Myeloid Leukemia Cell Line K562 Research Articles

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.

Venetoclax efficacy on acute myeloid leukemia is enhanced by the combination with butyrate

Venetoclax has been approved recently for treatment of Acute myeloid leukemia (AML). Venetoclax is a BH3-mimetic and induces apoptosis via Bcl-2 inhibition. However, venetoclax’s effect is still restrictive and a novel strategy is needed. In the present study, we demonstrate that sodium butyrate (NaB) facilitates the venetoclax’s efficacy of cell death in AML cells. As a single agent, NaB or venetoclax exerted just a weak effect on cell death induction for AML cell line KG-1. The combination with NaB and venetoclax drastically induced cell death. NaB upregulated pro-apoptotic factors, Bax and Bak, indicating the synergistic effect by the collaboration with Bcl-2 inhibition by venetoclax. The combined treatment with NaB and venetoclax strongly cleaved a caspase substrate poly (ADP-ribose) polymerase (PARP) and a potent pan-caspase inhibitor Q-VD-OPh almost completely blocked the cell death induced by the combination, meaning that the combination mainly induced apoptosis. The combination with NaB and venetoclax also strongly induced cell death in another AML cell line SKNO-1 but did not affect chronic myeloid leukemia (CML) cell line K562, indicating that the effect was specific for AML cells. Our results provide a novel strategy to strengthen the effect of venetoclax for AML treatment.

Asperuloside regulates the proliferation, apoptosis, and differentiation of chronic myeloid leukemia cell line K562 through the RAS/MEK/ERK pathway

ContextChronic myeloid leukemia (CML) is a malignant hematopoietic stem cell disease caused by excessive proliferation and abnormal differentiation of hematopoietic stem cells. Asperuloside (ASP) is considered to have good biological activity and may be a good anti-CML drug. ObjectiveThis study aimed to explore the effects and possible mechanisms of ASP on the biological behavior of K562 cells based on RNA-seq. Materials and methodsThe IC50 of ASP in K562 cells was calculated by the concentration-effect curve. Cell viability, apoptosis, and differentiation were detected by CCK8, flow cytometry, benzidine staining, and WB analysis, respectively. Further, RNA-seq was used to analyze the possible mechanism of ASP regulating K562 cells. ResultsASP significantly inhibited the proliferation, and promoted apoptosis and differentiation of K562 cells. A total of 117 differentially expressed genes were screened by RNA-seq, mainly involved in the RAS/MEK/ERK pathway. PD98059 was used to inhibit the RAS/MEK/ERK pathway in K562 cells, and results confirmed that PD98059 could not only inhibit the RAS/MEK/ERK pathway, but also inhibit the regulation of ASP on the proliferation and differentiation of K562 cells. ConclusionASP inhibited the proliferation, promoted apoptosis and differentiation of K562 cells by regulating the RAS/MEK/ERK pathway, and played a good anti-CML role.

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.

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.

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.

Discovery of CZS-241: A Potent, Selective, and Orally Available Polo-Like Kinase 4 Inhibitor for the Treatment of Chronic Myeloid Leukemia

Recent studies demonstrate that PLK4 has emerged as a therapeutic target for the treatment of multiple cancers owing to its indispensable role in cell division. Herein, starting from previously identified effective compound CZS-034, based on rational drug design strategies, tyrosine kinase receptor A (TRKA) selectivity- and metabolic stability-guided structure-activity relationship (SAR) exploration were carried out to discover a highly potent (IC50 = 2.6 nM) and selective (SF = 1054.4 over TRKA) PLK4 inhibitor B43 (CZS-241) with acceptable human liver microsome stability (t1/2 = 31.5 min). Moreover, compound B43 effectively inhibited leukemia cells in 29 tested cell lines, especially chronic myeloid leukemia (CML) cell lines K562 and KU-812. Pharmacokinetic characteristics revealed that compound B43 possessed over 4 h of half-life and 70.8% bioavailability in mice. In the K562 cells xenograft mouse model, a 20 mg/kg/day dosage treatment obviously suppressed tumor progression. As a potential and novel PLK4-targeted candidate drug for CML, compound B43 is undergoing extensive preclinical safety evaluation.

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.

Effect of Curcumin on the Proliferation, Apoptosis, and Cell Cycle of Human Acute Myeloid Leukemia Cell Line K562

To investigate the effects of curcumin on the proliferation, apoptosis, and cell cycle of human acute myeloid leukemia cell line K562. MTT method was used to detect the proliferation inhibition of logarithmic growth phase human acute myeloid leukemia K562 cells, flow cytometry was used to detect the cell cycle, Annexin V-FITC was used to detect the apoptosis rate, and real-time fluorescent quantitative PCR and Western blot were used to detect the expression of Bax, BCL-2 and caspase-3 mRNA and protein, respectively. The inhibition rate of cell proliferation in curcumin 10, 20, and 40 μmol/L group for 24 h and 48 h were higher than that in the control group (curcumin 0 μmol/L), and the cell proliferation inhibition rate was concentration-time dependent (r=0.879, r=0.914). The proportion of G0/G1 cells and apoptosis rate of K562 cells in the curcumin 10, 20, and 40 μmol/L group were higher than those in the control group, and showed drug concentration dependent (r=0.856, r=0.782). The expression of Bax and Caspase-3 mRNA in the curcumin 10, 20, and 40 μmol/L group was higher, while BCL-2 mRNA was lower than those in the control group, and showed drug concentration dependent (r=0.861, r=0.748, r=-0.817). The gray value of Bax protein expression in the curcumin 10, 20, and 40 μmol/L group was higher than that in the control group, while the gray value of BCL-2 and Caspase-3 protein expression was lower than that in the control group, and showed drug concentration dependent (r=0.764, r=-0.723, r=-0.831). Curcumin can inhibit the proliferation of human acute myeloid leukemia cell line K562 cells, block the cell cycle at G0/G1 phase, promote cell apoptosis, and induce apoptosis by regulating Bax, BCL-2, and Caspase-3.

Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.

TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.

Abstract 3737: DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia

Abstract Infants and young children (> 3 yrs.) harboring ETO2-GLIS2 have the worst outcomes amongst all pediatric acute myeloid leukemia (AML) subtypes, with poor responses to induction therapy, high incidence of relapse, and dismal 5-year survival rates. Previous studies demonstrated that the ETO2-GLIS2 fusion leads to imbalance in the expression of ETS-related transcription factor ERG and GATA1 with neo-super enhancer (SE) activities in a single oncogenic hit (Thirant et al. 2017). ERG was found to co-localize with ETO2-GLIS2 fusion at 90% (372/419) of H3K27ac marked SE-regions and was strongly upregulated (9.44-fold). Herein, we aimed to investigate cooperativity of DNA methylation and chromatin marks on ERG overexpression in this AML subtype. We analyzed transcriptomic data from pediatric ETO2-GLIS2 AML patients (n=40) from dbGAP (phs000465.v19. p8) in comparison to normal bone marrow (NBM) (n=71) samples, confirming upregulation (median log FC: 2.4-fold) in ERG expression in the ETO2-GLIS2 subgroup compared to NBM. Next, we performed an unbiased genome-wide methylation array using Infinium MethylationEPIC 850K-arrays (Illumina). Differences in mean methylation (β-value) at individual CpG sites between NBM and AML samples were considered significant at differences greater than ±10%. We observed reduced methylation at 72% (5/7) CpGs at the ERG-promoter (1500bp upstream of transcription start site) in ETO2-GLIS2 subgroup compared to NBM. Overall median methylation across the promoter was reduced by 12% in the AML subgroup. In comparison, 22 differentially methylated CpGs were observed in the body, of which methylation increased at 68% (15/22) of the CpGs, with median methylation similarly increased by 15% in the AML patients. This represents a classic example of methylation-expression relation, having a promoter demethylation and body-hypermethylation, linked to upregulation of the gene-expression. Next, we investigated the overlaps between CpGs and the distribution of DNAse clusters and H3K27ac marks based on ENCODE data on myeloid leukemia cell line K562. We observed frequent overlaps between demethylated CpGs and DNAse clusters, while H3K27ac broad peaks were coincident with both demethylated CpGs at promoter or hypermethylated CpGs at body. Based on our findings, we thus conclude that DNA-methylation both at the ERG promoter and body is tightly linked to the overlapping chromatin marks of the gene. DNA-methylation might aid in opening of adjacent chromatin on ERG to possibly facilitate SE enrichment and over-expression the gene. Future studies will investigate targeted alterations of DNA-methylation on the gene to test the impact on ERG expression and leukemic growth. Citation Format: Samrat Roy Choudhury, Jordan T. Bird, Stephanie Byrum, Jason E. Farrar. DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3737.

Evaluation of anti-tumor effect of the exopolysaccharide from new cold-adapted yeast, Rhodotorula mucilaginosa sp. GUMS16 on chronic myeloid leukemia K562 cell line

Recently, the development and application of fungal exopolysaccharides (EPS) as natural biopolymers are on the rise. The present study is based on the investigation of possible antiproliferative and antioxidant activities of EPS from the Rhodotorula mucilaginosa sp. GUMS16 on BCR-ABL positive cells (K562). The cytotoxicity, colony formation assays lactate and dehydrogenase (LDH) activity were performed to assess the possible cancer cell death. To elucidate the underlying antiproliferative mechanism of the EPS, cell cycle analysis following real-time PCR (gene expression assessment) were evaluated. The results indicated that, the EPS with an IC50 dose of 1500 μg/ml, reduced the viability of K562 cells without having toxic effects on normal cells as well as decrease in size and number of colonies in EPS-treated group (p < 0.0001). The increase of LDH was 2.75 times more than the control (p < 0.0001). Gene expression revealed up- and down-regulation of apoptotic and anti-apoptotic genes in EPS group compared with the control. Moreover, the DPPH scavenging activity of the EPS in treated cells was significantly higher than the control group (p < 0.0001). Taken together, we concluded that the EPS from GUMS16 strain is able to inhibit the growth of K562 cells besides having antioxidant activities.

Selective Chemical Activation of Piezo1 in Leukemia Cell Membrane: Single Channel Analysis.

Piezo1/2 are mechanosensitive calcium-permeable channels that can be activated by various modes of membrane deformation. The identification of the small molecule Yoda1, a synthetic Piezo1 agonist, revealed the possibility of chemical activation of the channel. Stimulating effects of Yoda1 on Piezo1 have been mainly documented using over-expressing cellular systems or channel proteins incorporated in artificial lipid bilayers. However, the activating effect of Yoda1 on native Piezo1 channels in the plasma membrane of living cells remains generally undefined, despite the increasing number of studies in which the agonist is utilized as a functional tool to reveal the contribution of Piezo1 to cellular reactions. In the current study, we used the human myeloid leukemia K562 cell line as a suitable model to examine chemically induced Piezo1 activity with the use of the patch-clamp technique in various specific modes. The functional expression of Piezo1 in leukemia cells was evidenced using a combinative approach, including single channel patch-clamp measurements. Utilizing our established single-current whole-cell assay on K562 cells, we have shown, for the first time, the selective real-time chemical activation of endogenously expressed Piezo1. Extracellular application of 0.5–1 µM Yoda1 effectively stimulated single Piezo1 currents in the cell membrane.

Evaluation of the Apoptotic Effects of the Humic Acid Treatment on Chronic Myeloid Leukemia Cell Line K562

Evaluation of the Apoptotic Effects of the Humic Acid Treatment on Chronic Myeloid Leukemia Cell Line K562

Stereochemistries and Biological Properties of Oligomycin A Diels-Alder Adducts.

Oligomycin A is a potent antibiotic and antitumor agent. However, its applications are restricted by its high toxicity and low bioavailability. In this study, we obtained Oligomycin A Diels-Alder adducts with benzoquinone and N-benzylmaleimide and determined their absolute configurations by combining 1H and ROESY NMR data with molecular mechanics conformational analysis and quantum chemical reaction modeling. The latter showed that adduct stereochemistry is controlled by hydrogen bonding of the Oligomycin A side-chain isopropanol moiety with the carbonyl group of the dienophile. Biological studies showed that the Diels-Alder modification of the Oligomycin A diene system resulted in a complex antiproliferative potential pattern. The synthesized adducts were determined to be more active against the triple-negative (ERα, PR, and HER2 negative) breast cancer cell line MDA-MB-231 and lung carcinoma cell line A-549 compared to Oligomycin A. Meanwhile, Oligomycin A was more potent against myeloid leukemia cell line K-562 and breast carcinoma cell line MCF-7 than its derivatives. Thus, modification of the diene moiety of Oligomycin A is a promising strategy for developing novel antitumor agents based on its scaffold.

Anti-chronic myeloid leukemia activity and quantitative structure-activity relationship of novel thiazole aminobenzamide derivatives

The anti-chronic myeloid leukemia activity of thiazole aminobenzamide derivatives in vitro was tested by a methanethiosulfonate (MTS)-based viability assay method, and the result showed that some compounds exhibited good inhibitory activities against human chronic myeloid leukemia cell line K562, imatinib-resistant strain K562/R and T135I mutant cell line BaF3-ABL-BCR-T315I. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to analyze the relationship between the structure of thiazole aminobenzamide derivatives and the inhibition of K562/R cell activity. In CoMFA, Q2 was 0.899 and R2 was 0.963; in CoMSIA, Q2 and R2 were 0.840 and 0.903, respectively. These data indicated that the selected test set showed suitable external predictive ability. Combined with the contour map results, we further analyzed the three-dimensional quantitative structure (3D-QSAR) model. The results demonstrated that in the backbone of the thiazole aminobenzamide derivative, the substitution of a small group at R1 position, or the introduction of a hydrophilic group at R2 position, or the introduction of a large-volume amino acid at R3 position may be beneficial to improve the anti-CML activity of the compound.

H2O2 mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.

Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition.

Numerous recurrent genetic mutations are known to occur in acute myeloid leukemia (AML). Among these common mutations, Fms-like tyrosine kinase 3 remains as one of the most frequently mutated genes in AML. We observed apparent marrow expansion of megakaryocytes in three out of six patients with Flt3-mutated AML following treatment with a recently FDA-approved Flt3 inhibitor, gilteritinib which possesses activity against internal tandem duplication and tyrosine kinase domain Flt3 mutations and also inhibits tyrosine kinase AXL. To assess whether biopsy findings can be attributed to promotion of megakaryocytic (Mk) differentiation with gilteritinib, we devised a cellular assay by overexpressing double mutated Flt3-ITDY591F/Y919F in chronic myeloid leukemia cell line K562 to study Mk differentiation in the presence of Flt3 and AXL inhibitors with non-mutually exclusive mechanisms. These experiments demonstrated the lack of direct effect Flt3 inhibitors gilteritinib and quizartinib on megakaryocytic differentiation at either transcriptional or phenotypic levels, and highlighted antileukemic effects of AXL receptor tyrosine kinase inhibitor and its potential role in megakaryocytic development.

Down-regulation of Survivin and Bcl-2 concomitant with the activation of caspase-3 as a mechanism of apoptotic death in KG1a and K562 cells upon exposure to a derivative from ciprofloxacin family

Ciprofloxacin derivatives belong to a family of antibiotics called fluoroquinolones. Recently, these compounds have been recommended for the treatment of cancer. In the present study, we assessed the cytotoxicity of several new synthetic ciprofloxacin derivatives and the apoptosis-inducing activity of the most efficient derivative in two human myeloid leukemia K562 and KG1-a cell lines. Among the prepared ciprofloxacin derivatives, 1-cyclopropyl-7-(4-(2-((3,7-dimethyloct-6-en-1-yl)oxy)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (4-DMOCP) was more active compound with IC50 of 19.56 and 22.13 μM for K562 and KG1-a, respectively. Apoptotic activity of the 4-DMOCP was examined morphologically through Hoechst 33258 staining, Annexin V/PI double staining, and caspase-3 activity assays. Changes in the expression level of some apoptosis-related genes and protein, including Bcl-2, Bax, Survivin, p53, Caspase-8 and Caspase-9 were evaluated by the real-time quantitative PCR (qRT PCR) and western blotting. The qRT PCR analysis showed that 4-DMOCP induces apoptosis in both cell lines via the down-regulation of Survivin and Bcl2, up-regulation of caspase-8 and -9, as well as a time-dependent increase in the Bax/Bcl2 transcripts. The mRNA level of p53 was also increased in both cell lines. In addition, western blot analysis revealed that treatment with the compound, down-regulated the protein expression levels of Bcl2 and Survivin and up-regulated the protein level of Bax in both cell lines. These findings suggest that these new compounds can be good candidates for the treatment of acute and chronic myeloid leukemia.

Checkpoint kinase‑1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA-mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK-8, and comet assay. The results demonstrated that shRNA-induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.