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IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

LL-37 can inhibit the growth of K562 cancer cells when it is conjugated with iron oxide nanoparticles. In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method and then modified with the LL-37 peptide through an NH2 bridge. The accuracy of the synthesis process was confirmed through various analytical tests, including FTIR, XRD, FESEM, and EDX. To assess the treatment's effectiveness, a viability test was carried out on K562 leukemia cells and normal peripheral blood mononuclear cells. In addition, flow cytometry and Hoechst staining were used to investigate the mechanism of action of the drug. The expression levels of the Bcl-2, Bax, and TP53 genes in the treated cells and the control group were measured using qRT-PCR. The results indicated that the size of the nanoparticles ranged between 34 and 40 nm. The NH2@LL-37@Fe3O4 nanoparticles more effectively inhibited the growth of cancer cells in a concentration-dependent manner, as compared to Fe3O4 alone. Further analysis revealed that apoptosis occurred through increased expression of TP53 and Bax genes compared to the Bcl-2 gene. Therefore, induction of apoptosis and inhibition of growth in K562 cells was attributed to the impact of iron oxide magnetic nanoparticles conjugated with the LL-37 peptide through the TP53/Bax/Bcl-2 pathway. [ABSTRACT FROM AUTHOR]

Abstract LL-37 can inhibit the growth of K562 cancer cells when it is conjugated with iron oxide nanoparticles. In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method and then modified with the LL-37 peptide through an NH2 bridge. The accuracy of the synthesis process was confirmed through various analytical tests, including FTIR, XRD, FESEM, and EDX. To assess the treatment's effectiveness, a viability test was carried out on K562 leukemia cells and normal peripheral blood mononuclear cells. In addition, flow cytometry and Hoechst staining were used to investigate the mechanism of action of the drug. The expression levels of the Bcl-2, Bax, and TP53 genes in the treated cells and the control group were measured using qRT-PCR. The results indicated that the size of the nanoparticles ranged between 34 and 40 nm. The NH2@LL-37@Fe3O4 nanoparticles more effectively inhibited the growth of cancer cells in a concentration-dependent manner, as compared to Fe3O4 alone. Further analysis revealed that apoptosis occurred through increased expression of TP53 and Bax genes compared to the Bcl-2 gene. Therefore, induction of apoptosis and inhibition of growth in K562 cells was attributed to the impact of iron oxide magnetic nanoparticles conjugated with the LL-37 peptide through the TP53/Bax/Bcl-2 pathway.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the role and mechanism of IL-39 in K562 cells. Methods: The expressions of IL-39R and STAT1/STAT3 in K562 cells were detected via qRT-PCR； the protein levels of phospho-STAT1/STAT3 and STAT1/STAT3 in K562 cells were detected by Western blot； the mRNA regulated by IL-39 in K562 cells was predicted by RNA sequencing. Results: rIL-39 significantly promoted the expression of IL-39R in K562 cells； IL-39 affected K562 cells through STAT1 pathway； IL-39 significantly regulated the expression of mRNA in K562 cells， thus potentially affecting a series of biological processes. Conclusion: IL-39 can directly act on K562 cells， alter gene transcriptional expression and inhibit tumor growth through the STAT1 pathway. IL-39 may similarly modulate gene expression profile in human leukemia cells. [ABSTRACT FROM AUTHOR]

Introduction: The Vel– phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin. [ABSTRACT FROM AUTHOR]

Oncological diseases represent a significant global health challenge, with high mortality rates. Early detection is crucial for effective treatment, and aptamers, which demonstrate superior specificity and stability compared to antibodies, offer a promising avenue for diagnostic advancement. This study presents the design, development and evaluation of a quartz crystal microbalance (QCM) sensor functionalized with the T2-KK1B10 aptamer for the sensitive and specific detection of Chronic Myeloid Leukemia (CML) K562 cells. The research focuses on optimizing the biorecognition layer by adjusting the aptamer conditions, demonstrating the sensor's ability to detect these CML cells with high specificity and sensitivity. The aptamer-modified QCM sensor operates on the principle of mass change detection upon binding of target cells. By employing the Langmuir isotherm model, the performance of the sensor was optimized for the capture of CML cells from biological samples with LOD of 263 K562 cells. The sensor was also successfully regenerated multiple times without sensitivity loss. Validation of the sensor's performance was conducted under controlled laboratory settings, followed by extensive testing utilizing human lyophilized plasma and clinical samples from patients. The sensor exhibited high sensitivity and specificity in the detection of CML cells within clinical specimens, thereby illustrating its potential for practical clinical deployment. This research presents a novel approach to the early diagnosis of CML, facilitating timely intervention and enhanced patient outcomes. The developed aptasensor demonstrates potential for broader application in cancer diagnostics and personalized medicine.

Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. In this study, we treated K562 cells with 40 μmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification. [ABSTRACT FROM AUTHOR]

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) of HLA‐E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA‐E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti‐tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA‐E mRNA transcription and membrane HLA‐E (mHLA‐E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA‐E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA‐E (sHLA‐E) was not influenced by both rs1264457 and rs76971248. The higher HLA‐E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA‐E molecules played a significant inhibitory effect on the killing activity of NK‐92MI cells (p < 0.05). In conclusion, the higher HLA‐E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK‐92MI cells' killing. [ABSTRACT FROM AUTHOR]

Background: Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. Aims: This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. Methods and results: After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48 h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). Conclusion: Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC. [ABSTRACT FROM AUTHOR]

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings.

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells. • Shrimp non-adherent, lymphocyte-like haemocytes have a cytotoxic activity against xenogenous mammalian and insect cells. • Shrimp non-adherent, lymphocyte-like haemocytes have a cytotoxic activity against homogenous WSSV-infected shrimp cells. • During killing activity, mRNA and protein expression of cytotoxic molecules in shrimp lymphocyte-like haemocytes increased. [ABSTRACT FROM AUTHOR]

Proteolysis targeting chimeras (PROTAC) are bifunctional chimeric molecules capable of directly degrading binding proteins through the ubiquitin-proteasome pathway. PROTACs have demonstrated significant potential in overcoming drug resistance and targeting previously untreatable targets. However, several limitations still need to be addressed, including their high molecular weight resulting in poor membrane permeability and bioavailability. In this study, we proposed that cancer-targeted penetrating peptides could enhance the cell permeability of PROTACs. We developed 26 novel targeted penetrating peptides for leukemia and lymphoma cells, among which C9C-f(3Bta) and Cyclo-C9C-R exhibited superior membrane permeability, targetability, and stability. By combining C9C-f(3Bta) and Cyclo-C9C-R with IMA-PROTAC, we effectively enhanced the anti-proliferative activity of IMA-PROTAC, facilitated degradation of Bcr-Abl protein in K562 cells, and reduced downstream STAT5 phosphorylation. Furthermore, the combined application promoted cell apoptosis while blocking G1 phase progression. HPLC-MRM-MS revealed that the combination of C9C-f(3Bta) or Cyclo-C9C-R with IMA-PROTAC significantly enhanced intracellular IMA-PROTAC content. In summary, our proof-of-concept study validated the hypothesis that combining PROTACs with targeted penetrating peptides can improve protein degradation efficiency as well as anti-proliferative capabilities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Venetoclax is a BH3 mimetic that was recently approved for the treatment of acute myeloid leukemia (AML) treatment. However, the effect of venetoclax on AML remains limited, and a novel strategy is required. Here, we demonstrate for the first time that the cytotoxic effect of venetoclax drastically increased when by combined with the naturally occurring flavonoid quercetin. Combined treatment with venetoclax and quercetin caused most of AML KG-1 cells to exhibit a condensed morphology. Cell cycle analysis revealed that the combination strongly induced cell death. Caspase inhibitor blocked this cell death, and the combination induced poly (ADP-ribose) polymerase (PARP) cleavage, indicating that apoptosis was the primary mechanism. These effects were also observed in another AML cell line Kasumi-1 but not in chronic myeloid leukemia (CML) K562 cells. Public data analysis demonstrated that B-cell/CLL lymphoma 2 (Bcl-2) expression is increased in AML cells compared to other malignant tumors, and the survival and the growth of AML cell line depends on Bcl-2. We found that quercetin increased Bcl-2-associated X protein (Bax) expression in KG-1. Our study provides a novel function for quercetin and presents a promising strategy for AML treatment using venetoclax., (© 2024. The Author(s).)

Background: Chronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic., Purpose: The identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML., Methods: Transcriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562 T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry., Results: Our multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562 T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death., Conclusion: This study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Erythropoiesis is a multistep process of erythroid cell production that is controlled by multiple regulatory factors. Ribosome rescue factor PELO plays a crucial role in cell meiotic division and mice embryonic development. However, the function of PELO in erythroid differentiation remains unclear. Here, we showed that knockdown of PELO increased hemin-induced erythroid differentiation of K562 and HEL cells, exhibiting a higher number of benzidine-positive cells and increased mRNA levels of erythroid genes. PELO knockdown inhibited the proliferation and cell cycle progression and promoted apoptosis of K562 cells. Mechanistically, PELO could regulate the expression of KLF10 through interaction with MYC. Moreover, KLF10 knockdown also enhanced erythroid differentiation of K562 and HEL cells induced by hemin. Collectively, our results demonstrated that PELO regulates erythroid differentiation and increases KLF10 expression levels by interacting with MYC., (© 2024 Federation of European Biochemical Societies.)

The design of novel chelators for therapeutic applications has been the subject of extensive research to address various diseases. Many chelators can manipulate the levels of metal ions within cells and effectively modulate the metal excess. In some cases, chelators show significant toxicity to cells. We investigated polyimidazole ligands by potentiometry and UV-Vis spectroscopy for their ability to form copper(II) complexes. We also compared the antiproliferative activity of the polyimidazole ligands and their copper(II) complexes with polypyridine ligands in CaCo-2 (colorectal adenocarcinoma), SH-SY5Y (neuroblastoma) and K562 (chronic myelogenous leukemia) cells and normal HaCaT (keratinocyte) cells. Polyimidazole ligands are less cytotoxic than their analogous polypyridine ligands. All polyimidazole ligands, except the tetraimidazole ligand for K562 cells, did not show any significant effect on the viability of cancer and normal cells. In contrast, the cytotoxic activity of polypiridine ligands was also observed in normal cells with IC 50 values similar to those of cancer cells. Tetraimidazole ligand, the only ligand active on the leukemic K562 cell line, induced caspase-dependent apoptosis and increased intracellular reactive oxygen species production with mitochondrial damage. The low cytotoxicity of the polyimidazole ligands, even if it limits their use as anticancer agents, could make them useful in other medical applications, such as in the treatment of metal overload, microbial infections, inflammation or neurodegenerative disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Graziella Vecchio reports financial support was provided by Italian Ministry of Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Different vanillin-based aldehydes were used to synthesize novel tetrahydropyrimidines (THPMs) via conventional Biginelli reaction. The THPMs were tested against human normal cells (MRC-5) and cancer cell lines (HeLa, K562, and MDA-MB-231). With IC 50 values of 10.65, 10.70, and 12.76 µM, compounds 4g, 4h, and 4i exerted the strongest cytotoxic effects against K562 cells. The best activity was achieved for 4g on MDA-MB-231 cells (IC 50  = 9.20 ± 0.14 µM). The effects of compounds 4g, 4h, and 4i on the cell-cycle phase distribution of K562 cells were analyzed. Principal component analysis was carried out for the chemometrics analysis to comprehend the relationship between the anticancer activity of the THPMs, pharmacokinetic properties, and partition coefficients, as well as the relationship between the chromatographic behavior and retention parameters. The highest retention rates are found for molecules 4g, 4h, and 4i, which have the longest carbon chains, indicating that the length of the alkyl chain positively affects the molecule's anticancer activity but only if the number of carbon atoms is not higher than seven. Additionally, molecular docking analysis was performed to determine the preferred binding modes of the investigated ligands (4g, 4h, and 4i) with a DNA dodecamer and bovine serum albumin., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Tetrahydropyrimidine (compound A = methyl 4-[4'-(heptyloxy)-3'-methoxyphenyl]-1,6-dimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate) was chosen for in vivo studies after exhibiting noteworthy in vitro activity against the K562 and MDA-MB-231 cell lines, with IC 50 values of 9.20 ± 0.14 µM and 12.76 ± 1.93 µM, respectively. According to experimental (fluorescence titration, viscosity, and differential scanning calorimetry) results, A interacts with DNA via the minor groove. In vivo, acute oral toxicity studies in Wistar albino rats proved no noticeable symptoms of either toxicity or death during the follow-up period. Genotoxic and antigenotoxic studies at three different concentrations of A (5, 10, and 20 mg/kg of body weight) in Wistar albino rats showed that the dose of 5 mg/kg body weight did not cause DNA damage and had a remarkable DNA protective activity against CCl 4 -induced DNA damage, with a percentage reduction of 78.7%. It is also important to note that, under the investigated concentrations of A, liver damage is not observed. Considering all experimental outcomes realized under various in vivo investigations (acute oral toxicity, genotoxicity, antigenotoxicity, and biochemical tests), compound A could be a promising candidate for further clinical testing., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations., Competing Interests: Competing interests statement:B.C.-B., X.L., and A.P.L. are inventors of a US patent application related to this work (US Patent App. 18/353,005, 2024).

Herein we report on the design, synthesis and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists. The structural prerequisites that shape both physicochemical and immunomodulatory profiles of desmuramylpeptide NOD2 agonists have been delineated. Within this context, we identified 3 , a butyrylated desmuramylpeptide, as a potent in vitro NOD2 agonist (EC 50 = 4.6 nM), exhibiting an almost 17-fold enhancement in potency compared to its unsubstituted counterpart 1 (EC 50 = 77.0 nM). The novel set of desmuramylpeptides demonstrate unique in vitro immunomodulatory activities. They elicited cytokine production in peripheral blood mononuclear cells (PBMCs), both alone and in conjunction with lipopolysaccharide (LPS). The spermine-decorated 32 also stimulated the LPS-induced cytotoxic activity (2.95-fold) of PBMCs against K562 cancer cells. Notably, the cholesterol-conjugate 26 displayed anti-inflammatory actions, highlighted by its capacity to convert the inflammatory monocyte subset into an anti-inflammatory phenotype. Finally, the eicosapentaenoylated derivative 23 augmented antigen presentation by mouse bone marrow-derived dendritic cells (BMDCs), thus highlighting its potential as a vaccine adjuvant.

Background: As an important downstream effector of various signaling pathways, mTOR plays critical roles in regulating many physiological processes including erythropoiesis. It is composed of two distinct complexes, mTORC1 and mTORC2, which differ in their components and downstream signaling effects. Our previous study revealed that the inhibition of mTORC1 by rapamycin significantly repressed the erythroid progenitor expansion in the early stage but promoted enucleation and mitochondria clearance in the late stage of erythroid differentiation. However, the particular roles and differences of mTORC1 and mTORC2 in the regulation of erythropoiesis still remain largely unknown. In the present study, we investigated the comparative effects of dual mTORC1/mTORC2 mTOR kinase inhibitor AZD8055 and mTORC1 inhibitor rapamycin on erythroid differentiation in K562 cells induced by hemin and erythropoiesis in β-thalassemia mouse model. Materials and Methods: In vitro erythroid differentiation model of hemin-induced K562 cells and β-thalassemia mouse model were treated with AZD8055 and rapamycin. Cell Counting Kit-8 was used to detect cell viability. The cell proliferation, cell cycle, erythroid surface marker expression, mitochondrial content, and membrane potential were determined and analyzed by flow cytometry and laser scanning confocal microscopy. Globin gene expression during erythroid differentiation was measured by RT-qPCR. The mTORC2/mTORC1 and autophagy pathway was evaluated using western blotting. Results: Both AZD8055 and rapamycin treatments increased the expression levels of the erythroid differentiation-specific markers, CD235a, α -globin, γ -globin, and ε -globin. Notably, AZD8055 suppressed the cell proliferation and promoted the mitochondrial clearance of hemin-induced K562 cells more effectively than rapamycin. In a mouse model of β -thalassemia, both rapamycin and AZD8055 remarkably improve erythroid cell maturation and anemia. Moreover, AZD8055 and rapamycin treatment inhibited the mTORC1 pathway and enhanced autophagy, whereas AZD8055 enhanced autophagy more effectively than rapamycin. Indeed, AZD8055 treatment inhibited both mTORC2 and mTORC1 pathway in hemin-induced K562 cells. Conclusion: AZD8055 is more effective than rapamycin in inhibiting proliferation and promoting mitochondrial clearance in erythroid differentiation, which might provide us one more therapeutic option other than rapamycin for ineffective erythropoiesis treatment in the future. These findings also provide some preliminary information indicating the roles of mTORC1 and mTORC2 in erythropoiesis, and further studies are necessary to dissect the underlying mechanisms., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Qian Liu et al.)

Platelets are essential blood components that maintain hemostasis, prevent excessive bleeding, and facilitate wound healing. Reduced platelet counts are implicated in various diseases, including leukemia, hepatitis, cancer, and Alzheimer's disease. Enhancing megakaryocytic differentiation is a promising strategy to increase platelet production. Compound K (CK), a major bioactive metabolite of ginsenosides from Panax ginseng , has demonstrated anti-cancer and neuroprotective properties. In this study, we investigated the effects of CK on megakaryocytic differentiation and apoptosis in chronic myeloid leukemia (CML) cell lines K562 and Meg-01. CK treatment significantly upregulated the mRNA expression of key megakaryocytic differentiation markers, including CD61, CD41, and CD42a, and promoted the formation of large, multinucleated cells in K562 cells. Additionally, flow cytometry analysis revealed that CK at 5 µM induced apoptosis, a critical process in thrombocytopoiesis, in both K562 and Meg-01 cells. RT 2 Profiler PCR array analysis further identified a marked increase in the expression of genes associated with the activation of the NLRP3 inflammasome in CK-treated K562 and Meg-01 cells. This study is the first to demonstrate that CK promotes megakaryocytic differentiation and apoptosis through the activation of the ERK/EGR1 and NLRP3 inflammasome pathways. These findings suggest that CK may enhance platelet production, indicating its potential as a therapeutic candidate for platelet-related disorders and other associated diseases.

Objective: To investigate the effect of feeder layer cells expressing interleukin (IL)-21 on the amplification of NK cells In Vitro ., Methods: The K562 cell line with IL-21 expression on its membrane was constructed by electroporation, and co-cultured with NK cells after inactivation. The proliferation of NK cells was observed. The killing function of the amplified NK cells In Vitro was evaluated by the lactate dehydrogenase (LDH) and interferon-γ (IFN-γ) release assay. A colorectal cancer xenograft model in NOD/SCID mice was established, and a blank control group, a NK cell group and an amplified NK cell group were set up to detect the tumor killing effect of amplified NK cells in vivo ., Results: K562 cells expressing IL-21 on the membrane were successfully constructed by electroporation. After co-culturing with K562 cells expressing IL-21 on the membrane for 17 days, the NK cells increased to 700 times, which showed an enhanced amplification ability compared with control group ( P < 0.001). In the tumor cell killing experiment In Vitro , there was no significant difference in the killing activity on tumor cells between NK cells and amplified NK cells, and there was also no significant difference in mice in vivo ., Conclusion: K562 cells expressing IL-21 on the membrane can significantly increase the amplification ability of NK cells In Vitro , but do not affect the killing function of NK cells In Vitro and in vivo . It can be used for the subsequent large-scale production of NK cells In Vitro .

Alternative splicing is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative acute myeloid leukemia (AML) samples from the Cancer Genome Atlas, focusing on the splicing factor RNA-binding motif protein 4 (RBM4) and its splicing mechanism on the target gene transcription factor EB (TFEB), which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transforming growth factor-beta (TGF-β), an immunosuppressive cytokine, is often elevated in various tumors and inhibits the immune system's ability to combat tumor cells. Despite promising results from TGF-β inhibitor therapies, their clinical efficacy remains limited., Purpose: This study aimed to enhance the antitumor capabilities of natural killer (NK) cells in the presence of TGF-β by exploring the potential of asiaticoside, a natural compound with established clinical safety., Study Design: The effects of asiaticoside on NK cells were investigated to determine its potential to counteract TGF-β-induced immunosuppression and elucidate the underlying mechanisms., Methods: Natural compounds were screened using a Luminex assay to identify those promoting Interferon-γ (IFN-γ) secretion from NK cells. Asiaticoside-pretreated NK cells' cytotoxicity was assessed against K562, OVCAR8, and A2780 cells using organoids from ascites-derived ovarian cancer (OC) cells. In vivo efficacy was evaluated with B16 melanoma lung metastasis and subcutaneous tumor models in C57BL/6 mice, using asiaticoside as a 50 mg/kg injection. The compound's ability to enhance NK cell-driven anti-neoplastic responses was further assessed in an OC murine model. Effects on TGF-β/SMAD pathways and mitochondrial functions were examined through various microscopy and metabolomic techniques. The involvement of the mTOR/DRP1 axis in asiaticoside-mediated restoration of mitochondrial oxidation in NK cells after TGF-β suppression was determined using the mTOR inhibitor rapamycin and the DRP1 inhibitor Mdivi-1., Results: Asiaticoside-treated NK cells retained their ability to suppress tumor growth and metastasis despite TGF-β presence. Asiaticoside downregulated TGF-β receptors 1 (TGFBR1) expression, impaired the protein stability of TGFBR1 and TGF-β receptors 2 (TGFBR2), and reduced SMAD2 phosphorylation, preventing SMAD2 translocation from the mitochondria. This preserved mitochondrial respiration and maintained NK cell antitumor activity., Conclusion: The study concludes that asiaticoside has significant potential as a strategy for "priming" NK cells in cellular immunotherapy. By demonstrating that asiaticoside degrades the TGF-β receptor, leading to reduced phosphorylation of SMAD2 and preventing its mitochondrial translocation, thereby maintaining mitochondrial integrity. Meantime, asiaticoside counteracts TGF-β-induced suppression of mitochondrial oxidative and aerobic respiration through the mTOR/DRP1 pathways. The research uncovers a previously unreported pathway for preserving mitochondrial respiration and NK cell functionality. A detailed mechanistic insight into how asiaticoside functions at the molecular level was explored. Its ability to counteract the immunosuppressive effects of TGF-β makes it a valuable candidate for enhancing the effectiveness of immunotherapies in treating a variety of tumors with elevated TGF-β levels., Competing Interests: Declaration of competing interest We are submitting our manuscript titled " Asiaticoside modulates human NK cell functional fate by mediating metabolic flexibility in the tumor microenvironment" for publication consideration in the esteemed "Phytomedicine". The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Chronic myeloid leukemia (CML), caused by BCR::ABL1 fusion gene, is known to regulate disease progression by altering the expression of genes. However, the molecular mechanisms underlying these changes are largely unknown. In this study, we identified RNA Exonuclease 5 (REXO5/LOC81691) as a novel gene with elevated mRNA expression levels in chronic myeloid leukemia (CML) patients. Additionally, using the REXO5 knockout (KO) K562 cell lines, we revealed a novel role for REXO5 in the DNA damage response (DDR). Compared to wild-type (WT) cells, REXO5 KO cells showed an accumulation of R-loops and increased DNA damage. We demonstrated that REXO5 translocates to sites of DNA damage through its RNA recognition motif (RRM) and selectively binds to R loops. Interestingly, we identified that REXO5 regulates R-loop levels by degrading mRNA within R-loop using its exonuclease domain. REXO5 KO showed ATR-CHK1 activation. Collectively, we demonstrated that REXO5 plays a key role in the physiological control of R-loops using its exonuclease domain. These findings may provide novel insights into how REXO5 expression changes contribute to CML pathogenesis., (© 2024. The Author(s).)

Purpose: Acute erythroleukemia (AEL) is a rare and highly aggressive subtype of acute myeloid leukemia (AML) with an extremely poor prognosis when treated with available drugs. Therefore, new investigational agents capable of inducing remission are urgently required., Methods: Bioinformatics analysis, western blot and qRT-PCR were used to reveal the potential biological mechanism of bryostatin 4 (B4), an antineoplastic macrolide derived from the marine bryozoan Bugula neritina. Then, in vivo experiments were conducted to evaluate the role of transforming growth factor (TGF)-β signaling in the progression of AEL., Results: Our results revealed that the proliferation of K562 cells and TF-1 cells was significantly inhibited by B4 at IC 50 values of 37 nM and 52 nM, respectively. B4 inhibited TGF-β signaling and its downstream pathway targets, particularly the phosphorylation of Smad2, Smad3, Ras, C-RAF, ERK1/2, and MEK. B4 also played an important role in cell invasion and migration in K562 cells and TF-1 cells by reducing the protein levels of the mesenchymal cell marker vimentin. Moreover, Flow cytometry and western blot analyses demonstrated that B4 induced apoptosis and initiated G0/G1 phase arrest by modulating mitochondrial dysfunction and cyclin-dependent kinase (CDK) expression., Conclusion: These findings indicated that B4 could inhibit the proliferation, migration, invasion, and TGF-β signaling pathways of AEL cells, thus suggesting that B4 possesses therapeutic potential as a treatment for AEL., (© 2024. Springer Nature Switzerland AG.)

Background: Acute promyelocytic leukemia (APL) is the sub-type of Acute myeloid leukemia (AML) which is described by differentiation block at promyelocytic stage and t(15; 17) translocation with All trans retinoic acid (ATRA) and arsenic trioxide (ATO) as standard treatments. Chronic myeloid leukemia (CML) translocation t (19; 22) causes a rise in granulocytes and their immature precursors in the blood. Different mutations cause resistance to first-line tyrosine kinase therapies in CML. Beside drug resistance, leukemia stem cells (LSC) are critical resources for relapse and resistance in APL and CML. The drug toxicity and resistant profile associated with LSC and current therapeutics of APL and CML necessitate the development of new therapies. Imidazoles are heterocyclic nitrogen compounds with diverse cellular actions. The purpose of this research was to assess the anti-leukemic properties of four novel imidazole derivatives including L-4, L-7, R-35, and R-NIM04., Methods and Results: Pharmacological and biochemical approaches were used which showed that all four imidazole derivatives interfere with the NB4 cells proliferation, an APL cell line, while only L-7 exhibit anti-proliferative activity against K562 cells, a CML cell line. The anti-proliferative effect of imidazole derivatives was linked to apoptosis induction. Further real-time polymerase chain reaction (RT-PCR) analysis revealed downregulation of AXL-Receptor Tyrosine Kinase (AXL-RTK) and target genes of Wnt/beta-catenin pathway like c-Myc, Axin2 and EYA3. An additive effect was observed after combinatorial treatment of L-7 with standard drugs ATRA or Imatinib on the proliferation of NB4 and K562 cells respectively which was related to further downregulation of target genes of Wnt/beta catenin pathway., Conclusion: Imidazole derivatives significantly reduce proliferation of NB4 and K562 cells by inducing apoptosis, down regulating of AXL-RTK and Wnt/β-catenin target genes., (© 2024. The Author(s).)

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response.

Coaggregation assays using K562 cells have been extensively employed to study how cell adhesion molecules mediate specificity between different populations. Here we describe how to prepare K562 cells, optimize electroporation conditions, calibrate antibodies used for protein detection, determine the surface expression of desired adhesion molecules, and considerations for the rotational force to be applied during the assay. We also detail procedures for analyzing coaggregates using our established CoAggregation (CoAg) Index. For complete details on the use and execution of this protocol, please refer to Bisogni et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Athenaea fasciculata , a Brazilian native species from the Solanaceae family, is recognized as a promising source of bioactive withanolides, particularly Aurelianolide A and B, which exhibit significant antitumoral activities. Despite its potential, research on the chemical constituents of this species remains limited. This study aimed to dereplicate extracts and partitions of A. fasciculata to streamline the discovery of bioactive withanolides., Methods: Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), various extracts-including n -hexane, methanol, and ethanol-were analyzed, and their mass spectrometry data were processed through the GNPS platform for the generation of molecular networking. The results indicated that crude extracts displayed comparable cytotoxicity against Jurkat cells, by treatment at 150 µg/mL, while alcoholic extracts achieved approximately 80% inhibition of K562 cells and K562-Lucena 1 at the same concentration. Notably, the dichloromethane partition exhibited the highest cytotoxicity across leukemia cell lines, particularly against Jurkat cells (IC 50 = 14.34 µg/mL). A total of 22 compounds were annotated by manual inspection and different libraries, with six of them demonstrating significant cytotoxic effects., Conclusions: This research underscores the therapeutic potential of A. fasciculata and highlights the effectiveness of integrating advanced analytical methods in drug discovery, paving the way for further exploration of its bioactive compounds.

A robust T-cell expansion process involves co-culturing T-cells with non-proliferating feeder cells combined with anti-CD3 antibody and IL-2. Although ionizing irradiation effectively inhibits feeder cell proliferation, the high operating costs limit cell therapy research to well-funded institutions. UVC, known for causing DNA damage-induced cell death and commonly used for environmental sterilization, presents a cost-effective alternative to ionizing irradiation for generating non-proliferating feeder cells. UVC irradiation of K562 artificial antigen presenting cells (aAPCs) resulted in significant DNA damage, evidenced by increased γ-H2AX phosphorylation within 15 minutes and elevated 8-OHdG levels at 24 hours. This indicates the occurrence of DNA double-strand breaks and oxidative damage. Following UVC irradiation, glucose uptake and ATP production were significantly reduced, whereas aCD3 retention at the surface of the cell increased twofold. Selective inhibition of glucose uptake and ATP production similarly enhanced aCD3 retention by approximately 10-fold and 6-fold, respectively. This suggests that UVC-induced energy deprivation dampens aCD3 internalization, potentially enhancing T-cell activation through prolonged aCD3 and T-cell receptor interaction. Tumor-infiltrating lymphocytes (TILs) expanded with UVC-irradiated PBMCs demonstrated comparable viability, expansion, immunophenotype, and effector function to those expanded with ionizing irradiation. UVC irradiation was equally effective in suppressing feeder cell proliferation and facilitating the expansion of functionally potent T-cells compared to traditional ionizing irradiation. Implementing UVC irradiation in T-cell expansion can significantly reduce costs, enhancing the accessibility and feasibility of cell therapy research across various institutions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Benavidez Arias, Nguyen, Ross, Eagerton and Ritthipichai.)

MYB is a key regulator of hematopoiesis and erythropoiesis, and dysregulation of MYB is closely involved in the development of leukemia, however the mechanism of MYB regulation remains still unclear so far. Our previous study identified a long noncoding RNA (lncRNA) derived from the -34 kb enhancer of the MYB locus, which can promote MYB expression, the proliferation and migration of human leukemia cells, and is therefore termed MY34UE-AS. Then the interacting partner proteins of MY34UE-AS were identified and studied in the present study. hnRNPA0 was identified as a binding partner of MY34UE-AS through RNA pulldown assay, which was further validated through RNA immunoprecipitation (RIP). hnRNPA0 interacted with MY34UE-AS mainly through its RRM2 domain. hnRNPA0 overexpression upregulated MYB and increased the proliferation and migration of K562 cells, whereas hnRNPA0 knockdown showed opposite effects. Rescue experiments showed MY34UE-AS was required for above mentioned functions of hnRNPA0. These results reveal that hnRNPA0 is involved in leukemia through upregulating MYB expression by interacting with MY34UE-AS, suggesting that the hnRNPA0/MY34UE-AS axis could serve as a potential target for leukemia treatment., Competing Interests: Declaration of Competing interest All authors declare that they have no conflicts of interest with the contents of this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Melibiosamine (Gal-α(1,6)-GlcNH 2 ), consisting of galactose and glucosamine linked by an α(1,6)-glycosidic bond, is an artificial disaccharide derivative that selectively inhibits the proliferation of K562 tumor cells relative to HUC-F2 normal cells. In this study, we employed a linkage-editing strategy to synthesize CH 2 - and CHF-linked melibiosamine analogs through chemo- and stereoselective hydrogenation of fluorovinyl- C -glycoside. ( R )-CHF-Melibiosamine exhibited more potent antiproliferative activity than O -linked melibiosamine, while ( S )-CHF-melibiosamine was less potent.

Cytarabine (Ara-C) is a commonly used chemotherapeutic drug for the treatment of leukemia, known for its significant tolerability. The down regulation of miR-203 in leukemia cells suggests its potential involvement in the pathogenesis of leukemia. In this study, we investigated the effects and possible mechanisms of miR-203 and Ara-C on proliferation and apoptosis of human leukemia K562 cells which were cultured with Ara-C and/or with transfection of miR-203 expression vectors. Our results showed that the combination of Ara-C and miR-203 synergistically inhibited the proliferation of K562 cells and the sensitivity of leukemia cells to Ara-C was increased by 2.5-fold with trasfection of miR-203. The proportion of apoptotic cells in the Ara-C and miR-203 combination group was higher than Ara-C or control plasmid group. Caspase-3 and caspase-9 activities were increased in Ara-C and miR-203 combination group. miR-203 down regulated the protein level of Bcr/abl in K562 cells compared with plasmid control. In conclusion, Ara-C in combination with miR-203 has a synergistic effect of proliferation inhibition and apoptosis induction in chronic myelogenous leukemia K562 cells, which may be associated with miR-203 down regulating Bcr/abl, thereby inhibiting cell proliferation and promoting cell apoptosis.

Overexpression of matrix metalloproteinase-2 (MMP-2) possesses a correlation with leukemia especially chronic myeloid leukemia (CML). However, no such MMP-2 inhibitor has come out in the market to date for treating leukemia. In this study, synthesis, biological evaluation, and molecular modeling studies of a set of biphenylsulfonamide derivatives as promising MMP-2 inhibitors were performed, focusing on their potential applications as antileukemic therapeutics. Compounds DH-18 and DH-19 exerted the most effective MMP-2 inhibition (IC 50 of 139.45 nM and 115.16 nM, respectively) with potent antileukemic efficacy against the CML cell line K562 (IC 50 of 0.338 µM and 0.398 µM, respectively). The lead molecules DH-18 and DH-19 reduced the MMP-2 expression by 21.3% and 17.8%, respectively with effective apoptotic induction (45.4% and 39.8%, respectively) in the K562 cell line. Moreover, both these compounds significantly arrested different phases of the cell cycle. Again, both these molecules depicted promising antiangiogenic efficacy in the ACHN cell line. Nevertheless, the molecular docking and molecular dynamics (MD) simulation studies revealed that DH-18 formed strong bidentate chelation with the catalytic Zn 2+ ion through the hydroxamate zinc binding group (ZBG). Apart from that, the MD simulation study also disclosed stable binding interactions of DH-18 and MMP-2 along with crucial interactions with active site amino acid residues namely His120, Glu121, His124, His130, Pro140, and Tyr142. In a nutshell, this study highlighted the importance of biphenylsulfonamide-based novel and promising MMP-2 inhibitors to open up a new avenue for potential therapy against CML., (© 2024 Wiley Periodicals LLC.)

Chronic myeloid leukemia is a hematological cancer, where disease relapse and drug resistance are caused by bone-hosted-residual leukemia cells. An innovative resolution is bone-homing and selective-active targeting of anticancer loaded-nanovectors. Herein, ivermectin (IVM) and methyl dihydrojasmonate (MDJ)-loaded nanostructured lipid carriers (IVM-NLC) were formulated then dually decorated by lactoferrin (Lf) and alendronate (Aln) to optimize (Aln/Lf/IVM-NLC) for active-targeting and bone-homing potential, respectively. Aln/Lf/IVM-NLC (1 mg) revealed nano-size (73.67 ± 0.06 nm), low-PDI (0.43 ± 0.06), sustained-release of IVM (62.75 % at 140-h) and MDJ (78.7 % at 48-h). Aln/Lf/IVM-NLC afforded substantial antileukemic-cytotoxicity on K562-cells (4.29-fold lower IC 50 ), higher cellular uptake and nuclear fragmentation than IVM-NLC with acceptable cytocompatibility on oral-epithelial-cells (as normal cells). Aln/Lf/IVM-NLC effectively upregulated caspase-3 and BAX (4.53 and 15.9-fold higher than IVM-NLC, respectively). Bone homing studies verified higher hydroxyapatite affinity of Aln/Lf/IVM-NLC (1 mg; 22.88 ± 0.01 % at 3-h) and higher metaphyseal-binding (1.5-fold increase) than untargeted-NLC. Moreover, Aln/Lf/IVM-NLC-1 mg secured 1.35-fold higher in vivo bone localization than untargeted-NLC, with lower off-target distribution. Ex-vivo hemocompatibility and in-vivo biocompatibility of Aln/Lf/IVM-NLC (1 mg/mL) were established, with pronounced amelioration of hepatic and renal toxicity compared to higher Aln doses. The innovative Aln/Lf/IVM-NLC could serve as a promising nanovector for bone-homing, active-targeted leukemia therapy., Competing Interests: Declaration of competing interest The authors confirm that there are no conflicts of interest associated with this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Acquired multidrug resistance is often blamed for the failure of chemotherapy in patients with malignant tumors, including chronic myeloid leukemia (CML). In this study, the authors investigated the role of circular RNA 0080145 (circ&#95;0080145) in imatinib (IM) resistance of CML. Materials and Methods: Quantitative real-time polymerase chain reaction was applied to measure the expression of circ&#95;0080145, microRNA-326 (miR-326), and PTPRF interacting protein alpha 1 ( PPFIA1 ) mRNA. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the half maximal inhibitory concentration (IC50) of IM and cell proliferation. Flow cytometry analysis was utilized to assess cell apoptosis. The levels of glucose uptake and lactate production were examined using specific kits. Protein levels were detected through Western blot assay. The targeting relationship between miR-326 and circ&#95;0080145 or PPFIA1 was verified by dual-luciferase reporter assay. The murine xenograft model was constructed to investigate the effect of circ&#95;0080145 in vivo . Results: circ&#95;0080145 was upregulated in IM-resistant CML patients and cells. circ&#95;0080145 silencing suppressed IM resistance, cell growth, and glycolysis and induced apoptosis in IM-resistant CML cells in vitro . Moreover, circ&#95;0080145 knockdown blocked tumor growth and IM resistance in vivo . miR-326 was a target of circ&#95;0080145, and miR-326 inhibition restored the effects of circ&#95;0080145 silencing on cell progression and IM resistance. In addition, PPFIA1 was a target gene of miR-326. The suppressive roles in IM resistance, cell growth and glycolysis, and the promotional role in apoptosis mediated by miR-326 were abolished by PPFIA1 overexpression in IM-resistant CML cells. Conclusion: circ&#95;0080145 contributes to IM resistance via modulating miR-326/ PPFIA1 axis, which might provide a novel avenue for CML therapy.

Herein, 17 previously undescribed polyacetylenes and 9 known ones were isolated from Tridax procumbens L. Their structures were identified using spectroscopic techniques (NMR, UV, IR, MS and optical rotation), the modified Mosher method, electronic circular dichroism (ECD) data and ECD calculation. The cytotoxicity of polyacetylenes on six human tumour cell lines (K562, K562/ADR, AGS, MGC-803, SPC-A-1 and MDA-MB-231) was evaluated. (3S,10R)-tridaxin B (2a), (3S,10S)-tridaxin B (2b) and tridaxin F (8) demonstrated substantial cytotoxic effects against the K562 cell line, with half-maximal inhibitory concentration (IC 50 ) values of 2.62, 14.43 and 17.91 μM, respectively. Cell and nucleus morphology assessments and Western blot analysis confirmed that the cytotoxicity of the three polyacetylenes on K562 cells was mediated through a dose-dependent apoptosis pathway. Furthermore, (3S,10R)-tridaxin A (1a) and tridaxin G (9) exhibited considerable inhibitory effects on lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages, with IC 50 values of 15.92 and 20.35 μM, respectively. Further investigations revealed that 9 exerted anti-inflammatory activities by impeding the nuclear translocation of NF-κB and down-regulating the expression of pro-inflammatory factors, including those of iNOS, COX-2, IL-1β and IL-6, in a concentration-dependent manner. The study provides evidence that polyacetylenes from T. procumbens may serve as a potential source of anti-tumour or anti-inflammatory agents for treating related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondrial transporters facilitate the translocation of metabolites between the cytoplasm and mitochondria and are critical for mitochondrial functional integrity. Although many mitochondrial transporters are associated with metabolic diseases, how they regulate mitochondrial function and their metabolic contributions at the cellular level are largely unknown. Here, we show that mitochondrial thiamine pyrophosphate (TPP) transporter SLC25A19 is required for mitochondrial respiration. SLC25A19 deficiency leads to reduced cell viability, increased integrated stress response (ISR), enhanced glycolysis and elevated cell sensitivity to 2-deoxyglucose (2-DG) treatment. Through a series of biochemical assays, we found that the depletion of mitochondrial NADH is the primary cause of the impaired mitochondrial respiration in SLC25A19 deficient cells. We also showed involvement of SLC25A19 in regulating the enzymatic activities of complexes I and III, the tricarboxylic acid (TCA) cycle, malate-aspartate shuttle and amino acid metabolism. Consistently, addition of idebenone, an analog of coenzyme Q10, restores mitochondrial respiration and cell viability in SLC25A19 deficient cells. Together, our findings provide new insight into the functions of SLC25A19 in mitochondrial and cellular physiology, and suggest that restoring mitochondrial respiration could be a novel strategy for treating SLC25A19-associated disorders., Competing Interests: Declaration of competing interest The author declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 50 μM 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. [Display omitted] • HQ inhibited erythroid differentiation and miR-451a expression in K562 cells. • miR-451a modulates erythroid differentiation by targeting BATF, SETD5, ARHGEF3. • c-Jun regulates miR-451a to participate in HQ-induced erythroid differentiation disorder. [ABSTRACT FROM AUTHOR]

• Ethanolic extract of E. gypsicola exerts cytotoxic effects on leukemia K562 cells. • The E. gypsicola extract inhibits cell migration and induces differentiation in K562 cells. • The extract leads to cellular oxidative stress in K562 cells. • The extract triggers apoptosis in K562 cells via up-regulation of BAX and down-regulation of Bcl2 and Survivin. The Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. The E. gypsicola extract exhibited potent medication properties with IC 50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. These findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia. [Display omitted] [ABSTRACT FROM AUTHOR]