Differentiation Of Human Myeloid Cells Research Articles

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

Ethanolic extract of Euphorbia Gypsicola induces differentiation and apoptosis in human myeloid leukemia K562 cells

IntroductionThe Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. MethodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. ResultsThe E. gypsicola extract exhibited potent medication properties with IC50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. ConclusionThese findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia.

Humanized Patient-derived Xenograft Models of Disseminated Ovarian Cancer Recapitulate Key Aspects of the Tumor Immune Environment within the Peritoneal Cavity.

huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine

BackgroundCeefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. MethodsU937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. ResultsCeefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. ConclusionsThese results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine.General significance:This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

Effects of olive oil-based propolis, caffeic acid phenethyl ester, and methylprednisolone on differentiation of human acute myeloid leukemia cells

Effects of olive oil-based propolis, caffeic acid phenethyl ester, and methylprednisolone on differentiation of human acute myeloid leukemia cells

Naturally Occurring Bicoumarin Compound Daphnoretin Inhibits Growth and Induces Megakaryocytic Differentiation in Human Chronic Myeloid Leukemia Cells.

Daphnoretin extracted from the stem and roots of Wikstroemia indica (L.) C.A. Mey has been shown to possess antiviral and antitumor activities. Herein, we hypothesized that daphnoretin might induce megakaryocytic differentiation, thereby inhibiting the proliferation of cells and serving as a differentiation therapy agent for chronic myeloid leukemia (CML). Daphnoretin-treated K562 and HEL cells were examined for growth inhibition, cell morphology, and megakaryocyte-specific markers. Potential mechanisms of megakaryocytic differentiation of daphnoretin-treated K562 cells were evaluated. The results showed that daphnoretin inhibited the growth of K562 and HEL cells in a dose- and time-dependent manner. Flow cytometry analyses revealed that daphnoretin treatment slightly increased the proportion of sub-G1 and polyploid cells compared to that of dimethyl sulfoxide (DMSO)-treated control cells. Morphological examination showed that daphnoretin-treated K562 and HEL cells exhibited enlarged contours and multinucleation as megakaryocytic characteristics compared to DMSO-treated control cells. Daphnoretin treatment also dramatically enhanced the expression of megakaryocytic markers CD61 and CD41. Under optimal megakaryocytic differentiation conditions, daphnoretin increased the phosphorylation of STAT3 but not STAT5. In summary, daphnoretin inhibited cell growth and induced megakaryocytic differentiation in K562 and HEL cells. The efficacy of daphnoretin in vivo and in patients with CML may need further investigations for validation.

Natural Products as Mcl-1 Inhibitors: A Comparative Study of Experimental and Computational Modelling Data

The human myeloid leukemia cell differentiation protein (hMcl-1) is an anti-apoptotic multi-partner protein, belonging to the B-cell lymphoma-2 (Bcl-2) family of proteins. Studies have linked hMcl-1 alleviated expression with resistance to hemopoietic chemotherapeutics, which makes it a key drug target in blood cancers. However, most of the developed small- to medium-sized hMcl-1 inhibitors have typical off-target activity towards other members of the Bcl-2 family. To improve the hMcl-1 inhibitor design, especially exploring a suitable scaffold with pharmacophoric features, we focused on natural hMcl-1 inhibitors. To date, seven classes of natural compounds have been isolated, which display a low micromolar affinity for hMcl-1 and have limited biophysical studies. We screened hMcl-1 co-crystal structures, and identified nine co-crystal structures of hMcl-1 protein, which were later evaluated by multiple receptor conformations (which indicates that the differences between hMcl-1 in crystal structures are low (RMSD values between 0.52 and 1.13 Å, average RMSD of 0.638–0.888 Å, with a standard deviation of 0.102–0.185Å)), and multiple ligand conformations (which led to the selection of the PDB structure, 3WIX (RMSD value = 0.879 Å, standard deviation 0.116 Å), to accommodate various Mcl-1 ligands from a range of co-crystal PDB files) methods. Later, the three adopted docking methods were assessed for their ability to reproduce the conformation bound to the crystal as well as predict trends in Ki values based on calculated RMSD and docking energies. Iterative docking and clustering of the docked pose within ≤1.0 Å was used to evaluate the reproducibility of the adopted docking methods and compared with their experimentally determined hMcl-1 affinity data.

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

6,7-Dimethoxycoumarin Influences the Erythroid Differentiation of Human Chronic Myelogenous Leukemia K562 Cells through Regulating FOXO3/p27 Signal Pathway.

Objective To study the pharmacological activity and the mechanism of action of natural compounds derived from 6,7-dimethoxycoumarin on the differentiation of human chronic myeloid leukemia K562 cells. Methods We use MTT assay (Sigma-Aldrich, USA) to detect cell viability; use flow cytometry to analyze DNA content for cell cycle analysis; use benzidine staining to synthesize hemoglobin to determine K562 cell differentiation; use western blot analysis and qPCR to detect the expression levels of FOX03, P27, CDK4, and their phosphorylation; and use the AOBS laser scanning confocal system (Leica, Wetzlar, Germany) to analyze and quantify the number of positive green spots. The statistical methods used are one-way analysis of variance (ANOVA) and Dunnett's test to analyze within and between groups. Results In order to explore the effect of 6,7-dimethoxycoumarin on the differentiation of K562 cell erythrocytes, it was concluded that 6,7-dimethoxycoumarin promotes the differentiation of K562 cell erythrocytes; the proliferation of K562 cells was detected by MTT method, and the results showed that 6,7-dimethoxycoumarin can inhibit the proliferation of K562 cells; to evaluate the effect of 6,7-dimethoxycoumarin on the proliferation of K562 cells, the results showed that 6,7-dimethoxycoumarin increased the expression of FOXO3, P27, CDK4, and CDK65, and decreased the phosphorylation of CDK4 and CDK6 proteins. To further explore the effect of knocking out FOXO3 on cell differentiation, the results show that 6,7-dimethoxycoumarin can reduce the differentiation and proliferation of K562 cells by increasing the expression of FOXO3. Conclusion This study extended the understanding of the pharmacological activity of 6,7-dimethoxycoumarin and may provide a potential new target for the treatment of chronic myelogenous leukemia. However, we still need to further study the specific molecular capabilities of 6.7 dimethylcoumarin to understand their possible capture mechanism.

MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.

Effect of Alpha-Fetoprotein on Differentiation of Myeloid Supressor Cells.

The effect of recombinant alpha-fetoprotein (AFP) on human myeloid suppressor cell (MDSC) differentiation was studied in vitro in the presence of cytokines IL-6 (10 ng/mL) and GM-CSF (10 ng/mL). It was found that AFP at concentrations of 50 and 100 IU/mL increased the number of MDSC (CD33+ HLA-DR-/lowCD11b+) in culture. Analysis of MDSC subpopulations showed that the increase was due to monocytic M-MDSC (HLA-DR-/lowCD33+CD11b+CD14+CD66b-). There was no modulating effect of AFP on granulocytic PMN-MDSC (HLA-DR-/lowCD33+CD11b+CD14-CD66b+). The effects of recombinant AFP on MDSC differentiation were thus demonstrated for the first time.

Effect of Graphene Oxide Nanoparticles on Differentiation of Myeloid Suppressor Cells.

We studied the effect of graphene oxide (GO) nanoparticles on differentiation of human myeloid suppressor cells (MDSC) in an in vitro system. Separated mononuclear cells of healthy donors were induced with cytokines (IL-6 and GM-CSF) into the MDSC phenotype (both polymorphonuclear (PMN-MDSC) and monocyte (M-MDSC) subsets of these cells were taken into account). Pegylated GO nanoparticles (GO-PEG; mean size 569±14 nm, PEG content ~20%) were used. GO-PEG in low concentrations (2.5 and 5 μg/ml) increased the percentage of MDSC in cultures, but reduced their content in high concentration (10 μg/ml). After exposure to GO-PEG (2.5 and 5 μg/ml), the MDSC content increased at the expense of M-MDSC, while the level of PMN-MDSC did not change. The decrease in MDSC levels after exposure to high doses of GO-PEG (10 μg/ml) was due to a decrease in PMN-MDSC. Thus, GO-PEG nanoparticles can oppositely regulate differentiation of MDSC by inhibiting or stimulating differentiation of these cells depending on the concentration.

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated differentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 induces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD inhibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.

Notopterol-induced apoptosis and differentiation in human acute myeloid leukemia HL-60 cells.

Purpose: This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms.Methods: Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright–Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined.Results: Notopterol obviously inhibited the growth of HL-60 cells with an IC50 value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14.Conclusion: This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.

O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis

The addition of a single β-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after β-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.

Induction of apoptosis and erythroid differentiation of human chronic myelogenous leukemia K562 cells by low concentrations of lidamycin.

Apoptosis induction and differentiation of promyelocytic leukemic cells into mature cells are major strategies for the drug-based treatment of leukemia. Lidamycin (LDM) which is a member of the enediyne antibiotic family exhibits extreme cytotoxicity. In the present study, the induction of apoptosis and differentiation in human chronic myeloid leukemia K562 cells by low concentrations of lidamycin were investigated. K562 cells were treated with lidamycin at various concentrations for 48h, and accumulated in the metaphase as determined in previous experiments. Cell viability was determined using a Cell Counting Kit-8 (CCK-8) assay and the IC50 value of lidamycin was 0.1±3.2nM. Induction of apoptosis was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) staining. Growth inhibition and apoptosis induction were observed in cells treated with low concentrations of lidamycin. In addition, western blot analysis revealed that treatment of the K562 cells with lidamycin at low concentrations upregulated the expression of caspase-8 and caspase-3. The induction of differentiation in human chronic myeloid leukemia K562 cells by lidamycin at low concentrations was also investigated. The nitroblue tetrazolium reduction ability of K562 cells was increased following treatment with lidamycin. Low concentrations of lidamycin triggered erythroid differentiation among K562 cells, indicated by morphological changes, increased hemoglobin content, and the expression of cell surface antigens such as CD71. Additionally the expression of GATA-binding factor1 (GATA-1) protein in low concentration lidamycin-treated K562 cells was increased. The results of the present study suggest that a low-concentration lidamycin exerts effects on apoptosis and erythroid differentiation induction by increasing the expression of caspases and GATA-1 protein. Lidamycin may serve a positive role in relevant targeted chemotherapy and may represent a potential candidate for chronic myelogenous leukemia differentiation-inducing treatment.

Phosphorylation of ELAVL1 (Ser219/Ser316) mediated by PKC is required for erythropoiesis

Elavl1 (also known as HuR), an RNA binding protein highly conserved between zebrafish and human, regulates gene expression by stabilizing target mRNA. Our previous studies have uncovered that the predominant isoform elavl1a is required for zebrafish embryonic erythropoiesis. However, the exact mechanism of how elav11 spatiotemporally stabilizes target mRNAs to regulate specific erythropoiesis is not yet understood. Here we show that phosphorylation of elavl1a at Ser219 and Ser316 by PKC is necessarily required for cytosolic shuttling from the nucleus to stabilize gata1 mRNA and thus promotes erythropoiesis. Knockdown of elavl1a resulted in the hindrance of erythropoiesis and Hemin-induced erythroid differentiation of human myeloid leukemia K562 cells. Interestingly, inhibition of PKC reproduced the phenotype seen during zebrafish embryogenesis and erythroid differentiation of myeloid leukemia. Mechanistically, Hemin induced elavl1a export from nuclear to cytoplasmic space in K562 cells in a manner dependent on phosphorylation on Ser219 and Ser316, as overexpression of elavl1a with mutations on Ser219 and Ser316 resulted in erythropoiesis failure. Additionally, co-administration of low doses of elavl1a morpholino (MO) and three PKC inhibitors showed a combined effect in zebrafish embryonic erythropoiesis dysplasia. In conclusion, our study reveals that PKC-mediated phosphorylation of elavl1a at Ser219 and Ser316 sites controls its nucleo-cytoplasmic translocation in zebrafish, thereby regulating embryonic erythropoiesis.

Total Synthesis of (±)-Prostratin

Summary Prostratin, a highly complex diterpene natural product, is a pre-clinical candidate in developing a cure for HIV-1 infection and also a potent and selective inhibitor of several tumors. Despite nearly 40 years of progress, prostratin and related natural products still present formidable challenges for synthetic chemists. Here, we report a total synthesis of (±)-prostratin that proceeds in 23 steps from cyclopentadiene. The synthetic strategy includes rapid assembly of the tricyclic core and highly controlled establishment of the densely functionalized C ring, featuring an array of stereoselective transformations including alkoxide-guided 1,4-additions and cyclopropane formation. This work should enable further explorations of chemical and biological spaces based on prostratin and related natural products.

Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells

4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been demonstrated its anti-tumor effect through inducing differentiation and inhibiting proliferation. Eukaryotic initiation factor 3a (eIF3a) plays a critical role in affecting tumor cell proliferation and differentiation. However, whether eIF3a is implicated in chronic myeloid leukemia cells differentiation remains unclear. Our results demonstrated that eIF3a could be suppressed by ATPR in K562 cells. The results also confirmed that ATPR could arrest cell cycle in G0/G1 phase and induced differentiation. Moreover, over-expression of eIF3a promoted not only protein expression of c-myc and cyclin D1, but also prevented the expression of p-Raf-1, p-ERK and the myeloid differentiation markers CD11b and CD14 and had an influence on inducing the morphologic mature. However, silencing eIF3a expression by small interfering RNA could have an adverse effect on K562 cells. In addition, PD98059 (a MEK inhibitor) could block cell differentiation of CML cells and contributed to the expression of c-myc and cyclin D1. In conclusion, these results indicated that eIF3a played an important role in ATPR-induced cell differentiation in K562 cells, its mechanism might be related to its ability in regulating the activation of ERK1/2 signaling pathway in vitro.