Novel Mechanism by a Bis-Pyridinium Fullerene Derivative to Induce Apoptosis by Enhancing the MEK-ERK Pathway in a Reactive Oxygen Species-Independent Manner in BCR-ABL-Positive Chronic Myeloid Leukemia-Derived K562 Cells

In the treatment of breakpoint cluster region-Abelson (BCR-ABL)-positive chronic myeloid leukemia (CML) using BCR-ABL inhibitors, the appearance of a gatekeeper mutation (T315I) in BCR-ABL is a serious issue. Therefore, the development of novel drugs that overcome acquired resistance to BCR-ABL inhibitors by CML cells is required. We previously demonstrated that a bis-pyridinium fullerene derivative (BPF) induced apoptosis in human chronic myeloid leukemia (CML)-derived K562 cells partially through the generation of reactive oxygen species (ROS). We herein show that BPF enhanced the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase (MEK-ERK) pathway in a ROS-independent manner. BPF-induced apoptosis was attenuated by trametinib, suggesting the functional involvement of the MEK-ERK pathway in apoptosis in K562 cells. In addition, the constitutive activation of the MEK-ERK pathway by the enforced expression of the BRAFV600E mutant significantly increased the sensitivity of K562 cells to BPF. These results confirmed for the first time that BPF induces apoptosis in K562 cells through dual pathways—ROS production and the activation of the MEK-ERK pathway. Furthermore, BPF induced cell death in transformed Ba/F3 cells expressing not only BCR-ABL but also T315I mutant through the activation of the MEK-ERK pathway. These results indicate that BPF is as an effective CML drug that overcomes resistance to BCR-ABL inhibitors.

Replication of Dengue Virus in K562-Megakaryocytes Induces Suppression in the Accumulation of Reactive Oxygen Species

Dengue virus can infect human megakaryocytes leading to decreased platelet biogenesis. In this article, we report a study of Dengue replication in human K562 cells undergoing PMA-induced differentiation into megakaryocytes. PMA-induced differentiation in these cells recapitulates steps of megakaryopoiesis including gene activation, expression of CD41/61 and CD61 platelet surface markers and accumulation of intracellular reactive oxygen species (ROS). Our results show differentiating megakaryocyte cells to support higher viral replication without any apparent increase in virus entry. Further, Dengue replication suppresses the accumulation of ROS in differentiating cells, probably by only augmenting the activity of the transcription factor NFE2L2 without influencing the expression of the coding gene. Interestingly pharmacological modulation of NFE2L2 activity showed a simultaneous but opposite effect on intracellular ROS and virus replication suggesting the former to have an inhibitory effect on the later. Also cells that differentiated while supporting intracellular virus replication showed reduced level of surface markers compared to uninfected differentiated cells.

Effect of D-mannose on Philadelphia chromosome-positive leukemia cells

BACKGROUND: Although Abelson (ABL) tyrosine kinase inhibitors (TKIs) have demonstrated potency against chronic myeloid leukemia (CML), resistance to ABL TKIs can develop in CML patients after discontinuation of therapy. OBJECTIVE: Glucose metabolism may be altered in CML cells because glucose is a key metabolite used by tumor cells. We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. METHODS: We investigated whether D-mannose treatment induced metabolic changes in CML cells and reduced CML growth in the presence of ABL TKIs. RESULTS: Treatment with D-mannose for 72 h inhibited the growth of K562 cells. Combined treatment using ABL TKIs and D-mannose induced a significantly higher level of cytotoxicity in Philadelphia chromosome (Ph)-positive leukemia cells than in control cells. In the mouse model, severe toxicity was observed as evidenced by body weight loss in the ponatinib and D-mannose combination treatment groups. CONCLUSION: Our results indicate that metabolic reprogramming may be a useful strategy against Ph-positive leukemia cells. However, caution should be exercised during clinical applications.

Antibody-free profiling of transcription factor occupancy during early embryogenesis by FitCUT&RUN

Key transcription factors (TFs) play critical roles in zygotic genome activation (ZGA) during early embryogenesis, while genome-wide occupancies of only a few factors have been profiled during ZGA due to the limitation of cell numbers or the lack of high-quality antibodies. Here, we present FitCUT&RUN, a modified CUT&RUN method, in which an Fc fragment of immunoglobulin G is used for tagging, to profile TF occupancy in an antibody-free manner and demonstrate its reliability and robustness using as few as five thousand K562 cells. We applied FitCUT&RUN to zebrafish undergoing embryogenesis to generate reliable occupancy profiles of three known activators of zebrafish ZGA: Nanog, Pou5f3 and Sox19b. By profiling the time-series occupancy of Nanog during zebrafish ZGA, we observed a clear trend toward a gradual increase in Nanog occupancy and found that Nanog occupancy prior to the major phase of ZGA is critical for the activation of a significant proportion of early transcribed genes. Our results further suggested that the sequential binding of Nanog may be controlled by replication timing and the presence of Nanog motifs.

Semi-Synthesis, Cytotoxic Evaluation, and Structure—Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2−16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC50 value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.

8-Hydroxydaidzein Downregulates JAK/STAT, MMP, Oxidative Phosphorylation, and PI3K/AKT Pathways in K562 Cells

A metabolite isolated from fermented soybean, 8-hydroxydaidzein (8-OHD, 7,8,4′-trihydroxyisoflavone, NSC-678112), is widely used in ethnopharmacological research due to its anti-proliferative and anti-inflammatory effects. We reported previously that 8-OHD provoked reactive oxygen species (ROS) overproduction, and induced autophagy, apoptosis, breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) degradation, and differentiation in K562 human chronic myeloid leukemia (CML) cells. However, how 8-OHD regulates metabolism, the extracellular matrix during invasion and metastasis, and survival signaling pathways in CML remains largely unexplored. High-throughput technologies have been widely used to discover the therapeutic targets and pathways of drugs. Bioinformatics analysis of 8-OHD-downregulated differentially expressed genes (DEGs) revealed that Janus kinase/signal transducer and activator of transcription (JAK/STAT), matrix metalloproteinases (MMPs), c-Myc, phosphoinositide 3-kinase (PI3K)/AKT, and oxidative phosphorylation (OXPHOS) metabolic pathways were significantly altered by 8-OHD treatment. Western blot analyses validated that 8-OHD significantly downregulated cytosolic JAK2 and the expression and phosphorylation of STAT3 dose- and time-dependently in K562 cells. Zymography and transwell assays also confirmed that K562-secreted MMP9 and invasion activities were dose-dependently inhibited by 8-OHD after 24 h of treatment. RT-qPCR analyses verified that 8-OHD repressed metastasis and OXPHOS-related genes. In combination with DisGeNET, it was found that 8-OHD’s downregulation of PI3K/AKT is crucial for controlling CML development. A STRING protein–protein interaction analysis further revealed that AKT and MYC are hub proteins for cancer progression. Western blotting revealed that AKT phosphorylation and nuclear MYC expression were significantly inhibited by 8-OHD. Collectively, this systematic investigation revealed that 8-OHD exerts anti-CML effects by downregulating JAK/STAT, PI3K/AKT, MMP, and OXPHOS pathways, and MYC expression. These results could shed new light on the development of 8-OHD for CML therapy.

UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.

Long noncoding RNAs transcribed downstream of the human β-globin locus regulate β-globin gene expression

The five β-like globin genes (ε, Gγ, Aγ, δ, and β) at the human β-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the β-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea, which induces differentiation of K562 cells, increased hemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by hydroxyurea. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited β-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the β-globin gene locus are key factors regulating globin gene ex pression.

TXNRD1: A Key Regulator Involved in the Ferroptosis of CML Cells Induced by Cysteine Depletion In Vitro

Cysteine metabolism plays a critical role in cancer cell survival. Cysteine depletion was reported to inhibit tumor growth and induce pancreatic cancer cell ferroptosis. Nevertheless, the effect of cysteine depletion in chronic myeloid leukemia (CML) remains to be explored. In this work, we showed that cysteine depletion can induce K562/G01 but not K562 cell death in the form of ferroptosis. However, the glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathways of the two CML cell lines were both blocked after cysteine depletion. This unexpected outcome guided us to perform RNA-Seq to screen the key genes that affect the sensitivity of CML cells to cysteine depletion. Excitingly, thioredoxin reductase 1 (TXNRD1), which related to cell redox metabolism, was significantly upregulated in K562/G01 cells after cysteine depletion. We further inferred that the upregulation is negatively feedback by the enzyme activity decrease of TXNRD1. Then, we triggered the ferroptosis by applying TXNRD1 shRNA and TXNRD1 inhibitor auranofin in K562 cells after cysteine depletion. In summary, we have reason to believe that TXNRD1 is a key regulator involved in the ferroptosis of CML cells induced by cysteine depletion in vitro. These findings highlight that cysteine depletion serves as a potential therapeutic strategy for overcoming chemotherapy resistance CML.