Erythroid Differentiation Of K562 Cells Research Articles

C-Jun targets miR-451a to regulate HQ-induced inhibition of erythroid differentiation via the BATF/SETD5/ARHGEF3 axis

Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50μM HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.

CRKL but not CRKII contributes to hemin-induced erythroid differentiation of CML.

Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.

Regulation of Erythroid Differentiation via the HIF1α-NFIL3-PIM1 Signaling Axis Under Hypoxia.

Aims: Erythropoiesis is controlled by several factors, including oxygen level under different circumstances. However, the role of hypoxia in erythroid differentiation and the underlying mechanisms are poorly understood. We studied the effect and mechanism of hypoxia on erythroid differentiation of K562 cells and observed the effect of hypoxia on early erythropoiesis of zebrafish. Results: Compared with normal oxygen culture, both hemin-induced erythroid differentiation of K562 cells and the early erythropoiesis of zebrafish were inhibited under hypoxic treatment conditions. Hypoxia-inducible factor 1 alpha (HIF1α) plays a major role in the response to hypoxia. Here, we obtained a stable HIF1α knockout K562 cell line using the CRISPR-Cas9 technology and further demonstrated that HIF1α knockout promoted hemin-induced erythroid differentiation of K562 cells under hypoxia. We demonstrated an HIF1-mediated induction of the nuclear factor interleukin-3 (NFIL3) regulated in K562 cells under hypoxia. Interestingly, a gradual decrease in NFIL3 expression was detected during erythroid differentiation of erythropoietin-induced CD34+ hematopoietic stem/progenitor cells (HSPCs) and hemin-induced K562 cells. Notably, erythroid differentiation was inhibited by enforced expression of NFIL3 under normoxia and was promoted by the knockdown of NFIL3 under hypoxia in hemin-treated K562 cells. In addition, a target of NFIL3, pim-1 proto-oncogene, serine/threonine kinase (PIM1), was obtained by RNA microarray after NFIL3 knockdown. PIM1 can rescue the inhibitory effect of NFIL3 on hemin-induced erythroid differentiation of K562 cells. Innovation and Conclusion: Our findings demonstrate that the HIF1α-NFIL3-PIM1 signaling axis plays an important role in erythroid differentiation under hypoxia. These results will provide useful clues for preventing the damage of acute hypoxia to erythropoiesis.

Abstract 5630: Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML

Abstract Acute Myeloid Leukemia (AML) represents a diverse category of blood cancers marked by the clonal proliferation of atypical myeloid precursor cells within the bone marrow. Lysine Specific Demethylase 1 (LSD1), an epigenetic regulator, exhibits elevated expression in AML. Consequently, inhibiting LSD1 has emerged as a promising therapeutic approach for addressing blood and bone marrow cancers. While initial emphasis centered on LSD1's enzymatic inhibition, compelling evidence highlights its pivotal role as a scaffolding protein, orchestrating transcriptional and chromatin remodeling complexes that control cellular differentiation and proliferation. However, our understanding of the precise mechanisms governing these complexes remains limited, necessitating further investigation. The K562 erythroleukemia cell line has served as a prominent model for investigating the molecular factors influencing cell fate determination. In response to specific cues, these cells exhibit a bi-potential capacity, mirroring the behavior of normal hematopoietic megakaryocyte/erythrocyte progenitors (MEP). Notably, when LSD1 is either depleted or inhibited, the erythroid differentiation of K562 cells is hindered, even when exposed to various pro-differentiating triggers, including the BCR-ABL inhibitor Imatinib. In order to gain deeper insights and identify potential new protein partners involved in the K562 differentiation process that interact with LSD1, we employed the erythroleukemic K562 cell model. Using unbiased, proteome-wide proximity labeling we discovered that LSD1 serves as a scaffold for recruitment of the ChAHP complex, comprised of ADNP, CBX3, and CHD4. The ChAHP complex serves fundamental roles in chromatin remodeling, RNA splicing, and transcriptional control. We confirmed these proximity relationships by immunoblotting biotinylated proteins generated in situ by the promiscuous biotin ligase, (BirA*) when anchored to LSD1. We show that depleting ADNP induces spontaneous erythroid differentiation in K562 erythroleukemia cells, which is augmented by BCR-ABL inhibitor Imatinib treatment. ADNP-deficient K562 cells exhibited accelerated erythroid differentiation, as evidenced by a four-fold increase in the surface expression of erythroid markers (CD71, CD235a) and a significant upregulation of beta-globin gene cluster markers. These findings collectively suggest an antagonistic functional relationship between ADNP and LSD1, potentially counterbalancing LSD1's role in lineage allocation. Furthermore, we demonstrate that the loss of the key ChAHP component, ADNP, substantially hampers proliferation and increases apoptosis in K562 cells. These results underscore the potential of ADNP/ChAHP as an appealing target to modulate the choice between erythroid and megakaryocytic fates and offer promise as a target for pro-differentiation therapies in AML Citation Format: Mehraju Din Lone, Mohd Sayeed, Alaa Habieb, Praveen Kumar, Michael E. Engel. Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5630.

Saracatinib prompts hemin-induced K562 erythroid differentiation but suppresses erythropoiesis of hematopoietic stem cells.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

Long Non-Coding RNA H19 Leads to Upregulation of γ-Globin Gene Expression during Erythroid Differentiation

Long noncoding RNAs (lncRNAs) are important because they are involved in a variety of life activities and have many downstream targets. Moreover, there is also increasing evidence that some lncRNAs play important roles in the expression and regulation of γ-globin genes. In our previous study, we analyzed genetic material from nucleated red blood cells (NRBCs) extracted from premature and full-term umbilical cord blood samples. Through RNA sequencing (RNA-Seq) analysis, lncRNA H19 emerged as a differentially expressed transcript between the two blood types. While this discovery provided insight into H19, previous studies had not investigated its effect on the γ-globin gene. Therefore, the focus of our study was to explore the impact of H19 on the γ-globin gene. In this study, we discovered that overexpressing H19 led to a decrease in HBG mRNA levels during erythroid differentiation in K562 cells. Conversely, in CD34+ hematopoietic stem cells and human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, HBG expression increased. Additionally, we observed that H19 was primarily located in the nucleus of K562 cells, while in HUDEP-2 cells, H19 was present predominantly in the cytoplasm. These findings suggest a significant upregulation of HBG due to H19 overexpression. Notably, cytoplasmic localization in HUDEP-2 cells hints at its potential role as a competing endogenous RNA (ceRNA), regulating γ-globin expression by targeting microRNA/mRNA interactions.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40μM hemin for 0-120h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40μM HQ for 72h, K562 cells were induced with 40μM hemin for 48h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and β-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Induction of the Erythroid Differentiation of K562 Cells Is Coupled with Changes in the Inter-Chromosomal Contacts of rDNA Clusters.

The expression of clusters of rDNA genes influences pluripotency; however, the underlying mechanisms are not yet known. These clusters shape inter-chromosomal contacts with numerous genes controlling differentiation in human and Drosophila cells. This suggests a possible role of these contacts in the formation of 3D chromosomal structures and the regulation of gene expression in development. However, it has not yet been demonstrated whether inter-chromosomal rDNA contacts are changed during differentiation. In this study, we used human leukemia K562 cells and induced their erythroid differentiation in order to study both the changes in rDNA contacts and the expression of genes. We observed that approximately 200 sets of rDNA-contacting genes are co-expressed in different combinations in both untreated and differentiated K562 cells. rDNA contacts are changed during differentiation and coupled with the upregulation of genes whose products are mainly located in the nucleus and are highly associated with DNA- and RNA-binding, along with the downregulation of genes whose products mainly reside in the cytoplasm or intra- or extracellular vesicles. The most downregulated gene is ID3, which is known as an inhibitor of differentiation, and thus should be switched off to allow for differentiation. Our data suggest that the differentiation of K562 cells leads to alterations in the inter-chromosomal contacts of rDNA clusters and 3D structures in particular chromosomal regions as well as to changes in the expression of genes located in the corresponding chromosomal domains. We conclude that approximately half of the rDNA-contacting genes are co-expressed in human cells and that rDNA clusters are involved in the global regulation of gene expression.

Regulation of erythroid differentiation in K562 cells by the EPAS1-IRS2 axis under hypoxic conditions.

Red blood cells (RBCs) produced in vitro have the potential to alleviate the worldwide demand for blood transfusion. Hematopoietic cell differentiation and proliferation are triggered by numerous cellular physiological processes, including low oxygen concentration (<5%). In addition, hypoxia inducible factor 2α (HIF-2α) and insulin receptor substrate 2 (IRS2) were found to be involved in the progression of erythroid differentiation. However, the function of the HIF-2α-IRS2 axis in the progression of erythropoiesis is not yet fully understood. Therefore, we used an in vitro model of erythropoiesis generated from K562 cells transduced with shEPAS1 at 5% O2 in the presence or absence of the IRS2 inhibitor NT157. We observed that erythroid differentiation was accelerated in K562 cells by hypoxia. Conversely, knockdown of EPAS1 expression reduced IRS2 expression and erythroid differentiation. Intriguingly, inhibition of IRS2 could impair the progression of hypoxia-induced erythropoiesis without affecting EPAS1 expression. These findings indicated that the EPAS1-IRS2 axis may be a crucial pathway that regulates erythropoiesis and that drugs targeting this pathway may become promising agents for promoting erythroid differentiation.

5613423 TREATMENT OF ERYTHROID PRECURSOR CELLS FROM β-THALASSEMIC PATIENTS WITH ISOXAZOLE DERIVATIVES: POTENT INDUCTION OF FETAL HEMOGLOBIN

Background: Increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients and several clinical trials in β-thalassemia and/or sickle-cell disease (SCD) are ongoing using HbF inducers, such as NCT01245179 (based on the HDAC inhibitor Panobinostat), NCT00790127 (based on 2,2-dimethylbutyrate, HQK-1001) and NCT03877809 (based on the mTOR inhibitor sirolimus). Methods: In this context, new molecules able to induce HbF are needed. In this communication, we have studied in deep a new class of promising HbF inducers characterized by an isoxazole chemical skeleton. These derivatives are 3,4-isoxazolediamide compounds recently synthesized in our laboratories. The original structures of two natural molecules, geldanamycin and radicicol, known to be natural Hsp (Heat Shot Protein) inhibitors, were modified to lead to a novel class of synthetic compounds, containing the isoxazole nucleus, and displaying potent and selective inhibition of Hsp90. Here we used erythroid precursors cells (ErPCs) isolated from β-thalassemic patients and treated with appropriate concentrations of 8 isoxazole derivatives previously demonstrated to be able to induce erythroid differentiation of K562 cells. Results: We demonstrated the high efficacy of the analyzed isoxazoles in inducing HbF. Some derivatives demonstrated an activity even higher than other HbF inducers used as positive controls, such as Hydroxyurea (HU) or Sirolimus. Most of the isoxazole derivatives displayed high effects in decreasing the excess of free α-globin chains in treated ErPCs. All the compounds were assayed for genotoxicity through the Ames test, employing the histidine-requiring Salmonella typhimurium mutant TA 97A, TA98, TA100 and TA1535 strains that allow to check frameshift mutation and base-pair substitution, with and without metabolic activation induced by S9 Mix. These assays excluded any drawbacks related to genotoxicity. Summary - Conclusion: We may therefore conclude that these novel isoxazoles could be proposed for clinical studies for possible therapeutic application for the treatment of β-thalassemia. These compounds have been recently patented as potential therapeutic agents for β-thalassemia (US Patent 11077116). Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z), by AIFA (AIFA-2016-02364887) and by the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thank A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo.

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.

In Vitro and In Vivo Studies for the Investigation of γ-Globin Gene Induction by Adhatoda vasica: A Pre-Clinical Study of HbF Inducers for β-Thalassemia.

Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of β-thalassemia. In this study, we have evaluated the HbF induction ability of A. vasica in vitro and in vivo, and the identification of potential therapeutic compounds through a bioassay-guided approach. In vitro benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by A. vasica extracts. Subsequently, an in vivo study with an aqueous extract of A. vasica (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of A. vasica. Following the bioassay-guided approach, two isolated compounds, vasicinol (1) and vasicine (2) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound 1 (0.1 µM) and compound 2 (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (1) and (2) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of A. vasica for fostering HbF production in clinical implications for blood disorders.

ETV6 Regulates Hemin-Induced Erythroid Differentiation of K562 Cells through Mediating the Raf/MEK/ERK Pathway.

As a member of transcription factor E-Twenty Six (ETS) family, ETS variant 6 (ETV6) plays significant role in hematopoiesis and embryonic development. ETV6 dysexpression also involved in the occurrence, development and progression of cancers and leukemia. In current work, we hypothesized that ETV6 plays a role in erythroid differentiation of chronic myeloid leukemia (CML). We found the protein expression level of ETV6 was significantly upregulated during hemin-induced erythroid differentiation of K562 cells. Moreover, overexpression of ETV6 inhibited erythroid differentiation in hemin-induced K562 cells with decreased numbers of benzidine-positive cells and decreased expression levels of erythroid differentiation specific markers glycophorin (GPA), CD71, hemoglobin A (HBA), α-globin, γ-globin and ε-globin. Conversely, ETV6 knockdown promoted erythroid differentiation in hemin-induced K562 cells. Furthermore, ETV6 expression level slightly positively with the proliferation capacity of K562 cells treated with hemin. Mechanistically, ETV6 overexpression inhibited fibrosarcoma/mitogen activated extracellular signal-regulated kinase/extracellular regulated protein kinase (Raf/MEK/ERK) pathway, ETV6 knockdown activated the Raf/MEK/ERK pathway. Collectively, the current work demonstrates that ETV6 plays an inhibitory role in the regulation of K562 cell erythroid differentiation via Raf/MEK/ERK pathway, it would be a potentially therapeutic target for dyserythropoiesis.

Effects of Oral Intake of Olive Leaf Extract on Hematological Parameters: A Double-Blinded, Randomized, Placebo-Controlled Trial in Progress

Background:Anemia is one of the major public health problems that affected 2.36 billion individuals worldwide in 2015. Half of all causes of anemia are iron or other nutrient deficiency anemia (WHO, 2015). Currently available treatment options for anemia include iron or other nutrient supplementation, nutritional interventions in the form of food fortification, blood transfusion, and erythropoietic agent infusion. However, conventional treatments are not without adverse effects. In this context, medicinal plants with bioactive components have been receiving considerable attention from researchers as natural hematinic agents complemented with or alternatives to conventional treatment.Olive leaf (Olea europaea L. Oleaceae) extract (OLE) is rich in bioactive compounds and have been reported to have several health benefits (Gorzynik-Debicka et al., 2018). Previously we showed that apigetrin and apigenin, components of OLE, induced erythroid differentiation in K562 cells (Tsolmon et al., 2011 and Isoda et al., 2014). Also, OLE and its phenolic compounds induced erythroid differentiation in hHSCs (Kondo et al., 2021, Samet et al., 2015).Additionally, we have reported that long-term consumption of OLE tea increased RBC count, Hb%, and hematocrit (Ht) in female volunteers aged between 40 - 70 years old (Ferdousi et al., 2019). However, none of the participants were anemic at the baseline. Therefore, in the present study, we aimed to investigate the effects of oral intake of OLE on hematological parameters in healthy to mildly anemic young adults.Study Design and MethodsA double-blinded, randomized, placebo-controlled study will be conducted in Bangladesh. Total 165 apparently healthy adults with Hb level at least 10g/dl for females (nonpregnant, non-breastfeeding) and 11g/dl for males, and aged between 18 to 59 years will be enrolled from the local community. Major exclusion criteria will include currently pregnant women or breastfeeding mothers, moderate to severe anemia (Hb concentration < 10 g / dL for women and < 11 g / dL for men), any medical condition, such as hematological diseases, malignant tumors, aplastic anemia, uncontrolled hypertension, chronic kidney disease, irritable bowel syndrome, and cardiovascular diseases, and currently taking medicine for diabetes or hypertension and taking regular iron or other supplements, etc.After providing informed written consent, study participants will be randomly assigned to placebo, 250 mg OLE and 500 mg OLE groups. Participants will take OLE or placebo capsules in two divided doses every day for 12 weeks. Hematological parameters, Hb concentration, RBC count, WBC count, Ht, MCV, MCH, MCHC, serum Fe and ferritin levels will be investigated at baseline and after 6 and 12 weeks of intervention. Liver and renal function tests will be conducted for safety assessment. Additionally, lipid profile will be investigated to understand how lipid parameters will be regulated if anemia condition is improved by OLE intervention (Araki et al., 2019). Given that fatigue is one of the most common symptoms of anemia, we will also evaluate the effect of OLE on the score of each item of the Fatigue Assessment Scale (FAS) questionnaire during the intervention.The analysis will be carried out on an intention-to-treat basis involving all the participants who will consume the test samples at least once. We will replace the missing data from dropouts using the baseline observation carried forward method. ANOVA with Bonferroni's post hoc test will be used for normally distributed data; otherwise, the Mann-Whitney U test or Kruskal-Wallis H(K) test will be used. Categorical variables will be compared by the Chi-square or Fisher's exact test. A one-way repeated-measures ANOVA with Bonferroni's post hoc test (Within-group) and ANOVA followed by Bonferroni's post hoc test (between-group) will be performed. Significance is at p < 0.05.This protocol is registered with UMIN-CTR Clinical Trial (UMIN000039023), and has been approved by University of Tsukuba Hospital Clinical Research Ethics Review Committee (R01-292) and the Ethical Review Committee of icddr,b (PR-21017). This study is funded by the Japan Society for the Promotion of Science (19K20106) and partly by the Japan Science and Technology Agency-SATREPS (JPMJSA1506). DisclosuresSuidasari: Nutrition Act Co. Ltd.: Current Employment. Yokozawa: Nutrition Act Co. Ltd.: Current Employment. Yamauchi: Nutrition Act Co. Ltd.: Current Employment.

MS4A3-HSP27 target pathway reveals potential for haematopoietic disorder treatment in alimentary toxic aleukia.

Alimentary toxic aleukia (ATA) is correlated with consuming grains contaminated by Fusarium species, particularly T-2 toxin, which causes serious hurt to human and animal health, chiefly in disorders of the haematopoietic system. However, the mechanism of haematopoietic dysfunction induced by T-2 toxin and the possible target pathway for the treatment of T-2 toxin-induced haematopoietic disorder of ATA remains unclear. In this study, genomes and proteomics were used for the first time to investigate the key differential genes and proteins that inhibit erythroid differentiation of K562 cells caused by T-2 toxin, and it was found that heat shock protein 27 (HSP27) and membrane-spanning 4-domains, subfamily A, member 3 (MS4A3) may play an important role in erythroid differentiation. Meanwhile, MS4A3 interference can inhibit the occurrence of erythroid differentiation of K562 cells and promote the phosphorylation of HSP27. Moreover, the binding of HSP27 to MS4A3 in natural state can activate the phosphorylation site of HSP27 (Ser-83), while T-2 toxin can abolish the activation of phosphorylation site by inhibiting the expression of MS4A3. These findings for the first time demonstrated that the MS4A3-HSP27 pathway may function an efficient therapeutic target pathway for treating T-2 toxin elicited haematopoietic disorders of ATA.

Effects of miR‑210‑3p on the erythroid differentiation of K562 cells under hypoxia.

GATA binding protein 1 (GATA-1) is one of the most important hematopoietic transcription factors in the production of blood cells, such as platelets, eosinophils, mast cells and erythrocytes. GATA-1 regulates the participation of microRNA (miRNAs/miRs) in erythroid differentiation under normoxia. However, GATA-1 expression and the regulation of miR-210-3p in the context of erythroid differentiation under hypoxia remain unknown. The present study examined the expression levels of GATA-1 and miR-210-3p in the model of erythroid differentiation in K562 cells under hypoxia, and determined the effects of GATA-1, miR-210-3p and SMAD2 on erythroid differentiation through lentivirus transfection experiments. The present study detected increased GATA-1 expression under hypoxia. Moreover, miR-210-3p was identified as a positive regulator of erythroid differentiation, which was upregulated both during erythroid differentiation and in GATA-1 overexpression experiments under hypoxia. Importantly, in the K562 cell model of erythroid differentiation under hypoxia, miR-210-3p was upregulated in a GATA-1-dependent manner. Using a double luciferase reporter assay, miR-210-3p was identified as a downstream target of GATA-1-mediated regulation of erythropoiesis. Gain- or loss-of-function analysis of miR-210-3p identified its importance in erythroid differentiation. Furthermore, it was found that SMAD2 may be a downstream target gene for miR-210-3p. Bioinformatics predictions suggested that SMAD2 mediated miR-210-3p-induced regulation of erythroid differentiation. Collectively, the present study provides novel insights into the miRNA regulation of erythroid differentiation.

Effect of MiR-451a on Erythroid Differentiation of K562 Cells under Hypoxia

To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ. K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a. The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05). Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.

Monoterpenes as therapeutic candidates to induce fetal hemoglobin synthesis and up-regulation of gamma-globin gene: An in vitro and in vivo investigation

Pharmacologically induced production of fetal hemoglobin (HbF) is a pragmatic therapeutic strategy for the reduction of globin chain imbalance and improving the clinical severities of patients with β-hemoglobinopathies. To identify highly desirable new therapeutic HbF-inducing agents, we screened functionally diverse ten monoterpenes, as molecular entities for their potent induction and erythroid differentiation ability in human erythroleukemia cell line (K562) and transgenic mice. Benzidine hemoglobin staining demonstrated six compounds to have significantly induced erythroid differentiation of K562 cells in a dose and time-dependent manner. This induction paralleled well with the optimal accumulated quantity of total hemoglobin in treated cultures. The cytotoxic studies revealed that three (carvacrol, 3-carene, and 1,4-cineole) of the six compounds with their maximal erythroid expansion ability did not affect cell proliferation and were found non-toxic. Four compounds were found to have high potency, with 4-8-fold induction of HbF at both transcriptional and protein levels in vitro. Subsequently, an in vivo study with the three active non-cytotoxic compounds showed significant overexpression of the γ-globin gene and HbF production. Carvacrol emerged as a lead HbF regulator suggested by the increase in expression of γ-globin mRNA content (5.762 ± 0.54-fold in K562 cells and 5.59 ± 0.20-fold increase in transgenic mice), accompanied by an increase in fetal hemoglobin (F-cells) levels (83.47% in K562 cells and 79.6% in mice model). This study implicates monoterpenes as new HbF inducing candidates but warrants mechanistic elucidation to develop them into potential therapeutic drugs in β-thalassemia and sickle cell anemia.

MiR-486-5p-directed MAGI1/Rap1/RASSF5 signaling pathway contributes to hydroquinone-induced inhibition of erythroid differentiation in K562 cells

Bone marrow failure is a characteristic effect of benzene exposure. Our previous study has shown that miR-486-5p is involved in benzene induced-suppression of erythroid differentiation. However, the mechanism of miR-486-5p to initiate the above process remains unclear. In this study, we used miRTar software to predict putative miRNA targets and pathway. We found that miR-486-5p may target Ras-associated protein-1 (Rap1) signaling pathway-associated genes. Our in vitro study further showed significant dose-dependent upregulation of MAGI1 and RASSF5 expressions in hydroquinone (HQ)-induced suppression of erythroid differentiation of K562 cells. Over-expression or down-regulation of miR-486-5p altered MAGI1 and RASSF5 expression and modified erythroid differentiation. Dual-luciferase reporter assay and fluorescence-based RNA electrophoresis mobility assay (FREMSA) further confirmed that miR-486-5p directly bound to the 3′-untranslated region (3’-UTR) of MAGI1 and RASSF5. In addition, the expressions of RAPGEF2 and RAP1A, which are downstream genes of MAGI1, were also significantly increased when HQ inhibited erythroid differentiation. Knockdown of MAGI1 reversed HQ-induced inhibition of erythroid differentiation via downregulation of RAPGEF2, RAP1A and RASSF5. Together, these data indicate that miR-486-5p directly targets MAGI1 and RASSF5 and integrates with Rap1 signaling to modify HQ-induced inhibition of erythroid differentiation in K562 cells.