Leukemia U937 Cells Research Articles

The effects of acetylated cordycepin derivatives on promoting vascular angiogenesis and attenuating myocardial ischemic injury

BackgroundEnhanced angiogenesis following myocardial infarction (MI) is beneficial to preserve cardiac function. The present study aimed to investigate whether acetylated derivatives of cordycepin altered its original antitumor properties and exerted cardioprotective effects by promoting angiogenesis in vitro and in vivo. MethodsCordycepin and its derivatives with single (DA), double (DAA), and triple acetyl groups (DAAA) were assessed. The cell viability of leukemia U937 cells, malignant hepatoma Huh-7 cells, and human umbilical vascular endothelial cells (HUVECs) treated with cordycepin, DA, DAA, and DAAA were determined. The expression of β-catenin in U937 cells, as well as the expression of p65, p38 and other related signal regulators in HUVECs elicited by lipopolysaccharides (LPS) were also observed. Angiogenesis was determined by tube formation in HUVECs and Matrigel plug assay in mice. Cardiac function following administration of DAAA was evaluated in mice MI model simulated by coronary artery ligation. ResultsThe inhibitory effects of cordycepin and its acetylated derivatives on U937 cells, Huh-7 cells, HUVECs, and the expression of β-catenin in U937 cells were mitigated with increasing acetylation. Intriguingly, DAAA preserved the cell viability of HUVECs compared to other acetylated derivatives. Although DAAA had a significantly diminished antitumor effect compared to cordycepin, it promoted angiogenesis in mice and tube formation in HUVECs and attenuated LPS-induced phosphorylation of p65 and p38. Additionally, administration of DAAA improved cardiac function following coronary artery ligation in mice. ConclusionDAAA could be considered a promising adjunctive therapy to prevent post-MI heart failure through promoting angiogenesis.

S-nitrosoglutathione (GSNO) induces necroptotic cell death in K562 cells: Involvement of p73, TSC2 and SIRT1

BackgroundNitric oxide and Reactive Nitrogen Species are known to effect tumorigenicity. GSNO is one of the main NO carrying signalling moiety in cell. In the current study, we tried to delve into the effect of GSNO induced nitrosative stress in three different myelogenous leukemic K562, U937 and THP-1 cell lines. MethodWST-8 assay was performed to investigate cell viability. RT-PCR and western-blot analysis were done to investigate mRNA and protein expression. Spectrophotometric and fluorimetric assays were done to investigate enzyme activities. ResultWe found that GSNO exposure led to reduced cell viability and the mode of cell death in K562 was non apoptotic in nature. GSNO promoted impaired autophagic flux and necroptosis. GSNO treatment heightened phosphorylation of AMPK and TSC2 and inhibited mTOR pathway. We observed increase in NAD+/ NADH ratio following GSNO treatment. Increase in both SIRT1 m-RNA and protein expression was observed. While total SIRT activity remained unaltered. GSNO increased tumor suppressor TAp73/ oncogenic ∆Np73 ratio in K562 cells which was correlated with cell mortality. Surprisingly, GSNO did not alter cellular redox status or redox associated protein expression. However, steep increase in total SNO and PSNO content was observed. Furthermore, inhibition of autophagy, AMPK phosphorylation or SIRT1 exacerbated the effect of GSNO. Altogether our work gives insights into GSNO mediated necroptotic event in K562 cells which can be excavated to develop NO based anticancer therapeutics. ConclusionOur data suggests that GSNO could induce necroptotic cell death in K562 through mitochondrial dysfunctionality and PTM of different cellular proteins.

Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo

Background aimsHu8F4 is a T-cell receptor–like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor–expressing cells. MethodsWe generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor–expressing cells in vivo. ResultsThe primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. ConclusionsHerein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding–induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Effects of PIM1 Gene on Proliferation, Apoptosis and JAK2/STAT3 Signaling Pathway of Acute Myeloid Leukemia U937 Cells

To investigate the effects of the serine/threonine kinase family member 1 (PIM1) gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and PIM1 mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing PIM1 mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without PIM1 mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 μmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of PIM1 mRNA and added with CoA1 at a concentration of 20 μmol/L). After culture for 24 h, the expressions of PIM1 mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate. The PIM1 mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia (P < 0.05). Compared with U937 group, PIM1 mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G 2/M phase proportions were decreased in Si-PIM1 group (all P < 0.05), while p27, Caspase-3 protein, G0/G1 phase proportion and apoptosis rate were increased (all P < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group (P >0.05). Knockdown of PIM1 gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.

Targeting Myeloid Epithelial Tyrosine Kinase (MERTK) Receptor in Acute Myeloid Leukemia Using a Novel Antibody Drug Conjugate, Rgx-019-MMAE

Background: Myeloid epithelial reproductive tyrosine kinase receptor (MERTK) is a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family. It is known to be overexpressed in various solid and hematological cancers. Overexpression of MERTK is associated with reduced apoptosis, increased metastasis, and drug resistance in tumor cells, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) developed by Inspirna, selectively binds to MERTK on tumor cells, which results in internalization and degradation of the receptor, and induces cytotoxicity through the release of the payload, MMAE, which disrupts the mitosis process of actively dividing cells. Several monoclonal antibodies targeting MERTK have shown promising results against various solid cancers, however, the effect of targeting MERTK in AML progression via RGX-019-MMAE is not known. Here, we hypothesize MERTK is overexpressed in distinct subsets of AML and associated with leukemia progression, therefore, targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: To determine the clinical/prognostic relevance of MERTK, we analyzed a gene expression dataset (OHSU) and segregated the patients into low and high MERTK mRNA expression groups based on their mean expression value. To identify the specific AML subset with the highest MERTK expression, MERTK protein expression was analyzed in 818 patients with AML by performing Reverse Phase Protein Arrays (RPPA). MERTK protein expression was also measured by flow cytometry in OCI-AML3, OCI-AML2, Kasumi-1, ThP-1, Molm-13, Molm-14, MV4-11, and U937 leukemic cell lines and in the peripheral blood or bone marrow of five AML patients. Further, we treated Kasumi-1 and OCI-AML3 AML cell lines with varying doses of RGX-019-MMAE or isotype control for 72 and 120 hours and measured its effect on AML cell viability using CellTiterGlo 2.0 assay. Likewise, we treated primary cells from 5 different AML patients and determined the anti-leukemic effect of RGX-019-MMAE in primary cells. Finally, we investigated the synergistic effect of RGX-019-MMAE with Venetoclax (BCL2 inhibitor) or 5-Azacytidine in vitro. Results: Analysis of AML OHSU dataset showed that patients with high MERTK mRNA expression had significantly worse overall survival than those having low MERTK mRNA expression (p=0.02). RPPA analysis revealed significantly higher MERTK protein expression in monocytic AML subtypes, such as M4 and M5, especially in those with PTPN11 mutation or t (9;11) translocation. Moreover, in the RPPA we noted that high expression of MERTK protein levels is significantly correlated with inferior survival. This finding suggests that MERTK could serve as a therapeutic target in AML, especially in patients with monocytic subtypes of leukemia. In addition, we observed varying degrees of MERTK protein expression in AML cell lines, with Kasumi-1 and OCI-AML3 expressing the highest amount of MERTK protein. Next, treatment of Kasumi-1 and OCI-AML3 cell lines with varying doses of anti-MERTK ADC, RGX-019-MMAE, resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p&amp;lt;0.01), suggesting that RGX-019-MMAE inhibits AML cell proliferation. Further, we validated this finding in primary AML cells with high MERTK expression. As expected, we observed that treatment with RGX-019-MMAE induced significantly greater cell death (˜80% apoptosis) in leukemic cells than the isotype control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated in combination with RGX-019-MMAE and either Venetoclax or 5-Azacytidine showed a synergistic effect with increased cytotoxicity in a dose-dependent manner. These findings suggest that RGX-019-MMAE targets MERTK-expressing leukemic cells, sensitizes cells to chemotherapies, and promotes overall cytotoxicity. Conclusion: RGX-019-MMAE significantly induced cell death in AML cell lines and primary AML patient samples. RGX-019-MMAE, combined with chemotherapeutic agents and targeted therapy, produces a synergistic anti-leukemic effect. Our data indicates that MERTK is a potential therapeutic target in AML patients with monocytic subtypes of leukemia.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved self‑digestion process that is critical for maintaining homeostasis in response to various stresses. The autophagy‑related protein family, including the GABA type A receptor‑associated protein (GABARAP) and microtubule‑associated protein 1 light chain 3 subfamilies, is crucial for autophagosome biogenesis. Although the regulatory machinery of autophagy in the cytoplasm has been widely studied, its transcriptional and epigenetic regulatory mechanisms still require more targeted investigations. The present study identified histone lysine demethylase 3B (KDM3B) as a crucial component of autophagy on a panel of leukemia cell lines, including K562, THP1 and U937, resulting in transcriptional activation of the autophagy‑related gene GABA type A receptor‑associated protein like 1 (GABARAPL1). KDM3B expression promoted autophagosome formation and affected the autophagic flux in leukemia cells under the induction of external stimuli. Notably, RNA‑sequencing and reverse transcription‑quantitative PCR analysis showed that KDM3B knockout inhibited the expression of GABARAPL1. Chromatin immunoprecipitation‑quantitative PCR and luciferase assay showed that KDM3B was associated with the GABARAPL1 gene promoter under stimulation and enhanced its transcription. The present findings demonstrated that KDM3B was critical for regulating the GABARAPL1 gene and influencing the process of autophagy in leukemia cells. These results provide a new insight for exploring the association between autophagy and KDM3B epigenetic regulation in leukemia.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia

To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Portulacerebroside a Inhibits Adhesion, Migration, and Invasion of Human Leukemia HL60 Cells and U937 Cells through the Regulation of p38/JNK Signaling Pathway [Retraction

[This retracts the article DOI: 10.2147/OTT.S117523.].

Inhibition of Ferulic Acid on U937 Cell Growth and Its Related Mechanism

To investigate the effects of ferulic acid (FA) on proliferation, apoptosis and Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway of human acute myeloid leukemia (AML) U937 cells. Different concentrations of FA (0、10、25、50、100、200 μmol/L) was used to treat human AML cell lines Kasumi-1, HL-60, and U937 cells respectively for 24 h. The cell survival rate was detected by cell counting kit-8 method, and the 50% inhibitory concentration (IC50) of each cell line was calculated. The U937 cells were divided into control group, FA group (50 μmol/L FA), FA+pcDNA group (50 μmol/L FA+transfected with empty plasmid), FA+pcDNA-TLR4 group (50 μmol/L FA+transfected with TLR4 overexpression plasmid). Flow cytometry was used to detect U937 cell cycle and cell apoptosis; plate clone formation test was used to detect U937 cell clone formation ability; fluorescence quantitative PCR was used to detect the TLR4, NF-κB p65 mRNA levels in U937 cells; Western blot was used to detect the expression levels of CyclinD1, CyclinE, Bcl-2 related X protein (Bax), B cell lymphoma-2 (Bcl-2), Caspase-3, TLR4, and NF-κB p65 protein in U937 cells. With the increase of FA concentration, the survival rates of Kasumi-1, HL-60 and U937 cells gradually decreased(r=-0.919, r=-0.909, r=-0.900), the IC50 of U937 cells was 50.25±2.23 μmol/L. Compared with the control group, after drug treatment of U937 cells, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA group were significantly increased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and protein were significantly decreased (P<0.05); compared with FA group and FA+pcDNA group, the ratio of G0/G1 phase cells, apoptosis rate, expression levels of Bax and Caspase-3 proteins in FA+pcDNA-TLR4 group were significantly decreased (P<0.05), the number of cell clones, the ratios of S phase and G2/M phase cells, expression levels of expression levels of CyclinD1, CyclinE and Bcl-2 proteins, and TLR4, NF-κB p65 mRNA and proteins were significantly increased (P<0.05). FA inhibits U937 cell proliferation and promotes cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway.

Isofraxidin enhances hyperthermia‑induced apoptosis via redox modification in acute monocytic leukemia U937 cells.

The cell‑killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat‑induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre‑treatment. Apoptosis was measured by Annexin V‑FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT‑induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase‑3, caspase‑8 and phosphorylated‑JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD‑FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT‑induced apoptosis via a reactive oxygen species mediated mitochondria/caspase‑dependent pathway in U937 cells.

Novel pyridine-containing histone deacetylase inhibitors strongly arrest proliferation, induce apoptosis and modulate miRNAs in cancer cells

After over 30 years of research, the development of HDAC inhibitors led to five FDA/Chinese FDA-approved drugs and many others under clinical or preclinical investigation to treat cancer and non-cancer diseases. Herein, based on our recent development of pyridine-based isomers as HDAC inhibitors, we report a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2′-aminoanilides 5–8 as anticancer agents. The hydroxamate 5d proved to be quite HDAC3/6-selective exhibiting IC50 values of 80 and 11 nM, respectively, whereas the congener 5e behaved as inhibitor of HDAC1-3, −6, −8, and −10 (class I/IIb-selective inhibitor) at nanomolar level. Compound 5e provided a huge antiproliferative activity (nanomolar IC50 values) against both haematological and solid cancer cell lines. In leukaemia U937 cells, the hydroxamate 5d and the 2′-aminoanilide 8f induced remarkable cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, showing a stronger effect with respect to SAHA and MS-275 used as reference compounds. In U937 cells, the highest dose- and time-dependent cytodifferentiation was obtained by the 2′-aminoanilide 8d (up to 35% of CD11c positive/propidium iodide negative cells at 5 μM for 48 h). The same 8d and the hydroxamates 5d and 5e were the most effective in inducing p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f increased mRNA expression of p21, BAX and BAK, downregulated cyclin D1 and BCL-2 and modulated pro- and anti-apoptotic microRNAs towards apoptosis induction. Finally, 5e strongly arrested proliferation in nine different haematological cancer cell lines, with dual-digit nanomolar potency towards MV4-11, Kasumi-1, and NB4, being more potent than mocetinostat, used as reference drug.

Guanidine Derivatives Containing the Chalcone Skeleton Are Potent Antiproliferative Compounds against Human Leukemia Cells.

In this study, we investigated the effects of eleven synthetic guanidines containing the 1,3-diphenylpropenone core on the viabilities of six human cancer cells. The most cytotoxic compound against human cancer cells of this series contains a N-tosyl group and a N-methylpiperazine moiety 6f. It was cytotoxic against leukemia cells (U-937, HL-60, MOLT-3, and NALM-6) with significant effects against Bcl-2-overexpressing U-937/Bcl-2 cells as well as the human melanoma SK-MEL-1 cell line. It exhibited low cytotoxicity against quiescent or proliferating human peripheral blood mononuclear cells. The IC50 value for the leukemia U-937 cells was 1.6 ± 0.6 µM, a similar value to that in the antineoplastic agent etoposide. The guanidine containing a N-phenyl substituent 6i was also as cytotoxic as the guanidine containing the N-tosyl substituent and the N-methylpiperazine group 6f against human U-937 leukemia cells and both synthetic guanidines were potent apoptotic inducers. Cell death was mediated by the activation of the initiator caspase-9 and the executioner caspase-3, and associated with the release of cytochrome c. These synthetic guanidines are potent cytotoxic compounds against several human leukemia cells and even the human melanoma cell line SK-MEL-1 and might be useful in the development of new strategies in the fight against cancer.

SLFN11 Is a Potential Biomarker and Modulator of Treatment Response in AML

Introduction: Despite recent advances in treatment for acute myeloid leukemia (AML), AML remains a deadly disease, with approximately 70% of patients succumbing to the disease within five years of diagnosis (SEER database 2021). Intensive induction chemotherapy carries a risk of morbidity and mortality, making it a risky treatment for patients who are unlikely to achieve a remission with DNA damaging-based treatments. Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DAC) are now frequently used in AML treatment as less intensive treatment options. Thus, biomarkers to predict response to DNA damaging agents in AML are needed. We recently found a significant association between AML patient survival and high expression of Schlafen11 (SLFN11), a dynamically inducible key effector of the DNA replication stress response. SLFN11 has been shown to be associated with response to certain chemotherapy agents in some solid tumors in vitro and in vivo (Conteduca et. al. JCO 2019; Shee et. al. PLoS One 2019), but its importance in leukemia has not been well defined. It is also unknown whether SLFN11 levels affect response to HMAs. We hypothesized that SLFN11 can be used as a predictive biomarker for treatment response to DNA-damaging agents in AML but that response to HMAs would be independent of HMAs. We also hypothesized that identifying pathways upregulated in SLFN11-low cells could lead to novel combination therapies to increase sensitivity to chemotherapeutics. Methods: The human leukemia cell lines HEL and U937 cells were used for these studies. U937- and HEL-SLFN11 knockout (SLFN11KO) cells were generated using CRISPR-Cas9 gene editing technology. Cell viability was assessed by WST-1 reagent assay. SLFN11 and other protein levels were measured by Western blotting. Results: SLFN11 protein expression was readily observed by immunoblotting of HEL and U937 cell lysates. U937- and HEL-SLFN11KO cells were incubated with increasing concentrations of cytarabine (AraC), which is the backbone of most chemotherapy regimens currently used in AML treatment. Both U937- and HEL-SLFN11KO cells were more resistant to AraC than their respective control cells. To examine the mechanism of resistance in U937-SLFN11KO cells, SLFN11KO and control cells were treated with AraC for up to 8 hrs, and lysates were subjected to immunoblotting for various DNA damage response proteins. There were higher baseline levels of multiple DNA damage-related proteins (including Chk1 and Chk2) in the SLFN11KO cells. There was also prolonged activation of the DNA damage pathway in U937-SLFN11KO cells, as evidenced by phosphorylation of the ATR substrate Chk1 (Ser345) through 8 hours, whereas phospho-Chk1 levels decreased in control cells by 8 hours. Interestingly, this prolonged activation occurred without a concomitant increase in γH2AX (a marker of DNA damage and double strand breaks). Because U937-SLFN11KO cells demonstrated a more pronounced activation of the ATR/Chk1 pathway in response to AraC, we subjected U937-SLFN11KO and control cells to increasing concentrations of AraC in combination with a small dose (0.3 μM) of the ATR inhibitor VE822. Notably, ATR inhibition restored sensitivity of SLFN11KO cells to AraC. These results suggest that SLFN11KO cells are resistant to DNA damaging agents via upregulation and activation of DNA repair proteins, and that adding inhibitors of these proteins can decrease resistance to chemotherapy agents in SLFN11KO cells. In contrast, SLFN11KO cells remained sensitive to AZA and DAC, suggesting that HMAs may be preferable to AraC for patients with SLFN11-low AML. Conclusions: Low SLFN11 levels predict poor response to standard chemotherapy agents (i.e. AraC) in AML cells, suggesting that SLFN11 may prove to be an important predictive biomarker for AML patients. In addition, SLFN11-low AML cells have an increased reliance on DNA repair pathways, and inhibition of DNA repair proteins such as ATR may enhance responses to AraC in these cells. This raises the possibility of patient selection based on SLFN11 levels for specific combination approaches for the treatment of AML. Figure 1. Kaplan Meier survival curves of AML patients in the TCGA (LAML) database with high (top 50th percentile) or low (bottom 50th percentile) SLFN11 expression using UCSC Xena (Goldman et. al. Nat Biotechnol 2020). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combined Inhibition of MCL1 By AZD5991 and BCL2/Bclxl By AZD4320 As Promising Strategies to Overcome Acute Leukemia Tumor Burden and Niche Chemoresistance

Background. Abnormal regulation of anti-apoptotic proteins belonging to the BCL2 family contributes to therapeutic resistance of many hematological neoplasms. Although Venetoclax, a selective inhibitor of the BCL2 protein, has proven to exert beneficial effects in acute leukemias, the activation of another anti-apoptotic protein, MCL1, a key factor to Venetoclax acquired resistance, frequently reduces its benefits. Additionally, it is known that BCL-xL plays an important pro-survival role in cancer cells by interacting with BCL-2 Hence, we hypothesize that the co-inhibition of MCL1, Bcl-2 and BCL-xL would represent an improved strategy for the treatment of patients with acute leukemia. Thus, in this study, we assessed the efficacy of specific inhibitors of MCL1, BCL2 and BCLxL antiapoptotic proteins. Aims. To investigate the in vitro, ex vivo and in vivo effects of AZD5991 (AstraZeneca), a novel MCL1 inhibitor compound, alone or in combination with AZD4320 (BCL-2/BCL-XL inhibitor compound), venetoclax, cytarabine and doxorubicin. Methods.MCL1 mRNA expression data were obtained from TCGA AML study, performed in mononuclear cells isolated from 163 AML patients (median age 57.5 years [range: 18-88]). Bone marrow (BM) aspirates (25) from healthy donors (HD), Myelodysplastic syndrome (MDS), de novo AML, and acute lymphoblastic leukemia (ALL) patients at diagnosis were submitted to analysis of MCL1 protein levels by flow cytometry and immunofluorescence (IF). A panel of myeloid (OCI-AML3, MOLM13, U937, HL60, KG1a, K562) and lymphoblastic (RS4;11, Jurkat) leukemia cell lines were treated with increasing concentrations of AZD5991 for 48h and the 50% inhibiting concentration and apoptosis (annexin-V stain) rates were determined. Mononuclear and hematopoietic stem (CD34+) cells from HD were submitted to cytotoxic assays. Leukemia cell lines were also tested for reactive oxygen species (ROS) and nitric oxide (NO) production (DAF dye), mitochondrial membrane potential (DilC1 probe) and protein expression (flow cytometry, Western blot) in a time course of 0 to 48h, alone or in combination. Moreover, we tested the AZD5991/AZD4320 action in malignant cells cultured in a 3D-system (in the presence of the mesenchymal cells) and in a monocyte monolayer (2D) in order to evaluate tumor microenvironment (TME) participation. We also developed a xenograft mouse model using U937 leukemia cell line. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. The TCGA study (CBIO Portal) analysis showed high MCL1 gene expression in AML patients was observed (p<.0001). Immunofluorescence and flow cytometry showed that MCL1, BCL-2 and BCL-xL protein levels higher in MDS, AML and ALL BM cells compared to normal cells. All leukemia cell lines were dose-dependent sensitive to AZD5991. Growth inhibiting concentrations were OCI-AML3/0.04μM, U937/4.3μM, RS4;11/3.5μM, Jurkat/0.3μM. Apoptosis was increased in a time and concentration-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to impair mitochondrial membrane potential with reduced mitochondrial ROS and NO production (p<.01) and decreased MCL1, Bax and Bak but increased Bim protein levels. Coculture in a 3D-system showed that AZD5991 treatment reduced patients’ cell growth, without modulating HD cells. In the 2D-coculture, programmed monocytes intensified apoptosis with synergistic effect between AZD5991 and AZD4320. In vitro experiments demonstrated an additive effect between AZD5991 and AZD4320, venetoclax, cytarabine or doxorubicin. In addition, xenotransplanted mice confirmed the efficacy of AZD5991 compound alone or in combination with AZD4320 due to reduction of tumor weight (fold decrease (FD): 3.8; 2.8) and tumor volume (FD: 6.5; 2.8) compared to vehicle-treated group, respectively. Conclusion. MCL1 overexpression in mononuclear cells from patients with acute leukemia and that present or not resistance to venetoclax treatment, is associated with poor prognosis and relapse disease. Our results demonstrated that co-targeting MCL-1, BCL-2 and BCL-xL, through the AZD5991 and AZD4320 compounds in the treatment of acute leukemias, may enhance therapeutic specificity, overcome chemoresistance and contribute with the cure of these aggressive malignant disorders. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

A direct comparison between AML1-ETO and ETO2-GLIS2 leukemia fusion proteins reveals context-dependent binding and regulation of target genes and opposite functions in cell differentiation.

The ETO-family transcriptional corepressors, including ETO, ETO2, and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor—the DBD binds to DNA, while the ETO moiety manifests transcriptional activity. A directly comparative study of these “homologous” fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in M2-and M7-subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs to bind DNA, they share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors, including the ETS-, bZIP- and bHLH-family proteins. AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as activator. The repressor-versus-activator functions of AML1-ETO might be determined by the abundance of cooperative transcription factors/cofactors on the target genes. Importantly, AML1-ETO and ETO2-GLIS2 differentially regulate key transcription factors in myeloid differentiation including PU.1 and C/EBPβ. Consequently, AML1-ETO inhibits, but ETO2-GLIS2 facilitates, myeloid differentiation of U937 cells. This function of ETO2-GLIS2 is reminiscent of a similar effect of MLL-AF9 as previously reported. Taken together, this directly comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context provides insights into context-dependent transcription regulatory mechanisms that may underlie how these seemingly “homologous” fusion transcription factors exert distinct functions to drive different subtypes of leukemia.