Human Leukemia K562 Cells Research Articles

Antitumor Activity and Mechanism of Action of the Antimicrobial Peptide AMP-17 on Human Leukemia K562 Cells.

Cancer is one of the most common malignant diseases in the world. Hence, there is an urgent need to search for novel drugs with antitumor activity against cancer cells. AMP-17, a natural antimicrobial peptide derived from Musca domestica, has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. However, its antitumor activity and potential mechanism of action in cancer cells remain unclear. In this study, we focused on evaluating the in vitro antitumor activity and mechanism of AMP-17 on leukemic K562 cells. The results showed that AMP-17 exhibited anti-proliferative activity on K562 cells with an IC50 value of 58.91 ± 3.57 μg/mL. The membrane integrity of K562 was disrupted and membrane permeability was increased after AMP-17 action. Further observation using SEM and TEM images showed that the cell structure of AMP-17-treated cells was disrupted, with depressions and pore-like breaks on the cell surface, and vacuolated vesicles in the cytoplasm. Furthermore, further mechanistic studies indicated that AMP-17 induced excessive production of reactive oxygen species and calcium ions release in K562 cells, which led to disturbance of mitochondrial membrane potential and blocked ATP synthesis, followed by activation of Caspase-3 to induce apoptosis. In conclusion, these results suggest that the antitumor activity of AMP-17 may be achieved by disrupting cell structure and inducing apoptosis. Therefore, AMP-17 is expected to be a novel potential agent candidate for leukemia treatment.

Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells

To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Novel 1,2,4-triazoles derived from Ibuprofen: synthesis and in vitro evaluation of their mPGES-1 inhibitory and antiproliferative activity.

Some novel triazole-bearing ketone and oxime derivatives were synthesized from Ibuprofen. In vitro cytotoxic activities of all synthesized molecules against five cancer lines (human breast cancer MCF-7, human lung cancer A549, human prostate cancer PC-3, human cervix cancer HeLa, and human chronic myelogenous leukemia K562 cell lines) were evaluated by MTT assay. In addition, mouse embryonic fibroblast cells (NIH/3T3) were also evaluated to determine the selectivity. Compounds 18, 36, and 45 were found to be the most cytotoxic, and their IC50 values were in the range of 17.46-68.76µM, against the tested cancer cells. According to the results, compounds 7 and 13 demonstrated good anti-inflammatory activity against the microsomal enzyme prostaglandin E2 synthase-1 (mPGES-1) enzyme at IC50 values of 13.6 and 4.95µM. The low cytotoxicity and non-mutagenity of these compounds were found interesting. Also, these compounds significantly prevented tube formation in angiogenesis studies. In conclusion, the anti-inflammatory and angiogenesis inhibitory activities of these compounds without toxicity suggested that they may be promising agents in anti-inflammatory treatment and they may be supportive agents for the cancer treatment.

Euphorane C, an unusual C17-norabietane diterpenoid from Euphorbia dracunculoides induces cell cycle arrest and apoptosis in human leukemia K562 cells

Four new polycyclic diterpenoids euphoranes A − D ( 1 – 4 ), together with 12 known terpenoids ( 5 – 16 ) were isolated from the whole plants of Euphorbia dracunculoides . Compound 2 represents the first example of aromatic 15,16,17-trinorabietane diterpenoid in Euphorbia species, and euphorane C ( 3 ) is an unusual 17-norabietane diterpenoid. The absolute configurations of 1 , 2 , and 4 were determined by single crystal X-ray diffraction, while that of 3 was established with the aid of ECD and 1D NMR quantum chemical calculation. Moreover, the absolute stereochemistry of 11 was demonstrated for the first time by crystallographic data. Compounds 1 – 16 were screened for antiproliferative activities against five human cancer cell lines, and 3 showed significant cytotoxicity against the human leukemia K562 cells with IC 50 value of 3.59 μM. Preliminary mechanistic investigation revealed that 3 could induce cell cycle arrest at G0/G1 phase and apoptosis in K562 cells.

PHYTOCHEMICAL CONSTITUENTS AND ANTILEUKEMIC EFFECTS OF JUNIPERUS OXYCEDRUS EXTRACT

Aim: Many genetic and environmental factors can be effective in the process of cancerization. Preventing the progression of leukemia may be possible by controlling the pathways involving mechanisms such as apoptosis and autophagy. When the literature is examined, there are studies showing the effects of various types of juniper on various cancer cell lines, including human chronic myeloid leukemia cells, but the signal pathways in which they act are not fully known. In this study, the anticancer effects of Juniperus oxycedrus extract on K-562 human chronic myeloid leukemia cells were investigated. Method: After the cells were treated with the Juniperus oxycedrus extract, cytotoxicity and gene expression analyzes were performed. Changes in the expression of Akt, the member of the PI3K/Akt/mTOR signaling pathway; caspase 3, which is one of the main effective genes in the pathways regulating apoptosis; and the apoptosis suppressor BCL-2 gene, which is an oncogene, were investigated. Results: According to the MTT test results, Juniperus oxycedrus extract showed over approximately 50% cell viability in K-562 cells at all doses. The most appropriate dose of Juniperus oxycedrus fruit extract in this research was determined as 50 µg/ mL considering cell viability. After the gene expression analysis, it was observed that Akt expression increased 1.092 times, BCL-2 expression decreased approximately 0.3 times, and caspase 3 expression increased 1.2 times. Conclusions: Constituents of Juniperus oxycedrus plant may have apoptotic effects on chronic myeloid leukemia cells.

A cytotoxic effect of human lactoferrin fusion with Fc domain of IgG.

Lactoferrin (LTF) is a natural iron-binding protein with a potential for clinical utility in many human immune disorders, including cancer. A fusion of LTF with the Fc domain of IgG2 (FcLTF) was designed with inherent properties of an extended the half-life in circulation. Furthermore, the effects of LTF and FcLTF were assessed for influence on the activity of natural killer (NK) cells isolated from human peripheral blood, on the NK-92 cell line, and on human monocytes. The NK cytotoxic activity induced by LTF and FcLTF was determined against the human leukemia K562 cell line, and also for monocytes, by measuring TNFα and granzyme B production, and in an assay for Jurkat cell viability. Selected gene expression in NK-92 cells and monocytes, induced by LTF and FcLTF, was performed by Real Time PCR. No significant difference was observed in NK-92 cytotoxicity stimulated by LTF and FcLTF. The effects on NK cells isolated from the human peripheral blood were varied, possibly due to the immunoregulatory nature of LTF sensing the immune status of donors. Furthermore, only the FcLTF group strongly stimulated production of TNFα and granzyme B in isolated monocytes. In addition, only supernatants from the monocyte cultures treated with FcLTF decreased the viability of Jurkat cells. The ability of FcLTF to induce TNFα in monocytes was strongly inhibited by anti-CD32 and moderately inhibited by anti-CD14 antibody. Lastly, it was demonstrated that FcLTF, strongly induced expression of PI3K, with subsequent activation of AKT/mTOR signaling pathway. Overall, it was demonstrated that this novel fusion molecule may be a perferred choice for clinical utility than the wild type LTF.

Targeted deletion of 5'HS2 enhancer of β-globin locus control region in K562 cells.

Human β-thalassemia is closely associated with aberrant expression of β-like globin genes. Human β-like globin genes are organized in the order of 5'-ε-Gγ-Aγ-δ-β-3' within the β-globin locus. The expression of β-like globin genes is regulated by 3'HS1 and five DNase I hypersensitive sites (5'HS5~5'HS1) in a locus control region. The 5'HS2 enhancer transcribes enhancer RNA and regulates the expression of ε-globin, γ-globin and β-globin. To further study the function of 5'HS2, we detected the local 3D genomic architecture via chromatin conformation capture experiments and used CRISPR/ Cas9-based DNA fragment editing to delete 5'HS2 in human K562 leukaemia cells. In this study, we found that 5'HS2-mediated chromatin interactions were enriched in a topologically associated domain that was bordered by 3'HS1 and 5'HS5. Within this topologically associated domain, 5'HS2 is highly close to the promoter regions of HBE1, HBG2 and HBG1. Upon deletion of the 5'HS2 enhancer, 91 genes were significantly down-regulated with reduced abundance of H3K27ac at their promoter regions. These down-regulated genes are mainly associated with oxygen transport, immune response, cell adhesion, anti-oxidant and thrombosis. These data suggested that many genes associated with functions of erythrocytes were decreased at transcriptional levels upon deletion of the 5'HS2 enhancer.

Synthesis, structural evaluation, antioxidant, DNA cleavage, anticancer activities and molecular docking study of metal complexes of 2-amino thiophene derivative

Novel heterocyclic ligand of 2-amino thiophene derivative and metal complexes with manganese, cobalt, nickel, copper and zinc bivalent ions were prepared. Compounds were analyzed through elemental analysis, molar conductance, magnetic moment, FT-IR, UV–Visible, NMR (1H and 13C), EPR and XRD. The above spectroscopic data confirmed that ligand coordinated to metal through enolate oxygen, nitrogen of azomethine and carbonyl group. EPR data confirmed the geometry of copper complex and evidenced that the metal–ligand bonds are reasonable covalent character. The XRD analysis explained the crystalline character of compounds and fluorescence spectroscopy showed considerable fluorescent properties. In view of biological activities, the antioxidant properties of samples were assayed by scavenging activity of DPPH radical and results to prove greater activity of all the compounds except Cu(II) complex. DNA cleavage ability of compounds was evaluated using gel electrophoresis method. It revealed that the compounds effectively cleaved the pUC18 plasmid DNA. The anticancer studies of all the compounds were screened against cell lines MCF-7 Human Breast Cancer cells and K-562 Human Leukemia cells using SRB assay. The IC50 values suggested that synthesized compounds are more active towards above cancer cell lines compared with Adriamycin. Highly potent compounds in anticancer activity were studied by molecular docking against receptor CDK2.

OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer.

Simple SummaryArginase 1 and 2 are drivers of multiple immunosuppressive mechanisms and tumour-specific metabolic adaptations. Pharmacological inhibition of extracellular ARG1 has shown antitumour efficacy in various syngeneic tumour models, however, the importance of ARG2 as a therapeutic target has only been demonstrated by genetic deletion studies. This is the first study validating the benefits of pharmacological inhibition of ARG2 in cancer. Our work describes OATD-02 as a potent dual ARG1/ARG2 inhibitor with a cellular activity (necessary for targeting ARG2) exhibiting immunomodulatory and direct antitumour efficacy in animal models. Our results present OATD-02 as an attractive option for combination with other immunotherapeutics, such as PD-1/PD-L1 antibodies or IDO1 inhibitors, especially in the therapy of particularly resistant hypoxic tumours. The presented findings provided the rationale for planning first-in-human clinical trials for OATD-02 in cancer patients.Background: Arginases play essential roles in metabolic pathways, determining the fitness of both immune and tumour cells. Along with the previously validated role of ARG1 in cancer, the particular significance of ARG2 as a therapeutic target has emerged as its levels correlate with malignant phenotype and poor prognosis. These observations unveil arginases, and specifically ARG2, as well-validated and promising therapeutic targets. OATD-02, a new boronic acid derivative, is the only dual inhibitor, which can address the benefits of pharmacological inhibition of arginase 1 and 2 in cancer. Methods: The inhibitory activity of OATD-02 was determined using recombinant ARG1 and ARG2, as well as in a cellular system using primary hepatocytes and macrophages. In vivo antitumor activity was determined in syngeneic models of colorectal and kidney carcinomas (CT26 and Renca, respectively), as well as in an ARG2-dependent xenograft model of leukaemia (K562). Results: OATD-02 was shown to be a potent dual (ARG1/ARG2) arginase inhibitor with a cellular activity necessary for targeting ARG2. Compared to a reference inhibitor with predominant extracellular activity towards ARG1, we have shown improved and statistically significant antitumor efficacy in the CT26 model and an immunomodulatory effect reflected by Treg inhibition in the Renca model. Importantly, OATD-02 had a superior activity when combined with other immunotherapeutics. Finally, OATD-02 effectively inhibited the proliferation of human K562 leukemic cells both in vitro and in vivo. Conclusions: OATD-02 is a potent small-molecule arginase inhibitor with optimal drug-like properties, including PK/PD profile. Excellent activity against intracellular ARG2 significantly distinguishes OATD-02 from other arginase inhibitors. OATD-02 represents a very promising drug candidate for the combined treatment of tumours, and is the only pharmacological tool that can effectively address the benefits of ARG1/ARG2 inhibition. OATD-02 will enter clinical trials in cancer patients in 2022.

Phenolic constituents with anti-inflammatory and cytotoxic activities from the rhizomes of Iris domestica

Four undescribed flavonoid glucosides (iridins B–C, tectoridin A and ampelopsinin A); one undescribed phenolic glucoside (diplostephioside B); one undescribed phenolic compound (phenanthrenetriol A); and seventeen known compounds were isolated from the rhizomes of Iris domestica. The chemical structures of the undescribed compounds were established by spectroscopic/spectrometric data interpretation using HRESIMS, NMR, and ECD. Tectoridin A, nigricin A and naringenin exhibited anti-inflammatory activities with inhibition rates of 53.71%, 57.68% and 88.71%, respectively, against the NF-κB signaling pathway at a concentration of 10 μM. 4′-O-methylnyasol (10 μM) exhibited 84.91% antiproliferative activity against the K562 human leukemia cell line with an IC50 value of 4.20 μM.

Synthesis and antitumor activity of a series of novel N-aryl-5-(2,2,2-trifluoroethoxy)-1,5-dihydro-2H-pyrrol-2-ones derivatives

With the help of the establishment of novel reaction methodology, a series of N-Aryl-5-(2,2,2-trifluoroethoxy)-1,5-dihydro-2H-pyrrol-2-one conjugates were designed and synthesized in 2–4 steps, and subsequent anticancer activity of these compounds was evaluated. Preliminary results showed that these compounds have moderate to potent activities against human acute leukemia cells K562, human lung cancer A549, human breast cancer MDA-MB-231, and human cervical cancer HeLa cancer cell lines. Among them, compounds 2d and 2k were the most potent against K562 cell line with IC50 values of 0.07 and 0.52 µM, respectively, and the toxicity of 2d to the normal of hepatocytes (LO2) cell line was low (the survival rate 81 %). Flow cytometry analysis showed that 2d arrested K562 cells in the G2/M phase potently, even much better than Combretastatin A4 (CA4). In addition, the results demonstrated the involvement of the caspase-dependent or independent pathways of apoptosis, evidenced by the upregulation of FADD, pro-caspase 3, cleaved-caspase 3, HTRA2/Omi, SMAC/Diablo and the ratio of Bax/Bcl-2.The biological effects founding of 2d in this work point to prospective uses against acute leukemia.

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.

Cytotoxic Activity of Bark of Oroxylon indicum Benth against U937 and K562 Human Leukemic Cell Lines

Cancer, a leading cause of death, is a condition where cells have lost control over their mode of growth. We evaluated the anti- proliferative and cytotoxic and apoptogenic activity of methanolic extract of Oroxylon indicum Bark (MEOIB) on U937, K562 cells and normal lymphocytes. The cell viability was studied by MTT assay. Cell morphology was determined by Fluorescence, Confocal microscope and DNA fragmentation by agarose gel electrophoresis study. MEOIB inhibited the viability of cells in a time &amp; concentration dependent manner. The Morphologically it was found that externalization of PS from inner leaflet to outer leaflet of the cell membrane and condensed chromatin, membrane blabbing, fragmentation of nuclei in all the treated cells than control and DNA bands appeared in the form of ladder after treatment with MEOIB. The present study reveals that the MEOIB possesses potent anti-leukemic activity against U937 and K562 cell line but there no toxicity in normal lymphocytes.

Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα.

Simple SummaryDNA topoisomerase IIα (170 kDa, TOP2α/170) resolves nucleic acid topological entanglements by generating transient double-strand DNA breaks. TOP2α inhibitors/poisons stabilize TOP2α-DNA covalent complexes resulting in persistent DNA damage and are frequently utilized to treat a variety of cancers. Acquired resistance to these chemotherapeutic agents is often associated with decreased TOP2α/170 expression levels. Studies have demonstrated that a reduction in TOP2α/170 results from a type of alternative polyadenylation designated intronic polyadenylation (IPA). As a consequence of IPA, variant TOP2α mRNA transcripts have been characterized that have resulted in the translation of C-terminal truncated TOP2α isoforms with altered biological activities. In this paper, an example is discussed where circumvention of acquired TOP2α-mediated drug resistance was achieved by utilizing CRISPR/Cas9 specific gene editing of an exon/intron boundary through homology directed repair (HDR) to reduce TOP2α IPA. These results illustrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant IPA.Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.

MiR-let-7c-3p targeting on Egr-1 contributes to the committed differentiation of leukemia cells into monocyte/macrophages.

In preliminary experiments, it was found that the expression of early growth response-1 (Egr-1) was upregulated during the committed differentiation of leukemia cells into monocytes/macrophages. The cross-analysis of gene chip detection and database prediction indicated that Egr-1 was associated with upstream microRNA (miR)-let-7c-3p, thus the present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of leukemia cells into monocytes/macrophages. Phorbol 12-myristate 13-acetate (PMA) was used to induce the directed differentiation of human K562 leukemia cells into monocytes/macrophages and the differentiation of K562 leukemia cells was determined by cell morphology observation and expression of differentiation antigens CD11b and CD14 by flow cytometry. The expression levels of Egr-1 and miR-let-7c-3p were detected by reverse transcription-quantitative PCR and the protein expression of Egr-1 was detected by western blotting. The effect of Egr-1 on the differentiation of K562 cells was detected by short interfering (si)RNA interference assay. A dual-luciferase reporter assay was used to detect target binding of miR-let-7c-3p on the 3′UTR of Egr-1. Cell transfection of miR-let-7c-3p mimics and inhibitors was used to modulate the expression of miR-let-7c-3p, as indicated by RT-qPCR assays. Western blotting was also used to examine the effect of miR-let-7c-3p on Egr-1 expression. The PMA-induced differentiation of K562 cells was transfected with miR-let-7c-3p and the expression of differentiation antigen was detected by flow cytometry. A differentiation model of K562 leukemia cells into monocytes/macrophages was induced by PMA, which was indicated by morphological observations and upregulation of CD11b and CD14 antigens. The gene or protein expression of Egr-1 was significantly higher compared with that of the control group, while the expression of miR-let-7c-3p was significantly lower compared with that of the control group. siRNA interference experiments showed that the expression of cell differentiation antigen CD14 in the 100 µg/ml PMA + si-Egr-1 group was significantly lower compared with that in the 100 µg/ml PMA + si-ctrl group. The dual luciferase reporter gene results showed that the luciferase activity of the co-transfected mimic and Egr-1 WT groups was significantly lower than that of the NC control group, while the luciferase activity of the co-transfected mimic and Egr-1 MUT groups was comparable to that of the NC control group. Therefore, the dual-luciferase reporter gene assay confirmed that miR-let-7c-3p can target Egr-1. Western blotting showed that the expression of Egr-1 following transfection with miR-let-7c-3p inhibitor was significantly higher compared with that of the negative control and the expression of Egr-1 after transfection with miR-let-7c-3p mimic was significantly lower than that of the negative control. Following exposure to PMA, the expressions of CD11b and CD14 in the miR-let-7c-3p inhibitor group were significantly higher than those in the miR-let-7c-3p NC group, as indicated by CD11b and CD14 respectively. In conclusion, miR-let-7c-3p could bind to the 3′UTR of Egr-1 and negatively regulated Egr-1 expression. The miR-let-7c-3p/Egr-1 signaling axis was closely associated with the committed differentiation of K562 cells from leukemia cells to monocytes/macrophages.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation

Iron‑sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron‑sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5′-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

A bioisosteric approach to the discovery of novel N-aryl-N′-[4-(aryloxy)cyclohexyl]squaramide-based activators of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation

Inhibition of translation initiation has emerging implications for the development of mechanism-based anticancer therapeutics. Phosphorylation of eIF2α is recognized as a key target that regulates the translation initiation cascade. Based on the bioisosteric replacement of urea-derived eIF2α phosphorylation activator 1, a novel series of N-aryl-N′-[4-(aryloxy)cyclohexyl]squaramide derivatives was designed and synthesized; their effects on the activation of eIF2α phosphorylation was assessed systematically. A brief structure–activity relationship analysis was established by stepwise structural optimization of the squaramide series. Subsequently, the antiproliferative activities of the selected analogues were determined in human leukemia K562 cells. We then identified 10 potent eIF2α phosphorylation activators with considerable anticancer activity. The most promising analogues 19 and 40 possessed higher cancer cell selectivity (SI = 6.16 and 4.83, respectively) than parent 1 (SI = 2.20). Finally, protein expression analysis revealed that compounds 19 and 40 induced eIF2α phosphorylation and its downstream effectors ATF4 and CHOP.

Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5' splice site: Generation of etoposide resistance in human leukemia K562 cells.

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5′ splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5′ splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.

6,7-Dimethoxycoumarin Influences the Erythroid Differentiation of Human Chronic Myelogenous Leukemia K562 Cells through Regulating FOXO3/p27 Signal Pathway.

Objective To study the pharmacological activity and the mechanism of action of natural compounds derived from 6,7-dimethoxycoumarin on the differentiation of human chronic myeloid leukemia K562 cells. Methods We use MTT assay (Sigma-Aldrich, USA) to detect cell viability; use flow cytometry to analyze DNA content for cell cycle analysis; use benzidine staining to synthesize hemoglobin to determine K562 cell differentiation; use western blot analysis and qPCR to detect the expression levels of FOX03, P27, CDK4, and their phosphorylation; and use the AOBS laser scanning confocal system (Leica, Wetzlar, Germany) to analyze and quantify the number of positive green spots. The statistical methods used are one-way analysis of variance (ANOVA) and Dunnett's test to analyze within and between groups. Results In order to explore the effect of 6,7-dimethoxycoumarin on the differentiation of K562 cell erythrocytes, it was concluded that 6,7-dimethoxycoumarin promotes the differentiation of K562 cell erythrocytes; the proliferation of K562 cells was detected by MTT method, and the results showed that 6,7-dimethoxycoumarin can inhibit the proliferation of K562 cells; to evaluate the effect of 6,7-dimethoxycoumarin on the proliferation of K562 cells, the results showed that 6,7-dimethoxycoumarin increased the expression of FOXO3, P27, CDK4, and CDK65, and decreased the phosphorylation of CDK4 and CDK6 proteins. To further explore the effect of knocking out FOXO3 on cell differentiation, the results show that 6,7-dimethoxycoumarin can reduce the differentiation and proliferation of K562 cells by increasing the expression of FOXO3. Conclusion This study extended the understanding of the pharmacological activity of 6,7-dimethoxycoumarin and may provide a potential new target for the treatment of chronic myelogenous leukemia. However, we still need to further study the specific molecular capabilities of 6.7 dimethylcoumarin to understand their possible capture mechanism.

Cytotoxic Activity of Aquilaria malaccensis Ethanolic Leaves Extract on Human Chronic Myeloid Leukaemia K-562 Cell Line

Aquilaria malaccensis, also known as “Pokok Karas” in Malaysia, has various benefits and widely used in Chinese herbal medicines. Natural products such as medicinal plant have been found to be effective anti-cancer agents, killing cancer cells with fewer side effects than chemotherapy. Therefore, the purpose of this study was to evaluate the cytotoxic activities of ethanolic extract of A. malaccensis on the human chronic myeloid leukaemia K-562 cell line. In this study, cytotoxic activities of ethanolic extract of A. malaccensis leaves on K-562 cells was determined by using (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) assay. K-562 cells were treated with 70% ethanolic extracts and incubated for 24, 48 and 72 hours. The half-maximal inhibitory concentration (IC50) of ethanolic extract of A. malaccensis leaves was observed at 72 hours of incubation; 90 µg/mL for 70% ethanolic extract. Moreover, the morphological changes of K-562 cells were observed by using acridine orange/ propidium iodide (AO/PI) dual staining assay. The results showed that the ethanolic extract of A. malaccensis leaves induced K-562 cells into apoptotic cell death mode manner. This finding suggested that the ethanolic extract of A. malaccensis has the potential to become an anti-cancer agent for chronic myeloid leukaemia K-562 cell line.