Untreated NB4 Cells Research Articles

Effect of Shikonin on Autophagy and Apoptosis of Human Promyelocytic Leukemia Cells

To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism. Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins. Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05). Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.

PML-RARα/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia

Many different molecular mechanisms have been associated with PML-RARalpha-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARalpha binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARalpha with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARalpha/RXR binding sites or at nearby target genes. Our results suggest that PML-RARalpha/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential.

Silencing of Bc1-2 gene by small hairpin RNA inhibits growth of NB4 cells

To investigate the effects of small hairpin RNA(shRNA) targeting Be1-2 on the growth of NB4 cell line. Two of pairs oligonucleotides for short hairpin expression targeting the coding region of Bc1-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector downstream of U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as a negative control. Recombinant expression vector was identified by enzyme cutting and sequencing. Bc1-2 shRNAs were transfected into NB4 cell with Lipofectamine 2000. Western-Blot of Bc1-2 protein expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a MTT assay. Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-Blot assay showed that the expression level of Bc1-2 protein in NB4 cells decreased after Bc1-2 shRNAs treatment. There was no difference in Bc1-2 protein levels between control shRNA and untreated cells. Viable cells at 72 and 96 h after treatment with Bc1-2 shRNAs were less than that after treatment with control shRNAs and untreated NB4 cells, respectively (P<0.05). Control shRNA had no significant effect on the growth of the cells. Bc1-2 shRNA could effectively inhibit the growth of NB4 cells. Bc1-2 shRNA might be an effective anti-leukemia candidate.

Rig-G Protein Links Retinoid and Interferon Signal Pathways To Inhibit Cell Cycle through Increased p27 and p21 Protein Levels.

Over the last two decades, the molecular mechanisms of all trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) cells have been greatly explored The Rig-G gene was first isolated from an ATRA-treated APL cell line NB4. The expression of Rig-G was undetectable in untreated NB4 cells, but could be dramatically induced by ATRA treatment. Interestingly, database research showed that Rig-G represented a member of interferon-inducible (IFI) gene family. Moreover, a synergistic induction of Rig-G mRNA in NB4 cells by ATRA and interferons (IFNs) indicated a possible role of Rig-G in crosstalk between these two signaling pathways. In this work, we show that Rig-G is indeed a direct target of STAT1 protein, a critical transcription factor in regulating IFN responses, and involved in both ATRA and interferon pathways. By stable transfection of Rig-G in U937 cells, we find that Rig-G protein can significantly accumulate the cells at G1/S transition and inhibit cell proliferation through enhancing some important cell cycle regulators. Further studies demonstrate that Rig-G can physically interact with JAB1 (Jun activating domain binding protein). It can decrease JAB1-enhanced AP-1 transactivities through preventing JAB1 from entering the nucleus, and impair JAB1-dependent p27 nuclear export and degradation, resulting in a nuclear accumulation of p27. In addition, we show that Rig-G can also downregulate c-Myc followed by an upregulation of p21, another major negative player of cell growth. Taken together, it seems that Rig-G may be one of the first experimentally proven molecular mediators responsible for IFNs-dependent growth-suppressive responses, and it can link both IFN- and ATRA-triggered intracellular pathways to synergistically inhibit cell growth in many cell types.

IL-8 and MCP-1 Regulate the Transmigration of Acute Promyelocytic Leukemia toward Alveolar Type II Cells.

All-trans retinoic acid (ATRA) can induces complete remission in patients with acute promyelocytic leukemia (APL), and can also induce retinoic acid (RA) syndrome in upto 30 % of patients. Massive infiltration of APL cells can be found in the alveolar spaces in the patients with RA syndrome, indicating that ATRA-treated APL cells can migrate from blood vessels into the alveolar space and cause a cascade of reactions which results a clinical picture similar to adult respiratory distress syndrome. In this study, we determine the role of IL-8 and monocytes chemotactic protein-1 (MCP-1) in the cross-talk between APL cells and alveolar cells, and their role in the pathophysiology of RA syndrome. In the transmigration assay, ATRA-treated APL cells (NB4 cells) can transmigrate toward alveolar type II cells (A549 cells) in a dose dependent manner (ATRA: 0.01 to 1 mM), which is also in parallel with increased granulocytic differentiation. Chemoattractant activity can be found in the conditioning medium (CM) of A549 cells in the time dependent manner (1-3 days). When exposed to either IL-8 or MCP-1, chemotactic effect can also be found in the ATRA -treated NB4 cells, but not in untreated NB4 cells. We further determined the concentration of IL-8 or MCP-1 in the CM by ELISA. Our data show that the levels of both cytokines were progressively increased in the CM of A549 cells. Synergistic effect was found when A549 cells were co-cultured with NB4 cells. In the presence of ATRA, the levels of IL-8 and MCP-1 were further increased in the CM of co-culture of NB4 cells and A549 cells (p<0.05 & p<0.05; respectively). The chemotactic activity in CM of A549 cells was partially inhibited when the CM was neutralized with anti-IL-8 or anti-MCP-1 antibodies, respectively. In binding assay by flowcytometry, we further demonstrated that both IL-8 and MCP-1 were bound by the ATRA-treated NB4 cells, but not in un-treated NB4 cells. We conclude that both IL-8 and MCP can induce ATRA-treated APL transmigrating toward alveolar space and causing cascade of reactions leading to RA syndrome.

Differential expression of dendritic cell markers by all- trans retinoic acid on human acute promyelocytic leukemic cell line

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for naive T cells and play an important role in cancer immunology. All- trans retinoic acid (ATRA) is known to be a differentiating agent in the treatment of acute promyelocytic leukemia (APL). In this study, we investigated whether ATRA can differentiate the retinoic acid (RA)-sensitive promyelocytic leukemic cell line, NB4, to DC-like cells and whether these differentiated cells can activate T cells. NB4 cells were differentiated to myeloid cells by 4, 6, and 8 days of ATRA treatment. NB4 cells up-regulated markers found in DCs, including HLA-DR, costimulatory molecules (CD80 and CD86), adhesion molecules (CD40), and chemokine receptors (CCR6) when cultured for 8 days in the presence of 1 μM ATRA. Upregulation of CD83 was also detected on the surface of ATRA-treated NB4 cells versus untreated cells. The addition of cytokines alone, such as GM-CSF or CD40 ligand, did not affect the expression of CD83 in untreated NB4 cells but they up-regulated CD83 in ATRA-treated cells. CD11b was coexpressed with CD80, CD83, and CD86 in ATRA-treated NB4 cells. In a functional assay, ATRA-treated NB4 cells stimulated T cell proliferation when challenged with Staphylococcus enterotoxin B. These results suggest that the differentiation of NB4 cells by ATRA causes the cells to express DC markers, and that ATRA-differentiated NB4 cells are able to present antigens to T cells.

Selective up-regulation of phospholipase C-β2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.

All-trans retinoic acid regulates adhesion mechanism and transmigration of the acute promyelocytic leukaemia cell line NB-4 under physiologic flow.

The success of all-trans retinoic acid (ATRA) in the therapy of acute promyelocytic leukaemia (APL) has received increased attention. Unfortunately, life-threatening multiorgan failure commonly occurs, i.e. retinoic acid syndrome, and is thought to be the result of organ infiltration by leukaemic cells. We hypothesized that ATRA-induced differentiation of APL cells leads to adhesion receptor alterations responsible for leucocyte extravasation from the blood into tissue. Changes in adhesive properties of the APL cell line NB-4 in response to ATRA were investigated using a parallel plate flow chamber under conditions that recapitulate physiologic flow conditions. Untreated NB-4 cells initially tether and roll on activated human umbilical vein endothelial cell monolayers using a combination of E-selectin, P-selectin and alpha4 integrin. After ATRA treatment, > 80% of initial NB-4 cell attachment to endothelial cells was E-selectin dependent. Stable arrest (firm adherence) of NB-4 cells on activated endothelium was also altered by ATRA treatment. Untreated NB-4 cells used alpha4 integrin to arrest on endothelium, but beta2 integrin dependent arrest was induced by ATRA. With the acquisition of beta2 integrin function, ATRA-treated cells acquired the ability to transmigrate through activated endothelium. Thus, ATRA dramatically altered the adhesion phenotype on NB-4 cells: ATRA induced rolling largely attributable to E-selectin, abrogated alpha4 integrin dependent rolling, and promoted acquisition of beta2 integrin dependent firm adherence and transmigration. These findings represent novel cellular and differentiation effects of ATRA, and, to our knowledge, are the first demonstration that a therapeutic agent differentially regulates alpha4 and beta2 integrin on the same leucocyte.

19-nor Vitamin-D Analogs: A New Class of Potent Inhibitors of Proliferation and Inducers of Differentiation of Human Myeloid Leukemia Cell Lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 × 10−9 to 4 × 10−11 mol/L when using the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 × 10−11mol/L; in contrast, the 1,25D3 produced an ED50of 10−9 mol/L with the HL-60 target cells. Ro 25-9716 (10−9 mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10−8 mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1v 48% of the untreated control cells). The p27kip-1, a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10−7 mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 × 10−11mol/L) and their expression of CD11b was enhanced (80% positive [10−9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10−9mol/L) and all-trans retinoic acid (ATRA, 10−7 mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 × 10−9 mol/L] and Kasumi-1 [ED50, 5 × 10−10 mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27kip-1. In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

19-nor Vitamin-D Analogs: A New Class of Potent Inhibitors of Proliferation and Inducers of Differentiation of Human Myeloid Leukemia Cell Lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 × 10−9 to 4 × 10−11 mol/L when using the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 × 10−11mol/L; in contrast, the 1,25D3 produced an ED50of 10−9 mol/L with the HL-60 target cells. Ro 25-9716 (10−9 mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10−8 mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1v 48% of the untreated control cells). The p27kip-1, a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10−7 mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 × 10−11mol/L) and their expression of CD11b was enhanced (80% positive [10−9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10−9mol/L) and all-trans retinoic acid (ATRA, 10−7 mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 × 10−9 mol/L] and Kasumi-1 [ED50, 5 × 10−10 mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27kip-1. In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes.

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we call RIG-G (for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression of RIG-G is undetectable. ATRA treatment induces the transcriptional expression of RIG-G relatively late (12-24 hr) in a protein synthesis-dependent manner, whereas IFN-alpha induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology between RIG-G and several IFN-stimulated genes in human (ISG54K, ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescence in situ hybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction of RIG-G expression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.

In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

In Vitro Studies on Cellular and Molecular Mechanisms of Arsenic Trioxide (As2O3) in the Treatment of Acute Promyelocytic Leukemia: As2O3 Induces NB4 Cell Apoptosis With Downregulation of Bcl-2 Expression and Modulation of PML-RARα/PML Proteins

It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.