Leukemia Cell Line K562 Research Articles

Sodium azulene sulfonate reverses multidrug resistance in K562/A02 cells

Overexpression of p-glycoprotein (p-gp) is the main cause of multidrug resistance and chemotherapy failure in leukemia. Sodium azulene sulfonate (SAS) was used to reverse the multidrug resistance of human leukemia adriamycin-resistant strain K562/A02, and the underlying mechanism was investigated. Human leukemia cell line K562 and drug-resistant cell line K562/A02 in logarithmic phase were used in this study. After 48 hours of treatment of K562/A02 cells with SAS, the intrinsic cytotoxicity of chlorogenic acid and its sensitivity to adriamycin (ADM) were determined with MTT assay. The degree of reversal was calculated. Using ADM accumulation and rhodamine 123 efflux experiments, the average fluorescence intensity of ADM and rhodamine 123 (Rh123) in chlorogenic acid-treated K562/A02 cells was determined flow cytometrically. The expressions of p-gp, t-Akt and p-Akt in K562/A02 cells were assayed using Western blotting. SAS had almost no cytotoxic effect, and the degree of inhibition was only about 20% at the highest concentration of 100 mu-M. The EC50 of MDR reversal by SAS was in the nano range (539±37nM), and it had a high selectivity index for normal cells (>185). The accumulation of ADM in drug-resistant cells was increased significantly after treatment with 1 and 5 mu-M SAS, while the efflux of Rh123 was significantly inhibited, suggesting that SAS reversed MDR by inhibiting p-gp function. Western blotting experiments showed that SAS downregulated the expression of p-gp by inhibiting PI3K/Akt signaling pathway. This contributed to the reversal of drug resistance.SAS effectively reverses multidrug resistance in vitro by inhibiting the function of p-gp in K562/A02 cells, through a mechanism involving downregulation of the P13K/Akt signaling pathway. Therefore, SAS may be a potential candidate drug for reversal of MDR.

Hexokinase II inhibition by 3-bromopyruvate sensitizes myeloid leukemic cells K-562 to anti-leukemic drug, daunorubicin.

An increased metabolic flux towards Warburg phenotype promotes survival, proliferation and causes therapeutic resistance, in leukemic cells. Hexokinase-II (HK-II) is expressed predominantly in cancer cells, which promotes Warburg metabolic phenotype and protects the cancer cells from drug-induced apoptosis. The HK-II inhibitor 3- Bromopyruvate (3-BP) dissociates HK-II from mitochondrial complex, which leads to enhanced sensitization of leukemic cells to anti-leukemic drugs. In the present study, we analyzed the Warburg characteristics viz. HK-II expression, glucose uptake, endogenous reactive oxygen species (ROS) level of leukemic cell lines K-562 and THP-1 and then investigated if 3-BP can sensitize the leukemic cells K-562 to anti-leukemic drug Daunorubicin (DNR). We found that both K-562 and THP-1 cells have multi-fold high levels of HK-II, glucose uptake and endogenous ROS with respect to normal PBMCs. The combined treatment (CT) of 3-BP and DNR showed synergistic effect on the growth inhibition (GI) of K-562 and THP-1 cells. This growth inhibitory effect was attributed to 3-BP induced S-phase block and DNR induced G2/M block, resulted in reduced proliferation due to CT. Further, CT resulted in low HK-II level in mitochondrial fraction, high intracellular calcium and elevated apoptosis as compared with individual treatment of DNR and 3-BP. Moreover, CT caused enhanced DNA damage and hyperpolarized mitochondria, leading to cell death. Taken together, these results suggest that 3-BP synergises the anticancer effects of DNR in the chronic myeloid leukemic cell K-562, and may act as an effective adjuvant to anti-leukemic chemotherapy.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Characterization of Telomerase (hTERT) in Solid and Hematopoietic Cancer Cell Lines Reveals Different Expression Patterns.

Overexpression of human telomerase reverse transcriptase (hTERT) allows disordered proliferation and immortality of malignant cells, which has been of interest for the development of targeted therapies. The present study aimed to characterize hTERT gene expression in a series of cancer cell lines. Leukemia cell lines K-562, its vincristine-resistant derivative K-562-Lucena1 and daunorubicin-resistant derivative FEPS; gastric adenocarcinoma lines AGP01, ACP02 and ACP03; melanoma SK-Mel-103 cells; and MN01 and MRC5, two non-neoplastic cell lines were analyzed by real-time polymerase chain reaction in order to evaluate hTERT gene expression. In leukemia cells, hTERT gene expression was significantly increased only in K-562 (p<0.05) and K-562-Lucena1 (p<0.001) when compared to the calibrator MRC5. For solid tumor types, only ACP03 presented a significant hTERT gene expression when compared to ACP02 (p<0.05). hTERT gene expression in K-562 and K-562-L ucena was significantly increased (p<0.05 to p<0.001) compared to all other cell lines except ACP03. In leukemia cell lines, hTERT gene overexpression was shown to be a potential target for pharmacological assays for drugs aiming to inhibit telomerase activity and control cell proliferation in oncohematological diseases.

A novel thiosemicarbazone as a promising effective and selective compound for acute leukemia.

Acute leukemias are a heterogeneous group of aggressive malignant neoplasms associated with severe morbidities due to the nonselectivity of current chemotherapeutic drugs to nonmalignant cells. The investigation of novel natural and synthetic structures that might be used for the development of new drugs with greater efficiency and selectivity to leukemic cells is mandatory. In this context, thiosemicarbazones have been well described in the literature by their several biological properties and their reaction is known as versatile, low-cost, and highly chemoselective. With this perspective, this study aimed to investigate the cytotoxic effect and the main death mechanisms of a novel thiosemicarbazone (LAP17) on acute leukemia cell lines K562 and Jurkat. The results show that the strong cytotoxic effect of LAP17 to leukemic cells is due to apoptosis induction, which resulted in caspase-3 activation and DNA fragmentation. Intrinsic apoptosis seems to be related to the inversion of Bax/Bcl-2 expression, ΔΨm loss, and AIF release, whereas extrinsic apoptosis was initiated by FasR. Gene-expression profiling of HL-60 cells treated with LAP17 by the microarray technique revealed a significant enrichment of gene sets related to cell cycle arrest at G2/M. Accordingly, K562 and Jurkat cells treated with LAP17 revealed a clear arrest at G2/M phase. Taking into consideration that LAP17 was not cytotoxic to nonhematological cells (peripheral blood mononuclear cell and erythrocytes), these results suggest that LAP17 is a promising new compound that might be used as a prototype for the development of new antileukemic agents.

Inhibitory Effect of NPM Gene Knockdown on Proliferation of Chronic Myeloid Leukemia Cell Line K562 and Its Mechanism

To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.

Effect of Inhibiting NCL Expression on Drug-Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Resistant Cells

To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ＜0.05), the apoptosis of cells increased significantly (P ＜0.05), and cell resistance to adriamycin was down-regulated. Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.

Two new 8-isopentenyl isoflavane derivatives from Astragalus dolichochaete diels

Astragali Radix (Huangqi) is one of the well-known traditional Chinese medicines and has been used in China for more than two thousand years. As a close species, Astragalus dolichochaete Diels was found to possess cytotoxicity during our preliminary study of Astragalus plants from Yunnan Province, China. To better understand the chemical foundation of the cytotoxicity, the major constituents of A. dolichochaete were studied. As a result, two new 8-isopentenyl isoflavane derivatives, dolichochaeteins A and B (1 and 2), together with ten known constituents were isolated, and their structures were elucidated by means of spectroscopy, mainly 1D and 2D NMR techniques. The cytotoxic activities were evaluated for the raw extract, ethyl acetate fraction, compound 2 and glyasperin H (3) against human gastric carcinoma cell line SGC-7901, human hepatoma cell line SMMC-7721 and human leukemic cell line K562. All the samples exhibited significant cytotoxicity.

Immunomodulatory activity of Humulus lupulus bitter acids fraction: Enhancement of natural killer cells function by NKp44 activating receptor stimulation

Abstract Humulus lupulus (Hop) contains numerous metabolites with anticancer potential. Despite this, their immunomodulatory activity, and in particular of bitter acids, is unknown. In this study, we demonstrated that a Hop pellet extract fraction containing bitter acids possesses immunomodulatory activity by enhancing Natural Killer (NK) cells function. After fractionation by semi-preparative Liquid Chromatography, three different fractions were obtained. The phytocomplex and the fractions were tested to verify the ability to modulate the NK compartment. Cytofluorimetric analysis revealed that a fraction containing bitter acids was able to up-regulate of NKG2D and NKp44 activating receptors. A further simplification yield a fraction mainly composed by Humulinones and Cohulupone derivatives, that at the concentration of 0.1 µg/mL induced selective activation of Nkp44 receptor and enhanced the cytolytic activity of NK cells against leukemia cell line K562. Those results point out the possible use of Hop bitter acids as natural immunostimulants and adjuvants in chemotherapy protocols.

Beta-2 microglobulin knockout K562 cell-based artificial antigen presenting cells for ex vivo expansion of T lymphocytes.

Aim: The human K562 leukemia cell line as a scaffold of artificial antigen presenting cells (aAPCs) for ex vivo lymphocyte expansion does not usually express major histocompatibility complex (MHC) molecules. However, when stimulated by supernatants from human T lymphocyte cultures, K562 cells upregulate β-2 microglobulin (B2M) and MHC class I expression, which would induce allo-specific T cells. Methods: We disrupted the B2M locus in K562 cells by CRISPR/Cas9 and achieved MHC class I-negative K562 cells. Results: We further generated K562-based MHC class I-negative aAPC line by zinc-finger nuclease mediated insertion of costimulatory molecules into the AAVS1 locus. This aAPC line could attenuate allogeneic immune responses but support robust antigen-independent and CD19 antigen-specific chimeric antigen receptor-T cell expansion in vitro. Conclusion: B2M-knockout K562 cells provide a new scaffold for aAPC construction and broader application in adoptive immunotherapies.

Deferasirox drives ROS-mediated differentiation and induces interferon-stimulated gene expression in human healthy haematopoietic stem/progenitor cells and in leukemia cells

BackgroundAdministration of the iron chelator deferasirox (DFX) in transfusion-dependent patients occasionally results in haematopoiesis recovery by a mechanism remaining elusive. This study aimed to investigate at a molecular level a general mechanism underlying DFX beneficial effects on haematopoiesis, both in healthy and pathological conditions.MethodsHuman healthy haematopoietic stem/progenitor cells (HS/PCs) and three leukemia cell lines were treated with DFX. N-Acetyl cysteine (NAC) and fludarabine were added as antioxidant and STAT1 inhibitor, respectively. In vitro colony-forming assays were assessed both in healthy and in leukemia cells. Intracellular and mitochondrial reactive oxygen species (ROS) as well as mitochondrial content were assessed by cytofluorimetric and confocal microscopy analysis; mtDNA was assessed by qRT-PCR. Differentiation markers were monitored by cytofluorimetric analysis. Gene expression analysis (GEA) was performed on healthy HS/PCs, and differently expressed genes were validated in healthy and leukemia cells by qRT-PCR. STAT1 expression and phosphorylation were assessed by Western blotting. Data were compared by an unpaired Student t test or one-way ANOVA.ResultsDFX, at clinically relevant concentrations, increased the clonogenic capacity of healthy human CD34+ HS/PCs to form erythroid colonies. Extension of this analysis to human-derived leukemia cell lines Kasumi-1, K562 and HL60 confirmed DFX capacity to upregulate the expression of specific markers of haematopoietic commitment. Notably, the abovementioned DFX-induced effects are all prevented by the antioxidant NAC and accompanied with overproduction of mitochondria-generated reactive oxygen species (ROS) and increase of mitochondrial content and mtDNA copy number. GEA unveiled upregulation of genes linked to interferon (IFN) signalling and tracked back to hyper-phosphorylation of STAT1. Treatment of leukemic cell lines with NAC prevented the DFX-mediated phosphorylation of STAT1 as well as the expression of the IFN-stimulated genes. However, STAT1 inhibition by fludarabine was not sufficient to affect differentiation processes in leukemic cell lines.ConclusionsThese findings suggest a significant involvement of redox signalling as a major regulator of multiple DFX-orchestrated events promoting differentiation in healthy and tumour cells. The understanding of molecular mechanisms underlying the haematological response by DFX would enable to predict patient’s ability to respond to the drug, to extend treatment to other patients or to anticipate the treatment, regardless of the iron overload.

Effective anticancer activities of an acyclic symmetrical compartmental Schiff base ligand and its Co(II), Cu(II) and Zn(II) complexes against the human leukemia cell line K562

Syntheses of new N2O5 donor type compartmental Schiff base ligand, 2,2′-((((((2-hydroxypropane-1,3-diyl)bis(oxy))bis(2,1-phenylene))bis(methylene))bis(azanylylidene))bis(methanylylidene))bis(4-nitrophenol) (H3L) and its cobalt(II), copper(II) and zinc(II) complexes, [Co(HL)] (1), [Cu2(L)(μ-1,3-OAc)] (2) and [Zn(HL)] (3), are reported. The synthesized compounds were characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies and X-ray single-crystal diffraction. In the structure of 1 and 3, the metal ion has a N2O2 coordination environment with tetrahedral geometry. Compound 2 is a binuclear complex, in which copper ions are bonded by NO4 donor atoms set in square planar coordination geometry. The ability of all compounds to interact with ten selected biomacromolecules (BRAF kinase, CatB, DNA gyrase, HDAC7, rHA, RNR, TrxR, TS, Top II and B-DNA) are investigated by the docking calculations. In continue, the in vitro activities of all compounds against the human leukemia cell line K562 were investigated by evaluation of IC50 values and mode of cell death (apoptosis). The experiments revealed that the cytotoxicity of the studied complexes is higher than or comparable with cisplatin.

PS1013 IS QUERCETIN ABLE TO REVERT THE APOPTOTIC RESISTANCE TRIGGERED BY THE SHORT ISOFORM OF GATA‐1 IN MYELOID LEUKEMIA CELLS?

Background:Dysregulated trans‐acting factors are key elements in the leukemogenic process. Aberrant expression of GATA‐1, a master regulator of several hematopoietic genes has been reported in myeloid leukemias. Maintenance of a balanced expression of the two isoforms of the transcription factor GATA‐1, the full‐length protein (GATA‐1FL) and a shorter isoform (GATA‐1S), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors (WY Lee et al. 2018; JD Crispino et al. 2017). Elevated GATA‐1S levels have been recognized as a poor prognostic factor thus further emphasizing the pro‐leukemic role of this isoform in hematological malignancies.Aims:We evaluated the oxidative stress associated with different GATA‐1 isoforms over‐expressed in the myeloid leukemia cell line K562 and their resistance to pro‐apoptotic stimuli.Methods:K562 cells were transiently transfected with expression vectors for GATA‐1FL and GATA‐1S isoforms. Cytoplasmatic ROS levels were evaluated by flow citometry with the CellROX Deep Red reagent (Thermo Fisher Scientific). Antioxidant capacity was evaluated by measurements of Cu/ZnSOD and MnSOD protein levels and of total and oxidized glutathione (GSH/GSSG‐Glo Assay kit, Promega). Cell viability and apoptosis rate were determined with MTT (Sigma Aldrich) and Annexin V/Pi (BD Biosciences) assays on cells over‐expressing GATA‐1 isoforms and treated with cis‐platin alone or co‐treated with quercetin and cis‐platin.Results:Cytoplasmatic ROS resulted to be significantly increased in cells over‐expressing GATA‐1FL (GATA‐1FL cells) compared to GATA‐1S cells. Total GSH levels were found reduced in GATA‐1FL cells and, conversely, increased in GATA‐1S cells in which we also observed an higher GSH/GSSG ratio. According to the different cytoplasmatic ROS levels detected in these cells, these findings further reinforce the evidence of an enhanced antioxidant capacity in GATA1S cells and of an higher oxidative stress status in GATA‐1FL cells. Accordingly, we also found a significant decrease in the MnSOD protein levels in GATA1S cells (Fig. 1A‐B). Furthermore, we were able to demonstrate a correlation between these data and the lower cell viability and higher apoptotic rate detected in GATA‐1FL cells exposed to a 10 microM dose of the pro‐apoptotic drug cis‐platin, in contrast with the strong resistance to apoptotis shown by GATA‐1S cells. The relationship between changes in proliferation and apoptosis with cell redox states was also demonstrated by analyzing the antioxidant effects of quercetin, a natural flavonoid compound, on transfected cells. According to cell viability data, after 24 hours of 50 and 150 microM quercetin treatment, GATA‐1S cells showed an enhanced apoptotic rate with respect to the GATA‐1FL counterpart. To confirm the changes in the apoptotic susceptibility induced by quercetin in GATA‐1FL and GATA‐1S cells, apoptosis was also evaluated after co‐treatment with 10 microM cis‐platin. Results clearly demonstrated the enhancer activity of quercetin on the apoptosis susceptibility in both cell types and, more interestingly, its ability to revert the resistance to apoptosis shown by GATA‐1S cells when treated with cis‐platin alone (Fig. 1C‐D).Summary/Conclusion:Our study highlights a mechanism to possibly circumvent the apoptotic resistance triggered by aberrant expression of GATA‐1 isoforms in myeloid leukemia cells. Expectedly, a better understanding of this mechanism can eventually lead to design more effective ROS‐based therapies in myeloid leukemias.image

Regulatory Effect of MiR-543 on Proliferation and Apoptosis of Human Leukemia Cell Line K562 and Its Mechanism

To investigate the regulatory effect of miR-543 on proliferation and apoptosis of human leukemia cell line K562 and its mechanism. 46 CML patients treated in our hospital from 2018.2-2018.9 was selected and enrolled in CML group, and 30 healthy persons were selected and enrolled in control group at the same time. After Lipofectamine 2000 liposome was used to transfer the miR-543 mimic and negative control (Scramble mimic) to K562 cells, CCK8 assay was used to detect the effect of miR-543 on proliferation of K562 cells; Luciferase reporter assay was used to report the targeting relationship of miR-543 to Wnt gene. Flow cytometry was used to detect the effect of miR-543 on apoptosis of K562 cells; Western blot method was used to detect the Wnt, β-catenin, BCL-2, c-MYC and BAX expression level. The level of miRNA-543 in CML patients was significantly lower than that in healthy controls (P＜0.05). The expression level of miR-543 in mimic group was significantly higher than that in blank control group (P＜0.05). The Wnt gene expression level in mimic group was significantly lower than that in blank control group (P＜0.05). The expression of luciferase of Wnt wild plasmid was decreased by miR-543 mimic (P＜0.05), and the luciferase activity of Wnt mutant plasmid was not inhibited by miR-543 mimic after mutation of binding site (P＜0.05). There was no significant difference in the proliferation ability of K562 cells between the blank control group and the negative control (Scramble mimic) group (P＞0.05). The proliferation level of K562 cells in mimic group was significantly lower than that in blank control group and negative control (Scramble mimic) group (P＜0.05). Apoptotic level of K562 cells in miR-543 mimic group was significantly higher than that in blank control group and negative control (Scramble mimic) group (P＜0.05). Western blot assay showed that Wnt, β-catenin, BCL-2 and c-MYC protein levels in miR-543 mimic group were significantly lower than those in blank control group and negative control (Scramble mimic) group (P＜0.05); BAX protein level in miR-543 mimic group was higher than that in blank control group and negative control (Scramble mimic) group (P＜0.05). miR-543 can target Wnt protein to inhibit the activity of Wnt signaling pathway, thereby inhibiting the proliferation of leukemia cells and increasing the level of apoptosis.

Study of cytotoxicity and cell death of an active compound from prazinoic acids in chronic myeloid leukemia K562 cell line

Background: Nowadays, more than 100 types of cancer are known worldwide, which leukemia is one of the most common type. Chronic myeloid leukemia (CML) is one of the most recognized forms of leukemia, accounting for between 15% and 20% of total leukemia. Efforts to find new synthetic compounds are one of the basic strategies for finding new drugs for CML treatment. The purpose of this study was to investigate the effects of growth suppression and induction of apoptosis (family of pyrazinoic acid) on the cancer cell line of K562 leukemia. Methods: In this study, the effect of a certain concentration of 2-oxo-1, 2-diphenylethyl-pirazine-2-carboxylate (2 DP.P) was evaluated on the growth, cell cycle and induction of apoptosis of K562 cells using various techniques. MTT test was used to investigate the effects of 2-DP.P on cell viability. For this purpose, 5×104 of K562 cells were cultured at concentrations of 25-100 μM from the 2-DP.P for 24, 48 and 72 hours intervals. Electrophoresis, fluorescence microscopy and flow cytometry were used for analysis of apoptosis. Results: The 2-DP.P inhibited dose and time dependently growth in K562 cells. IC50 of the compound was calculated 25 μM. Also, data from fluorescence microscopy, DNA fragmentation and cell cycle analysis confirmed the reduction of viability and induction of apoptosis by the compound. Conclusion: due to the effects of growth inhibition and induction of apoptosis by the 2-DP.P, this compound can be suggested as a new and effective compound for further studies in treating patients with leukemia.

Synthesis, antimicrobial and antiproliferative properties of epi-oligomycin A, the (33S)-diastereomer of oligomycin A

abstractWe describe the synthesis of epi-oligomycin A, a (33S)-diastereomer of the antibiotic oligomycin A. The structure of (33S)-oligomycin A was determined by elemental analysis, spectroscopic studies, including 1D and 2D NMR spectroscopy, and mass spectrometry. Isomerization of C33 hydroxyl group led to minor changes in the potency against Aspergillus niger, Candida spp., and filamentous fungi whereas the activity against Streptomyces fradiae decreased by approximately 20-fold compared to oligomycin A. We observed that 33-epi-oligomycin A had the same activity on the human leukemia cell line K562 as oligomycin A but was more potent for the multidrug resistant subline K562/4. Non-malignant cells were less sensitive to both oligomycin isomers. Finally, our results pointed at the dependence of the cytotoxicity of oligomycins on oxygen supply.

The total synthesis of (−)-indolactam I

The first total synthesis of (−)-indolactam I (1) is reported. An efficient synthetic route was established to furnish the natural product in 8 steps. This strategy employed a copper-catalyzed amino acid arylation, peptide coupling, and Lewis acid-mediated macrocyclization to yield 1 from readily available building blocks. In addition, the cell growth inhibition activity of the natural product was examined through assays with the K562 leukemia cell line.

Design and synthesis of novel 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4-thiadiazol- 2-yl)urea derivatives with potent anti-CML activity throughout PI3K/AKT signaling pathway.

In this investigation, a series of 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea receptor tyrosine kinase inhibitors were synthesized by a simple and efficient structure-based design. Structure-activity relationship (SAR) analysis of these compounds based on cellular assays led to the discovery of a number of compounds that showed potent activity against human chronic myeloid leukemia (CML) cell line K562, but very weak or no cellular toxicity through monitoring the growth kinetics of K562 cell during a period of 72 h using the real-time live-cell imaging. Among these compounds, 1-(5-((6-((3-morpholinopropyl) amino)pyrimidin-4-yl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (7) exhibited the least cellular toxicity and better biological activity in cellular assays (K562, IC50: 0.038 μM). Compound 7 also displayed very good induced-apoptosis effect for human CML cell line K562 and exerted its effect via a significantly reduced protein phosphorylation of PI3K/Akt signal pathway by Human phospho-kinase array analysis. In vitro results indicate that 1-phenyl-3-(5-(pyrimidin-4-ylthio)-1,3,4- thiadiazol-2-yl)urea derivatives are lead molecules for further development as treatment of chronic myeloid leukemia and cancer.

GATA-1 isoforms differently contribute to the production and compartmentation of reactive oxygen species in the myeloid leukemia cell line K562.

Maintenance of a balanced expression of the two isoforms of the transcription factor GATA‐1, the full‐length protein (GATA‐1FL) and a shorter isoform (GATA‐1 S), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors thereby eventually leading to a variety of hematopoietic disorders. Although it is well established that these isoforms play opposite roles in these remarkable processes, most of the molecular pathways involved remain unknown.Here, we demonstrate that GATA‐1FL and GATA‐1S are able to differently influence intracellular redox states and reactive oxygen species (ROS) compartmentation in the erythroleukemic K562 cell line, thus shedding novel mechanistic insights into the processes of cell proliferation and apoptosis resistance in myeloid precursors. Furthermore, given the role played by ROS signaling as a strategy to escape apoptosis and evade cell‐mediated immunity in myeloid cells, this study highlights a mechanism through which aberrant expression of GATA‐1 isoforms could play a role in the leukemogenic process.

Myelodysplastic Syndrome-Associated SRSF2 Mutations Cause Splicing Changes by Altering Binding Motif Sequences.

Serine/arginine-rich splicing factor 2 (SRSF2) is a member of the SR protein family that is involved in both constitutive and alternative mRNA splicing. Mutations in SRSF2 gene are frequently reported in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). It is imperative to understand how these mutations affect SRSF2-mediated splicing and cause MDS. In this study, we characterized MDS-associated SRSF2 mutants (P95H, P95L, and P95R). We found that those mutants and wild-type SRSF2 proteins showed nuclear localization in HeLa cells. In vitro splicing reaction also revealed that mutant proteins associated with both precursor and spliced mRNAs, suggesting that the mutants directly participate in splicing. We established the human myeloid leukemia K562 cell lines that stably expressed myc-tagged wild-type or mutant SRSF2 proteins, and then performed RNA-sequence to analyze the splicing pattern of each cell line. The results revealed that both wild-type and mutants affected splicing of approximately 3,000 genes. Although splice site sequences adjacent to the affected exons showed no significant difference compared to the total exons, exonic motif analyses with both inclusion- and exclusion-enhanced exons demonstrated that wild-type and mutants have different binding sequences in exons. These results indicate that mutations of SRSF2 in MDS change binding properties of SRSF2 to exonic motifs and this causes aberrant splicing.