Human Myeloid Leukemia Cell Line Research Articles

Study on the inhibitory and pro-apoptotic effects of different concentrations of total tanshinone alone and in combination with tyrosine kinase inhibitors on human myeloid leukemia cell lines

Objective: To explore the effect and investigate the molecular mechanism of different concentrations of total tanshinones alone and in combination with tyrosine kinase inhibitors (TKIs) on the proliferation inhibition and apoptosis of human myeloid leukemia cell lines. Methods: K562 and Kasumi-1 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the TKIs-resistant strain K562/T315I cell line was constructed in Molecular Medicine Research Center, Beijing Lu Daopei Institute of Hematology. Logarithmic growth phase cells were taken and divided into intervention groups with total tanshinone of 0, 2.19, 4.38, 8.75, 17.50 and 35.00 μg/ml intervention groups, which were inoculated in 96-well plates at a density of 1×104 cells/well and exposed to the drug for 24 h, and a control group treated with dimethyl sulfoxide was also set up simultaneously. All experiments were repeated independently 3-5 times. The proliferative activity of the cells was assessed using the CCK-8 assay, the apoptotic rates were measured by flow cytometry, and the expression levels of apoptosis-regulating proteins Bcl-2 and Bax were analyzed by Western blotting. The cell lines treated and untreated with total tanshinone were subjected to transcriptome sequencing and gene set enrichment analysis to identify differentially expressed genes. Results: The half-inhibitory concentration (IC50) values of 8.75 μg/ml total tanshinone at 24 h for K562, K562/T315I and Kasumi-1 cells were (4.11±0.02), (4.95±0.04) and (3.98±0.01) μg/ml, respectively. When combined with 0.25 μmol/L imatinib, 8.75 μg/ml total tanshinone could enhance the induction of apoptosis effects on K562 and K562/T315I cell lines. After being treated with 4.38, 8.75, and 17.50 μg/ml of total tanshinone for 24 h, compared with the control group, total tanshinone upregulated the expression level of Bax protein, downregulated the expression level of Bcl-2 protein, and decreased the Bcl-2/Bax ratio (all P<0.05). Total tanshinone inhibited the proliferation-related signaling pathway and DNA damage repair pathway of myeloid leukemia cell lines, and activated the signaling pathway that induces apoptosis in leukemia cells. Conclusion: Different concentrations of total tanshinoneinhibites proliferation and promote apoptosis in K562, Kasumi-1 and TKIs-resistant K562/T315I cell lines, and further enhance the anti-leukemic effect when combined with TKIs.

Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

Interrupting an IFN-γ-dependent feedback loop in the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne

ObjectivesTo study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1.MethodsGene knock-out and knock-in mice were...

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

Anticancer activity features of imidazole-based ionic liquids and lysosomotropic detergents: in silico and in vitro studies.

Long-chain imidazole-based ionic liquids (compounds 2, 4, 9) and lysosomotropic detergents (compounds 7, 3, 8) with potent anticancer activity were synthesized. Their inhibitory activities against neuroblastoma and leukaemia cell lines were predicted by the new in silico QSAR models. The cytotoxic activities of the synthesized imidazole derivatives were investigated on the SK-N-DZ (human neuroblastoma) and K-562 (human chronic myeloid leukaemia) cell lines. Compounds 2 and 7 showed the highest in vitro cytotoxic effect on both cancer cell lines. The docking procedure of compounds 2 and 7 into the NAD+ coenzyme binding site of deacetylase Sirtuin-1 (SIRT-1) showed the formation of protein-ligand complexes with calculated binding energies of -8.0 and - 8.1kcal/mol, respectively. The interaction of SIRT1 with compounds 2, 7 and 9 and the interaction of Bromodomain-containing protein 4 (BRD4) with compounds 7 and 9 were also demonstrated by thermal shift assay. Compounds 2, 4, 7 and 9 inhibited SIRT1 deacetylase activity in the SIRT-Glo assay. Compounds 7 and 9 showed a moderate inhibitory activity against Aurora kinase A. In addition, compounds 3, 4, 8 and 9 inhibited the Janus kinase 2 activity. The results obtained showed that long-chain imidazole derivatives exhibited cytotoxic activities on K562 leukaemia and SK-N-DZ neuroblastoma cell lines. Furthermore, these compounds inhibited a panel of molecular targets involved in leukaemia and neuroblastoma tumorigenesis. All these results suggest that both long-chain imidazole-based ionic liquids and lysosomotropic detergents may be an effective alternative for the treatment of neuroblastoma and chronic myeloid leukemia and merit further investigation.

NAMPT Haploinsufficiency Is a Therapeutic Vulnerability in High-Risk Myeloid Malignancies

Monosomy 7 (−7) and del7q are the most common high-risk cytogenetic abnormalities and occur across the spectrum of myeloid disorders. -7/del(7q) also co-occur with other high-risk factors such as mutations or deletion of TP53. Standard, non-specific chemotherapy drugs are less effective in these high-risk patients, resulting in a median survival rate of approximately one year. To identify novel, targeted agents for these neoplasms, we sought “collateral lethal” genes, i.e. essential, druggable genes encoded on chromosome 7 that are haploinsufficient upon their loss of heterozygosity. To this end, we first identified essential genes located on chromosome 7. We mined genome-wide CRISPR/Cas9 screen data from 27 human acute myeloid leukemia (AML) cell lines within the DepMap database and identified 65 chromosome 7-encoded, candidate essential genes in AML. To determine those essential genes with known inhibitors, we intersected these 65 candidates with the Drug-Gene Interaction and canSAR.ai databases. We found that nicotinamide phosphoribosyltransferase ( NAMPT), the rate-limiting enzyme that catalyzes the first step of NAD + biosynthesis from nicotinamide, was one of four essential and druggable targets on chromosome 7. Notably, NAMPT was the only essential gene on chromosome 7 with a first-in-class orally bioavailable inhibitor, KPT 9274, currently in clinical trials (NCT04914845). Moreover, NAMPT DNA copy number, mRNA, and protein levels all negatively correlated with cell line sensitivity to NAMPT loss, implicating NAMPT as a collateral lethal candidate gene. We confirmed that isogenic human AML cell lines CRISPR-engineered to partially reduce NAMPT DNA copy led to reduced NAMPT protein levels, indicating NAMPT is a haploinsufficient gene. Cells with ~50% NAMPT levels were significantly more sensitive to the KPT 9274 treatment, and became less viable and more apoptotic, compared to control locus edited cells. To determine whether TP53 status could alter this vulnerability, we generated cell lines with both TP53 loss and 50% NAMPT loss. We found TP53 mutational status did not attenuate the observed effects of NAMPT inhibition on cell survival. Viability assays with primary patient AML samples confirmed that -7/del(7q) status imparts significantly elevated sensitivity to KPT 9274 treatment than samples with diploid chromosome 7 alleles. In summary, our findings reveal NAMPT heterozygosity as a therapeutic vulnerability in high-risk myeloid neoplasms that warrants clinical follow-up. Further, we provide a framework centered on data-mining for uncovering collateral lethal genes in other cancers with recurrent chromosomal arm level deletions.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

Effects of exposure to alternating low-intensity, intermediate-frequency electric fields on the differentiation of human leukemic cell line U937.

Studying the bioeffects of electric fields have been the subject of ongoing research which led to promising therapeutic effect, particularly in cancer treatment. Here, we investigated the impact of low-intensity, intermediate-frequency alternating electric fields on the differentiation of human myeloid leukemia cell line U937. The results showed a near twofold increase in differentiation of U937 cells treated for 24 h by alternating 600 kHz, 150 V/m electric fields, in comparison to their control groups. This measure was evaluated by latex bead phagocytosis assay, nitro blue tetrazolium test, and cell cycle analysis which revealed a significant shift in the number of cells from G2 +M to G0 +G1 phases. The simulation result for the intracellular field intensity showed around 50% attenuation with respect to the applied external field for our setup which ruled out masking of the applied field by the internal electric noise of the cell. Based on previous studies we postulate a possible calcium-related effect for the observed differentiation, yet the exact underlying mechanism requires further investigation. Finally, our results may offer a potential therapeutic method for leukemia in the future.

Tumor suppressor miR-520a inhibits cell growth by negatively regulating PI3K/AKT signaling pathway in acute myeloid leukemia.

Short regulatory RNAs, called microRNAs (miRNAs), have been found to possess regulatory functions in cancer and, as such, have recently been evaluated for their therapeutic role against various human malignancies. The present work aimed to investigate whether miR-520a can play a therapeutic role in the treatment of human acute myeloid leukemia. Human myeloid leukemia cell lines (Kasumi-1, Kasumi-3, Kasumi-6, BDCM, and K562) and a normal myeloid cell line (NCI-H5N6) were used for the study. Cell lines were subjected to real-time quantitative polymerase chain reaction (RT-qPCR), evaluation of cell viability and proliferation by MTT assay and colony formation assays. Dual acridine orange (AO)/ethidium bromide (EB) staining was applied for transfected K562 cells with miR-negative control (NC) or miR-520a mimics, and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining and flow cytometry were performed to analyze cancer cell apoptosis followed by western blot. Cancerous cell lines exhibited lower gene expression of miR-520a, and its overexpression significantly reduced (p < 0.05) the proliferation and viability of cancer cells. Cancer cells demonstrated the induction of Bax/Bcl-2-mediated apoptosis following miR-520a overexpression. The miR-520a was shown to target the PI3K/AKT signaling pathway in human acute myeloid leukemia to exercise its regulatory role in cancer. The study showed that miR-520a actively regulated cell proliferation in acute myeloid leukemia and illustrated the mechanism by which it exerts its regulatory role, emphasizing the possibility of targeting miR-520a as an efficient therapeutic strategy against human acute myeloid leukemia.

Asteriquinones from Aspergillus sp. GZWMJZ-258 and Their Derivatives.

A new asteriquinone, ochrindole F (1), and five previously reported analogues (2-6) were isolated from the culture of the fungus Aspergillus sp. GZWMJZ-258, an endophyte of Garcinia multiflora. The structure of compound 1 was determined by a spectroscopic analysis. Furthermore, eight new derivatives (7-14) were synthesized from major metabolites 2 and 3. These compounds showed selective antiproliferative activity against the human acute myeloid leukemia (AML) cell line MV4-11, among which compound 12 showed the strongest activity with an IC50 value of 0.14 μM and the highest selectivity with a selectivity index greater than 710. An initial probe of the mechanism of action showed that compounds 12 and 14 could inhibit the expression of FLT-3 in the MV4-11 cell line.

2-Substituted 1,5-benzothiazepine-based HDAC inhibitors exert anticancer activities on human solid and acute myeloid leukemia cell lines

Herein, we report the development of a new series of histone deacetylase inhibitors (HDACi) containing a 2-substituted 1,5-benzothiazepine scaffold. First, a virtual combinatorial library (∼1.6 × 103 items) was built according to a convenient synthetic route, and then it was submitted to molecular docking experiments on seven HDACs isoforms belonging to classes I and II. Integrated computational filters were used to select the most promising ones that were synthesized through an optimized approach, also amenable to generating both racemic and enantioenriched benzothiazepine-based derivatives. The obtained compounds showed potent HDAC inhibitory activity, especially those containing the sulphone moiety, endowed with IC50 in the nanomolar range. In addition, in vitro outcomes of our synthesized compounds demonstrated a cytotoxic effect on U937 and HCT116 cell lines and an arrest in the G2/M phase (13 ≤ IC50 ≤ 18 µM). Finally, Western blot analyses outlined the modulation of the histone acetyl markers such as H3K9/14, acetyl-tubulin, and the apoptotic indicator p21 in both cancer cell lines, disclosing a good HDAC inhibitor activity exerted by the designed items. Given the key role of HDACs in many cellular pathways, which makes these enzymes appealing and “hot” drug targets, our findings highlighted the importance of these 2-substituted 1,5-benzothiazepine-based compounds (both in the reduced and oxidized version) for the development of novel epidrugs.

KOENZİM Q0 İNSAN KRONİK MYELOİD LÖSEMİ K562 HÜCRELERİNİN PROLİFERASYONUNU ENGELLER VE MAPK VE AKT SİNYAL YOLAKLARINI MODÜLE EDER

Objective: This study evaluated the antiproliferative and pro-apoptotic effects of coenzyme Q0 (CoQ0) in human chronic myeloid leukemia K562 cell line. Material and Method: The cytotoxic effect of CoQ0 on human chronic myeloid leukemia cell line, K562 was determined by MTT test. The activity of caspase-3, expression of proteins involved in apoptosis, MAPK and AKT signaling pathways were determined with enzymatic assay and western blot analysis, respectively. Result and Discussion: Results showed that CoQ0 inhibited cell viability of K562 cells at 5 μM and higher concentrations and Bax protein expression was significantly decreased at 12.5 μM concentration of CoQ0. However, CoQ0 did not significantly affect caspase 3 activity and Bcl-2 protein expression. p-c-Raf (Ser259) protein expression was significantly decreased at 12.5 μM of CoQ0. Treatment with 10 μM of CoQ0 induced significantly phosphorylation of p38 MAPK and 12.5 μM CoQ0 caused a nonsignificant decrease in p-ERK1/2 protein expression in K562 cell line. Interestingly, in K562 cells, phosphorylation of Akt (Ser473) was diminished at 12.5 μM of CoQ0, with no change observed in p-Akt (Thr308) protein expression among groups. In conclusion, CoQ0 inhibited cell proliferation and suppressed phosphorylation of c-Raf (Ser259), Akt (Ser473), but not ERK1/2 in K562 cells. There is still a need for new insights into the anticancer mechanisms of CoQ0 and develop treatment strategies for chronic myeloid leukemia.

Scaffold-hopping of Linifanib to Design 6-Phenylisoxazolo[3,4-b]pyridin-3-amine Derivatives as FLT3 Inhibitors for Treating Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the most common type of blood cancer. Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase family. Overexpression of FLT3 was found in 70-100% of patients with acute myeloid leukaemia. FLT3 internal tandem duplication alteration (ITD) and the tyrosine kinase domain (TKD) are the most common molecular alteration in AML, and FLT3 has become a promising drug target for AML. Objective: A series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives F1–F15 with amide bonds as FLT3 inhibitors were designed and synthesized in order to find a new lead compound to treat AML. Methods: We designed an original scaffold-hopping protocol by combing the RECAP tool with the Gilde-Based Core-Hopping tool to design novel FLT3 inhibitors based on Linifanib. Inhibitors assembled were ranked by the docking scores generated by Glide. Compounds undisclosed among the top 10 were selected to design a series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives as FLT3 inhibitors. The kinase inhibitory activities of the fifteen compounds were assayed on FLT3 and FLT3-ITD. The antitumor activities of the structurally modified compounds F1–F15 were evaluated against MOLM-13 and MV4-11, typical FLT3-dependent human AML cells carrying FLT3-ITD mutants and the FLT3-independent human cervical carcinoma cell line HL-60 (harboring wide-type FLT3). Results: Structure–activity relationship (SAR) analysis showed that F14 could inhibit FLT3 and FLT3-ITD by 52% and 45.55%, respectively, at the concentration of 1 ΜF14 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines, MOLM-13, and MV4-11 (harboring FLT3-ITD mutant) with IC50 values of 2.558 μM and 1.785 μM, respectively. Conclusion: F14 could be used as a novel lead compound to further develop FLT3 inhibitors against AML with FLT3-ITD mutant

Utilization of Dairy By-Products as a Source of Functional and Health Compounds-The Role of Ovine Colostrum and Milk Whey on Chronic Myeloid Leukemia Cells.

Nowadays, the search for food products that promote consumers' health has gained interest, and dairy by-products, due to their biological quality, could have a prominent position among products with health benefits. However, little is known about their activity on cancer cells. This study aimed to provide evidence about the effect of ovine colostrum and milk whey on K562 cells, a model of the human chronic myeloid leukemia cell line. The exposure of K562 cells to a single administration of sheep by-products at different concentrations for three days and three treatments for three days was carried out. Using a flow cytometric approach, we found that CD235a expression remained stable in the cells exposed to ovine whey (milk and colostrum) at concentrations ranging from 1 ng/mL to 100 μg/mL, after three days from one or three administrations, respectively. A significant reduction in fluorescent cells was observed in the populations exposed to 1 mg/mL of both milk and colostrum at the same time points. In these conditions, the size and granularity of the leukemic cells also changed, with a substantial reduction in the number of actively dividing cells in the S phase of the cell cycle. This phenomenon was highlighted by the Annexin V/PI cytofluorimetric test, which is able to provide quantitative results regarding the population of cells in early or late apoptosis or necrotic cells after exposure to a single dose or three doses of colostrum or sheep whey for three days, respectively. This report showed that both colostrum and milk whey were able to modify the phenotypic profile and cell cycle of the K562 cell line, inducing apoptosis at the highest concentration.

Development and evaluation of novel artemisinin-isatin hybrids with potential anti-leukemic cytotoxicity.

Twenty-one novel ester tethered artemisinin-isatin hybrids were designed, synthesized and screened against human myeloid leukemia cell lines (K562 and K562/ADR), human acute lymphoblastic leukemia cell line (CCRF-CEM) as well as normal human peripheral blood mononuclear cells (PBMCs) for their cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The structure-activity relationships (SARs) were also discussed to facilitate further rational design of more effective candidates. The preliminary results showed that most of the ester tethered artemisinin-isatin hybrids (IC50: 0.32-29.35 µM) exhibited promising activity against CCRF-CEM cells, and some of them (IC50: 1.23-49.84 µM) were also active against K562 and K562/ADR human myeloid leukemia cell lines. Among them, hybrid 7d (IC50: 0.32, 2.67 and 1.23 µM) not only possessed profound activity against the three tested leukemia cell lines and excellent safety and selectivity profiles, but also showed promising pharmacokinetic properties. Accordingly, hybrid 7d could be considered as a potential lead molecule for the development of novel anti-leukemic agents with minimal untoward events to normal human cells.

Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 μM) close to that of Nelarabine (IC50 = 3 μM) and was evaluated as active.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(β-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulatorapproved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORMcan induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h,respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Novel indole retinoid derivative induces apoptosis and cell cycle arrest and modulates AKT and ERK signaling in HL-60 cells.

Chemotherapy with targeted drugs is the first line therapy option for acute and chronic myeloid leukemia. However, hematopoietic stem cell transplantation may be used in high-risk patients or patients with failed responses to chemo drugs. Discovery and development of more effective new agents with lower side effects is the main aim of leukemia treatment. In this study, a novel retinoid compound with tetrahydronaphthalene ring was synthesized and evaluated for anticancer activity in human chronic and acute myeloid leukemia cell lines K562 and HL-60. Novel N-(1H-indol-1-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide was synthesized based on molecular hybridization of the two different bioactive structures retinoid head and indole. The effects of the synthesized carboxamide compound, which was referred to as compound 5, were determined in K562 chronic myeloid leukemia and HL-60 acute myeloid leukemia cell lines and L929 fibroblast cell line, which served as a control. Colorimetric MTT and caspase3 activity tests, flow cytometry, western blot, and microscopic examinations were used to evaluate biological activity. Compound 5 more effectively induced cell death in HL60 cells in comparison to K562 cells and L929 fibroblast cells. Therefore, further mechanism of cell death was investigated in HL60 cell line. It was found that compound 5 induced remarkable cytotoxicity, caspase3 activation, and PARP fragmentation in HL60 cells. Flow cytometric staining showed that the percentage of cells arrested in G0/G1 was also increased with compound 5 treatment. Important modulator proteins of cell proliferation p-ERK, p-AKT, and p-m-TOR were also found to be inhibited with compound 5 treatment. Collectively, our results reveal compound 5, which is a novel indole retinoid compound as a potential active agent for the treatment of acute promyelocytic leukemia.

Convenient synthesis, characterization and biological evaluation of novel 1-phenylcyclopropane carboxamide derivatives

Small, strained ring molecules of phenylcyclopropane carboxamide have rigid, defined conformations and unique electronic properties. For these reasons many groups, seek to use these subunits to form biologically active compounds. Herein we report a generally applicable approach for preparing a small cyclopropane ring containing 1-phenylcyclopropane carboxamide derivatives to a wide range of the different aromatic compounds by α-alkylation of 2-phenyl acetonitrile derivatives with 1, 2-dibromo ethane in good yields followed by the conversion of cyano group to acid group by the reaction with concentrated hydrochloric acid. This obtained acid derivative undergoes acid amine coupling with various Methyl 2-(aminophenoxy)acetate to form 1-Phenylcyclopropane Carboxamide. These compounds possess distinct effective inhibition on the proliferation of U937, pro-monocytic, human myeloid leukaemia cell line while these compounds did not show cytotoxic activity on these cells. The structure-activity relationships of these compounds are discussed.

Discovery of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine derivatives as novel FLT3 covalent inhibitors for the intervention of acute myeloid leukemia.

Small molecule covalent drugs have proved to be desirable therapies especially on drug resistance related to point mutations. Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitors resistance which further causes the failure of treatment. Herein, a series of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine covalent derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. Among these derivatives, compound F15 displayed potent inhibition activities against FLT3 (IC50 = 123 nM) and FLT3-internal tandem duplication (ITD) by 80% and 26.06%, respectively, at the concentration of 1 μM. Besides, F15 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines MOLM-13 (IC50 = 253 nM) and MV4-11 (IC50 = 91 nM), as well as BaF3 cells with variety of secondary mutations. Furthermore, cellular mechanism assays indicated that F15 inhibited phosphorylation of FLT3 and its downstream signaling factors. Notably, F15 could be considered for further development as potential drug candidate to treat AML.