μg/mL to 12.5µg/mL. Two pure compounds, 1-(5-chloro-2-hydroxyphenyl)-3-methyl-1-butanone and 2,5-bis(methoxycarbonyl)terephthalic acid were isolated from the petroleum ether fractions and identified using 1H NMR and 13C NMR spectra analysis. The MIC value for 1-(5-chloro-2-hydroxyphenyl)-3-methyl-1-butanone was 12.5–50µg/mL and the MIC value for 2,5-bis(methoxycarbonyl)terephthalic acid was 6.25–25µg/mL. Both two compounds showed weak cytotoxicity against K562 with IC50<200mM. ConclusionsThis study revealed that the extracts from petroleum ether contribute to the anti-H. pylori activity. The compounds obtained from petroleum ether extracts, 1-(5-chloro-2-hydroxyphenyl)-3-methyl-1-butanone and 2,5-bis(methoxycarbonyl)terephthalic acid, inhibit the growth of H. pylori.