Acute Promyelocytic Leukemia Cells Research Articles

ATPR induces acute promyelocytic leukemia cells differentiation and cycle arrest via the lncRNA CONCR/DDX11/PML-RARα signaling axis

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a high mortality rate, and the production of PML-RARα fusion protein is the cause of its pathogenesis. Our group has synthesized a novel compound, 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), by structural modification of All-trans retinoic acid (ATRA), which has strong cell differentiation-inducing effects and inhibits the expression of PML-RARα. In this study, acute promyelocytic leukemia NB4 cells before and after ATPR induction were analyzed by whole transcriptome microarray, and the expression of lncRNA CONCR was found to be significantly downregulated. The role of CONCR in ATPR-induced cell differentiation and cycle arrest was explored through overexpression and silencing of CONCR. And then the database was used to predict that CONCR may bind to DEAD/H-Box Helicase 11 (DDX11) protein to further explore the role of CONCR binding to DDX11. The results showed that ATPR could reduce the expression of CONCR, and overexpression of CONCR could reverse the ATPR-induced cell differentiation and cycle blocking effect, and conversely silencing of CONCR could promote this effect. RNA immunoprecipitation (RIP) experiments showed that CONCR could bind to DDX11, the protein expression levels of DDX11 and PML-RARα were elevated after overexpression of CONCR. These results suggest that ATPR can regulate the expression of DDX11 through CONCR to affect the expression of PML-RARα fusion protein, which in turn induces the differentiation and maturation of APL cells.

HSPA8 Chaperone Complex Drives Chaperone-Mediated Autophagy Regulation in Acute Promyelocytic Leukemia Cell Differentiation

Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

Abstract 264: Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem cells

Abstract Background The “Differentiation therapy” has been emerging as a promising and more effective strategy against acute leukemia relapses. Objective In extension to the revolutionising therapeutic outcomes of All Trans Retinoic Acid (ATRA) to induce terminal differentiation of Acute Promyelocytic Leukemic (APL) blast cells, we decipher the potential effect of a natural compound “Esculetin” to serve as a differentiating agent in Acute Myeloid Leukemia (AML) as well as solid tumour cells. Underlaying role of Wnt signaling pathways in esculetin mediated blast cell differentiation was also evaluated. Methods Human acute myeloid leukemic cells (Kasumi-1) with t(8;21/AML-ETO) translocation were used as a model system. Growth inhibitory and cytotoxic activity of esculetin were analysed using growth kinetics and MTT assay. Morphological alterations, cell scatter characteristics, NBT reduction assay and cell surface marker expression pat- terns were analysed to detect terminally differentiated phenotypes. We employed RT2profiler PCR array system for the analysis of transcriptome profile of Wnt signaling components. Calcium inhibitors (TMB8 and Amlodipine) and Transforming growth factor beta (TGF-β) were used to modulate the Wnt signaling axes. To investigate whether esculetin exerts the similar effects on the invasive solid tumour cancer stem cells subset, we used a cellular model system of colon carcinoma HCT116 cells reflecting EMT phenotype. Results We illustrate cytotoxic as well as blast cell differentiation potential of esculetin on Kasumi-1 cells. Morphological alterations akin to neutrophilic differentiation as well as the corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blast cells. Exposure to esculetin also resulted in downregulation of canonical Wnt axis while upto ~ 21 fold upregulation of non-canonical axis associated genes. Esculetin also showed potential to revert the CSC marker expressions and concurrent EMT consistent with reduced functional aggressiveness in colon cancer cells. Conclusions Our study highlights the importance of selective use of calcium pools as well as “axis shift” of the canonical to non-canonical Wnt signaling upon esculetin treatment which might abrogate the inherent proliferation to release maturation arrest and induce the differentiation in leukemic blast cells as well as CSC stem cells. The current findings provide further therapeutic interventions to consider esculetin as a potent differentiating agent to counteract relapses. Citation Format: Ankit Mathur, Daman Saluja. Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 264.

Exploring the Therapeutic Potential of Ganoderma lucidum in Cancer.

Triterpenoids, such as ganoderic acid, and polysaccharides, including β-D-glucans, α-D-glucans, and α-D-mannans, are the main secondary metabolites of the medicinal fungus Ganoderma lucidum. There is evidence of the effects of ganoderic acid in hematological malignancies, whose mechanisms involve the stimulation of immune response, the macrophage-like differentiation, the activation of MAP-K pathway, an IL3-dependent cytotoxic action, the induction of cytoprotective autophagy, and the induction of apoptosis. In fact, this compound has been tested in twenty-six different human cancer cell types and has shown an anti-proliferative activity, especially in leukemia, lymphoma, and myeloma lines. Moreover, research clarified the capability of molecules from Ganoderma lucidum to induce mitochondrial damage in acute promyelocytic leukemia cells, without cytotoxic effects in normal mononuclear cells. Active lipids extracted from the spores of this fungus have also been shown to induce apoptosis mediated by downregulation of P-Akt and upregulation of caspases-3, -8, and -9. Among in vivo studies, a study in BALB/c mice injected with WEHI-3 leukemic cells suggested that treatment with Ganoderma lucidum promotes differentiation of T- and B-cell precursors, phagocytosis by PBMCs, and NK cell activity. Our review presents data revealing the possibility of employing Ganoderma lucidum in hematological malignancies and incorporating it into clinical practice.

Targeting inhibition of TCTP could inhibit proliferation and induce apoptosis in AML cells

Translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein, which participates in many important physiological processes. Recently, the roles of TCTP in cell proliferation and apoptosis, especially its close relationship with various tumors, have attracted widespread attention. In this study, we found that the protein level of TCTP was significantly reduced in acute promyelocytic leukemia cell line NB4 transfected with retinoic acid-induced gene G (RIG-G). The RIG-G was found in our previous work as a key mediator of anti-proliferative activity in retinoid/interferon-related pathways. Here, we tried to further explore the function of TCTP in the development of acute myeloid leukemia (AML) from different levels. Our results showed that inhibiting TCTP expression could attenuate AML cells proliferation and induce apoptosis both in AML cell lines and in xenograft of NOD-SCID mice. In addition, either compared with patients in complete remission or non-leukemia patients, we detected that the expression of TCTP was generally high in the fresh bone marrow of AML patients, suggesting that there was a certain correlation between TCTP and AML disease progression. Taken together, our study revealed the role of TCTP in AML development, and provided a potential target for AML treatment.

Fucosylation inhibitor 6-alkynylfucose enhances the ATRA-induced differentiation effect on acute promyelocytic leukemia cells

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1β, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.

Identification of triciribine as a novel myeloid cell differentiation inducer.

Differentiation therapy using all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is well established. However, because the narrow application and tolerance development of ATRA need to be improved, we searched for another efficient myeloid differentiation inducer. Kinase activation is involved in leukemia biology and differentiation block. To identify novel myeloid differentiation inducers, we used a Kinase Inhibitor Screening Library. Using a nitroblue tetrazolium dye reduction assay and real-time quantitative PCR using NB4 APL cells, we revealed that, PD169316, SB203580, SB202190 (p38 MAPK inhibitor), and triciribine (TCN) (Akt inhibitor) potently increased the expression of CD11b. We focused on TCN because it was reported to be well tolerated by patients with advanced hematological malignancies. Nuclear/cytoplasmic (N/C) ratio was significantly decreased, and myelomonocytic markers (CD11b and CD11c) were potently induced by TCN in both NB4 and acute myeloid leukemia (AML) M2 derived HL-60 cells. Western blot analysis using NB4 cells demonstrated that TCN promoted ERK1/2 phosphorylation, whereas p38 MAPK phosphorylation was not affected, suggesting that activation of the ERK pathway is involved in TCN-induced differentiation. We further examined that whether ATRA may affect phosphorylation of ERK and p38, and found that there was no obvious effect, suggesting that ATRA induced differentiation is different from TCN effect. To reveal the molecular mechanisms involved in TCN-induced differentiation, we performed microarray analysis. Pathway analysis using DAVID software indicated that "hematopoietic cell lineage" and "cytokine-cytokine receptor interaction" pathways were enriched with high significance. Real-time PCR analysis demonstrated that components of these pathways including IL1β, CD3D, IL5RA, ITGA6, CD44, ITGA2B, CD37, CD9, CSF2RA, and IL3RA, were upregulated by TCN-induced differentiation. Collectively, we identified TCN as a novel myeloid cell differentiation inducer, and trials of TCN for APL and non-APL leukemia are worthy of exploration in the future.

20(S)-ginsenoside Rh2 ameliorates ATRA resistance in APL by modulating lactylation-driven METTL3

Background20(S)-ginsenoside Rh2(GRh2), an effective natural histone deacetylase inhibitor, can inhibit acute myeloid leukemia (AML) cell proliferation. Lactate regulated histone lactylation, which has different temporal dynamics from acetylation. However, whether the high level of lactylation modification that we first detected in acute promyelocytic leukemia (APL) is associated with all-trans retinoic acid (ATRA) resistance has not been reported. Furthermore, Whether GRh2 can regulate lactylation modification in ATRA-resistant APL remains unknown. MethodsLactylation and METTL3 expression levels in ATRA-sensitive and ATRA-resistant APL cells were detected by Western blot analysis, qRT-PCR and CO-IP. Flow cytometry (FCM) and APL xenograft mouse models were used to determine the effect of METTL3 and GRh2 on ATRA-resistance. ResultsHistone lactylation and METTL3 expression levels were considerably upregulated in ATRA-resistant APL cells. METTL3 was regulated by histone lactylation and direct lactylation modification. Overexpression of METTL3 promoted ATRA-resistance. GRh2 ameliorated ATRA-resistance by downregulated lactylation level and directly inhibiting METTL3. ConclusionsThis study suggests that lactylation-modified METTL3 could provide a promising strategy for ameliorating ATRA-resistance in APL, and GRh2 could act as a potential lactylation-modified METTL3 inhibitor to ameliorate ATRA-resistance in APL.

Mechanisms of PML::Rara and GATA2 Protein Interactions in Hematopoietic Cells

Despite excellent outcomes for Acute Promyelocytic Leukemia (APL) patients with combined ATRA and Arsenic Trioxide therapy, the underlying molecular mechanisms by which the PML::RARA fusion oncoprotein initiates APL are not yet clear. We recently found that the ability of PML::RARA to initiate aberrant serial replating (a surrogate for self-renewal) is dependent upon its ability to bind to DNA, since PML::RARA with a missense mutation (C88A) that prevents the binding of RARA to DNA eliminates aberrant replating. Currently, however, there is no consensus regarding the genomic binding sites of PML::RARA in myeloid progenitor cells and APL cells. Also, the transcriptional and epigenetic consequences of PML::RARA expression in primary hematopoietic cells remain unclear. To define the genomic binding sites of PML::RARA, we generated V5 epitope tagged PML::RARA constructs in MSCV-based retroviral vectors, and transduced mouse hematopoietic stem progenitor cells (HSPCs) with retroviruses containing V5-PML::RARA WT, V5-PML::RARA C88A, or an empty vector. Anti-V5 ChIP-seq revealed that PML::RARA binds to 2,677 distinct loci in primary mouse hematopoietic cells, and 2,064 sites in primary human hematopoietic cells. We previously demonstrated that Gata2 expression is induced with PML::RARA, and indeed, we identified strong PML::RARA binding to the distal Gata2 enhancer in both human and mouse HSPCs. Surprisingly, ATAC-seq revealed that the vast majority of PML::RARA binding sites are found in regions that are chromatin-accessible even in WT promyelocytes, suggesting that these regions are “open” prior to PML::RARA expression, perhaps because of the normal binding of “pioneer” transcription factors to these regions in WT cells. Indeed, evaluation of transcription factor binding motifs in the regions occupied by PML::RARA revealed significant enrichment for RARA binding motifs, and also GATA motifs. We therefore performed ChIP-seq on GATA2 in HSPCs, which confirmed enrichment in binding at the same sites. These data suggested that PML::RARA and GATA2 may bind in close proximity at hundreds of loci in the genome. To test this, we performed proximity labeling studies followed by mass spectrometry using PML::RARA WT vs. PML::RARA C88A fused to the biotin ligase TurboID in primary HSPCs. This approach revealed that the interactions of several proteins with PML::RARA (including GATA2) are dependent on the binding of PML::RARA to DNA; this suggests that DNA may in fact act as a scaffold to stabilize these protein:protein interactions ( Figure 1). To determine whether Gata2 may induce increased chromatin accessibility following PML::RARA expression, we performed ATAC-seq following Gata2 or PML::RARA overexpression in WT hematopoietic progenitor cells. 3,157 regions shared increased accessibility with either PML::RARA or Gata2 overexpression. Moreover, at a transcriptional level, we found that 535 (27.4%) of PML::RARA differentially expressed genes were coordinately regulated by Gata2 in WT cells. This suggests that PML::RARA indirectly regulates many of its gene targets via the upregulation of Gata2, and that Gata2 may be required for PML::RARA to initiate its transcriptional program. To test this, we performed serial replating assays using cells where Gata2 was knocked out either before or after PML::RARA expression. Consistent with our previous study, knock out of Gata2 following PML::RARA expression led to increased self-renewal capacity. However, knock out of Gata2 before PML::RARA expression prevented self-renewal. These data suggest a model in which GATA2 “opens” specific chromatin regions in early myeloid cells, which are then accessible to RARA or PML::RARA. The two proteins interact at these sites, tethered by their mutual interactions to their respective DNA binding motifs. PML::RARA binding promotes a pre-leukemic transcriptional program mediated in part by positively regulating Gata2. Gata2 is therefore initially required for PML::RARA to establish its transcriptional program. However, once that program is established, Gata2 paradoxically acts as a tumor suppressor, performing a normal role to limit proliferative excess in hematopoietic stem and progenitor cells.

Phase Separation of PML/RARα Microspeckles Governs Transcriptional Dysregulation through Genomic Rewiring of BRD4 in Acute Promyelocytic Leukemia

Introduction: Abnormal nuclear morphologies are frequently observed in cancer cells, which have been implicated in biological reactions such as gene regulation and DNA damage repair. However, the nature of altered nuclear structures, particularly molecular components determining the specificity, regulatory functions and oncogenic activity of nuclear compartments, has remained largely enigmatic and awaits characterization. In acute promyelocytic leukemia (APL), the PML/RARα fusion oncoprotein, which is produced by t(15;17), has been long observed under the microscope for its ability in destroying PML nuclear bodies (NBs) and subsequently forming microspeckles. Restoration of PML NBs has been reported during the treatment with retinoic acid (RA) or arsenic trioxide (ATO). Despite being long observed, how PML/RARα-mediated microspeckles are formed and linked to APL leukemogenesis remains a mystery. To address this knowledge gap, we present a comprehensive analysis of the driving forces and constituent components of PML/RARα-assembled microspeckles and their functional implications in transcriptional dysregulation and leukemogenesis. Methods: High-resolution imaging techniques, multi-omics strategies, integrative bioinformatics analysis, and in-depth functional validation are used in this study. We employ Immunofluorescence, Fluorescence recovery after photobleaching and in vitro droplet formation assays to determine the chemical and physical properties and constituent components of PML/RARα microspeckles. Co-IP and mass spectrometry are performed to identify interactome of PML/RARα. ChIP-seq is utilized to determine the exact genomic regions where PML/RARα microspeckles occur and to evaluate the impact on chromatin occupancy of PML/RARα and its co-factors upon genetic or pharmacological perturbation. RNA-seq is performed to evaluate the transcriptional output of repression of condensates mediated co-factor activity. Functional experiments, including CCK-8 assay and Flow cytometry, are used to assess the pathological functions of PML/RARα assembled and BRD4 recruited microspeckles. Results: We uncover the biophysical mechanism of liquid-liquid phase separation (LLPS) underlying the assembly of PML/RARα microspeckles and elucidate their role in APL leukemogenesis. Our findings reveal that PML/RARα co-assembles with BRD4 to form de novo nuclear phase-separated condensates, which distinguish them from PML nuclear bodies. PML/RARα and BRD4 co-assembled condensates exhibit preferential occupancy on super-enhancers and broad-promoters, targeting genes essential for APL leukemogenesis. Mechanically, PML/RARα incorporates BRD4 into nuclear condensates, thereby facilitating its chromatin binding and redistribution. Importantly, blockage of the condensate-mediated co-activator BRD4 activity suppresses APL cell proliferation and induces apoptosis, thereby impairing PML/RARα-driven leukemogenesis. Finally, perturbation of LLPS depletes the chromatin co-occupancy of PML/RARα and BRD4 and attenuates their target gene activation, reinforcing the importance of LLPS in transcriptional dysregulation. Conclusions: In this study, we have provided a comprehensive analysis of PML/RARα-assembled microspeckles, shedding light on their driving forces, constituent components, and regulatory function. Our findings have resolved the long-standing cognitive stagnation regarding the morphological characteristics of PML/RARα microspeckles. We have demonstrated that PML/RARα forms de novo phase-separated condensates, which selectively recruit the co-activator BRD4 to gene loci with both super enhancers and broad promoters. This aberrant recruitment leads to dysregulated transcriptional programs and the development of malignant phenotypes. Importantly, our present study is the first to reveal the biophysical nature of PML/RARα-mediated microspeckles and elucidate the fundamental regulatory mechanism underlying their formation. We have also highlighted the role of PML/RARα phase separation in the pathogenesis of APL leukemogenesis. Therefore, our study provides valuable biophysical insights into the molecular basis for PML/RARα to exert its oncogenic activity.

Leukemia cells accumulate zinc for oncofusion protein stabilization

Acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) are both hematological malignancies characterized by genetic alterations leading to the formation of oncofusion proteins. The classical chromosomal aberrations in APL and CML result in the PML-RARα and BCR-ABL1 oncofusion proteins, respectively. Interestingly, our flow cytometric analyses revealed elevated free intracellular zinc levels in various leukemia cells, which may play a role in stabilizing oncofusion proteins in leukemia and thus support cell proliferation and malignancy. Long-term zinc deficiency resulted in the degradation of PML-RARα in NB4 cells (APL cell line) and of BCR-ABL1 in K562 cells (CML cell line). This degradation may be explained by increased caspase 3 activity observed in zinc deficient cells, whereas zinc reconstitution normalized the caspase 3 activity and abolished zinc deficiency-induced oncofusion protein degradation. In NB4 cells, fluorescence microscopic images further indicated enlarged and enriched lysosomes during zinc deficiency, suggesting increased rates of autophagy. Moreover, NB4 cells exhibited increased expression of the zinc transporters ZIP2, ZIP10 and ZnT3 during zinc deficiency and revealed excessive accumulation of zinc in contrast to healthy peripheral blood mononuclear cells (PBMCs), when zinc was abundantly available extracellularly. Our results highlight the importance of altered zinc homeostasis for some characteristics in leukemia cells, uncover potential pathways underlying the effects of zinc deficiency in leukemia cells, and provide potential alternative strategies by which oncofusion proteins can be degraded.

Induction of ferroptosis cell death in acute promyelocytic leukemia cell lines (NB4 and HL-60) using hydrothermally synthesized ZnO NPs in the presence of black cardamom extract

This paper reports an effective, one-step, and eco-friendly method for producing ZnO nanoparticles (NPs) via the hydrothermal procedure. In the synthesis procedure, where 1,8-cineole is the dominant component, an optimized concentration of black cardamom extract is utilized as a capping and reducing agent. The decision to use this extract can be justified because it has a quick reducing capacity. Scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Dynamic Light Scattering (DLS), and Fourier-transform infrared spectroscopy (FT-IR) have all been used to analyze the NPs. Furthermore, healthy cells (PBMCs) and the Acute Promyelocytic Leukemia cell lines (NB4 and HL-60) were treated to varied concentrations of ZnO NPs for 24 and 48 h. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) test was then used to determine the cell viability. Oxidative and ferroptosis markers were evaluated using commercial kits. Gene expression and cell death were measured using qRT-PCR and flow cytometry. Also, the binding energy and van der Waals interactions of ZnO NP-protein complexes were calculated using molecular docking. On leukemic cells, ZnO NPs displayed growth-inhibiting effects in a time and dose-dependent manner. By considerably boosting lipid-ROS, intracellular iron, ACSL4, and p53 while significantly lowering GSH and GPx activity levels, as well as SLC7A11 and GPx4 mRNA, the ZnO NPs accelerated the ferroptotic cell death. On the other hand, ZnO NPs showed no toxicity to healthy cells. So, based on our research, synthesized ZnO NPs can induce ferroptotic cell death in leukemic cells.

Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA

To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

MiR-603 promotes cell proliferation and differentiation by targeting TrkB in acute promyelocytic leukemia.

Arsenic trioxide (ATO) treatment effectively prolongs the overall survival of patients with acute promyelocytic leukemia (APL). Mutations in the oncogene PML::RARA were found in patients with ATO-resistant and relapsed APL. However, some relapsed patients do not have such mutations. Here, we performed microarray analysis of samples from newly diagnosed and relapsed APL, and found different microRNA (miRNA) expression patterns between these two groups. Among the differentially expressed miRNAs, miR-603 was expressed at the lowest level in relapsed patients. The expression of miR-603 and its predicted target tropomyosin-related kinase B (TrkB) were determined by PCR and Western blot. Proliferation was measured using an MTT assay, while apoptosis, cell cycle and CD11b expression were analyzed using flow cytometry. In APL patients, the expression of miR-603 was negatively correlated with that of TrkB. miR-603 directly targeted TrkB and downregulated TrkB expression in the APL cell line NB4. miR-603 increased cell proliferation by promoting the differentiation and inhibiting the apoptosis of NB4 cells. This study shows that the miR-603/ TrkB axis may be a potent therapeutic target for relapsed APL.

MiR-125b-5p-MAPK1-C/EBPα feedback loop regulates all-trans retinoic acid resistance in acute promyelocytic leukemia

BackgroundVarious studies have highlighted the significance of miR-125b-5p in tumour chemotherapy resistance; However, whether miR-125b-5p is associated with all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukemia (APL) has not been reported. MethodsDrug-resistance-related factors in APL were predicted using the DRESIS database. The expression levels of miR-125b-5p in ATRA-sensitive and ATRA-resistant APL cells were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A nitrotetrazolium blue (NBT) reduction assay and flow cytometry (FCM) were used to detect the effect of miR-125b-5p on ATRA resistance in APL cells. An APL xenograft tumour mouse model was established to determine the effect of miR-125b-5p on ATRA resistance. A dual-luciferase gene reporter assay, qRT-PCR, and western blotting verified the regulation by miR-125b-5p of its target gene, MAPK1, and the MAPK1 downstream factor, C/EBPα. An NBT reduction assay and FCM were used to detect the effect of C/EBPα on ATRA resistance in APL cells. Western blotting and qRT-PCR were used to assess the regulation of miR-125b-5p and MAPK1 by C/EBPα. ResultsmiR-125b-5p expression levels were dramatically increased in ATRA-resistant APL cells. Both in vitro and in vivo experiments revealed that miR-125b-5p overexpression enhanced ATRA resistance in APL. miR-125b-5p promoted ATRA resistance by sponging MAPK1. C/EBPα was negatively regulated by miR-125b-5p, which in addition, regulated ATRA resistance in APL cells. C/EBPα also regulated the miR-125b-5p-MAPK1 axis. ConclusionThe findings of this study indicate that the miR-125b-5p-MAPK1-C/EBPα feedback loop regulated ATRA resistance in APL. Thus, miR-125b-5p may be a promising target for treating ATRA resistance in APL.

Gallium(III)- and Thallium(III)-Encapsulated Polyoxopalladates: Synthesis, Structure, Multinuclear NMR, and Biological Activity Studies.

Three gallium(III)- and thallium(III)-containing polyoxopalladates (POPs) have been synthesized and structurally characterized in the solid state and in solution, namely, the phosphate-capped 12-palladate nanocubes [XPd12O8(PO4)8]13- (X = GaIII, GaPd12P8; X = TlIII, TlPd12P8) and the 23-palladate double-cube [Tl2IIIPd23P14O70(OH)2]20- (Tl2Pd23P14). The cuboid POPs, GaPd12P8 and TlPd12P8, are solution stable as verified by the respective 31P, 71Ga, and 205Tl nuclear magnetic resonance (NMR) spectra. Of prime interest, the spin-spin coupling schemes allowed for an intimate study of the solution behavior of the TlIII-containing POPs via a combination of 31P and 205Tl NMR, including the stoichiometry of the major fragments of Tl2Pd23P14. Moreover, biological studies demonstrated the antitumor and antiviral activity of GaPd12P8 and TlPd12P8, which were validated to be as efficient as cis-platinum against human melanoma and acute promyelocytic leukemia cells. Furthermore, GaPd12P8 and TlPd12P8 exerted inhibitory activity against two herpetic viruses, HSV-2 and HCMV, in a dose-response manner.

Apoptotic Cell-Derived CD14(+) Microparticles Promote the Phagocytic Activity of Neutrophilic Precursor Cells in the Phagocytosis of Apoptotic Cells.

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell-cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells.

Syntheses, characterization of Imidazo[4,5-f][1,10]phenanthroline derivative and As (III) complex, In vitro evaluation, the determine of apoptosis mechanism; theoretical and quantum studies

In the present work, a novel ligand, (4-amino-3-methoxybenzaldehyde)-1H-imidazo[4,5-f][1,10] phenanthroline (AMIP) based on the formation of an imidazole ring and [As(AMIP)](Cl)]Cl2 was synthesized and characterized through Fourier-transform infrared (FT-IR) spectroscopy. In addition, the anticancer activity of [As(AMIP)](Cl)]Cl2 complex was tested against human acute promyelocytic leukemia cells (HL-60) and granulocytic differentiation of human promyelocytic leukemia cells (NB4). The MTT results showed IC50 values of 7.31 ± 15 and 8.4 ± 62 μM for As (III) complex against NB4 and HL-60, respectively. In a study on both cell lines, As (III) reduced gene expression Bcl-2 and Bcl-xl, increased Bax and Bad, and activated caspase-3, resulting in apoptosis. The dye Hoechst 33342 showed increased apoptosis induction in the NB4 cell line than in the HL-60 cell line. BrdU test exhibited an inhibitory effect on DNA synthesis. The protein levels of caspase-3 were enhanced in a dose-dependent manner in both HL-60 and NB-4 cell lines. Based on the Annexin V-FITC/PI assay, apoptosis was the primary mechanism of cell death. The real-time polymerase chain reaction (real-time PCR) results demonstrated that the expression level of Atg 12, Atg 7, Lc 3, and Beclin-1 genes, antiapoptotic genes including Bcl-2 and Bcl-xl, and apoptotic genes containing BAD and BAX was improved. Furthermore, the hemolysis assay showed the anti-hemolytic activity of the complex. Moreover, quantum chemical calculations were utilized to optimize all structures, and the output results were employed to study molecular docking of compounds with caspase-3 tethered to irreversible inhibitor (PDB ID: 1NMS) and DNA with sequence d(CCGTCGACGG) (PDB ID:423D). The results showed that As (III) complex had the lowest-energy conformation, suggesting the optimal interaction.

Angiogenesis, coagulation, and fibrinolytic markers in acute promyelocytic leukemia (NB4): An evaluation of melatonin effects.

Melatonin is a powerful biological agent that has been shown to inhibit angiogenesis and also exerts anti-inflammatory effects. It is well known that new blood vessel formation (angiogenesis) has become an urgent issue in leukemia as well as solid tumors. Acute promyelocytic leukemia (APL) is a form of liquid cancer that manifests increased angiogenesis in the bone marrow of patients. Despite high-rate curable treatment with all-trans-retinoic acid (ATRA) and recently arsenic-trioxide (ATO), early death because of hemorrhage, coagulopathy, and Disseminated intravascular coagulation (DIC) remains still a concerning issue in these patients. It is, therefore, urgent to seek treatment strategies with antiangiogenic capabilities that also diminish coagulopathy and hyperfibrinolysis in APL patients. In this study, a coculture system with human umbilical vein endothelial cells (HUVECs) and NB4 APL cells was used to investigate the direct effect of melatonin on angiogenesis and its possible action on tissue factor (TF) and tissue-type plasminogen activator-1 (TPA-1) expression. Our experiments revealed that HUVEC-induced angiogenesis by cocultured NB4 cells was suppressed when melatonin alone or in combination with ATRA was added to the incubation medium. Melatonin at concentrations of 1 mM inhibited tube formation of HUVECs and also decreased interleukin-6 secretion and VEGF mRNA expression in HUVEC and NB4 cells. Taken together, the results of this study demonstrate that melatonin inhibits accelerated angiogenesis of HUVECs and ameliorates the coagulation and fibrinolysis indices stimulated by coculturing with NB4 cells.

Critical role of MXRA7 in differentiation blockade in human acute promyelocytic leukemia cells

The biology of the matrix remodeling associated 7 (MXRA7) gene had been ill-defined. Inspired by public datasets and domestic findings concerning MXRA7, we hypothesized that MXRA7 might be involved in the pathogenesis of leukemia. This study aimed to investigate the role and effect of MXRA7 expression in acute promyelocytic leukemia (APL) cells differentiation and behavior. Bioinformatics of public datasets revealed MXRA7 mRNA was expressed highly in acute myeloid leukemia (AML), especially in APL. High expression of MXRA7 was associated with poor overall survival of AML patients. We confirmed MXRA7 expression was upregulated in APL patients and cell line. Knockdown or overexpression of MXRA7 did not affect the proliferation of NB4 cells directly. Knockdown of MXRA7 in NB4 cells promoted drug induced cell apoptosis, while overexpression of MXRA7 had no obvious influence on the drug induced cell apoptosis. Lowering MXRA7 protein levels in NB4 cells promoted ATRA-induced cell differentiation possibly through decreasing PML-RARα level and increasing PML and RARα levels. Correspondingly, overexpression of MXRA7 showed the consistent results. We also demonstrated that MXRA7 altered the expression of genes involved in leukemic cell differentiation and growth. Knockdown of MXRA7 upregulated the expression levels of C/EBPB, C/EBPD and UBE2L6, and downregulated the expression levels of KDM5A, CCND2 and SPARC. Moreover, knockdown of MXRA7 inhibited the malignancy of NB4 cells in NOD-SCID mice model. In conclusion, this study demonstrated that MXRA7 influences the pathogenesis of APL via regulation of the cell differentiation. The novel findings about the role of MXRA7 in leukemia not only shed lights on the biology of this gene, but also proposed this gene as a new target for APL treatment.