Chronic Myelogenous Leukemia K562 Cells Research Articles

Inhibition of human chronic myelogenous leukemia K562 cell growth following combination treatment with resveratrol and imatinib mesylate.

To investigate the effect of treatment with resveratrol combined with imatinib mesylate on human chronic myelogenous leukemia K562 cell growth inhibition and apoptosis, in vitro cultured human chronic myelogenous leukemia K562 cells were incubated with different concentrations of resveratrol and imatinib mesylate when the cells were in the logarithmic phase. Next, the cell growth inhibition was evaluated using the MTT assay and cellular morphology observation. Apoptosis was determined using Annexin V fluorescein isothiocyanate/propidium iodide double staining. The results demonstrated that treatment with resveratrol (concentration-dependent) and imatinib mesylate showed significantly greater inhibition of K562 cell growth and a higher apoptosis rate of K562 cells than imatinib mesylate medication alone and the control group (P < 0.01). The imatinib mesylate medication alone group showed significant inhibition of K562 cell growth and apoptosis rate of K562 cells compared to the control group (P < 0.01). Our findings indicate that imatinib mesylate and resveratrol are potent drug treatments for human chronic myelogenous leukemia, offering a promising means of inhibiting cell growth and apoptosis.

Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis.

The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML.

Abstract 4186: An integrative genomic pipeline to target the NCOR1 cistrome with precision

Abstract NCOR1 is a co-regulator of multiple transcription factors (TFs) and is mutated in major cancers. We mined publically available data-sets to reveal the diversity, function and druggability of the NCOR1 cistrome. From ENCODE, in K562 chronic myelogenous leukemia cells, NCOR1 ChIP-Seq data were integrated with H3K9me3 (a functional consequence), CpG methylation levels and open and closed chromatin states in a matrix of 45 interactions that were combined with RNA-Seq data. Boot-strapping approaches then revealed significantly affected gene networks. Surprisingly, only the combination of NCOR1, H3K9me3 and an intermediate level of CpG methylation, in open chromatin, significantly associated with a network of 212 repressed genes (lo_gene). By contrast, 15 different NCOR1 combinatorial binding patterns associated with significantly elevated gene expression. For example, NCOR1 and low CpG methylation associated with a network of 908 significantly elevated genes (hi_gene). A simple linear regression model was built on the correlation of NCOR1 and 11 different histone modifications with RNA-seq values, across 81 bins spanning gene loci, to reveal the optimal bin predictor of gene expression. NCOR1 binding at the TSS significantly and positively correlated with gene expression, and mirrored the patterns of the activating histone mark H3K4me3. Transcription factor and GO enrichment in the lo_gene and hi_gene networks revealed these significant associations with different TFs and GO terms. In the lo_genes network the most significantly associated TF was regulatory factor X (RFX1), (FDR=1e-4), whereas in the hi_gene network it was Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), (FDR=2.7e-23). GO analyses revealed these cohorts were most associated with skeletal development (FDR, 2e-3) and transcription regulation (FDR 1.8e-2) terms respectively, underscoring their diverse functions. Mining the NCI60 database, combined with bootstrap approaches, identified that the NCOR1 hi_gene (upregulated) network was most significantly enriched for genes associated with sensitivity towards HDAC inhibitors, including vorinostat, compared to other drug classes including RTKi. Finally, mining a GEO microarray data set of 59 CML patients revealed that significantly up-regulated genes in Imatinib resistant patients were significantly enriched for targets associated with vorinostat sensitivity in the NCI60. Together this integration pipeline in R, from ENCODE, GEO and NCI60, forms a highly flexible pipeline that can be applied to any DNA binding protein or epigenetic state as a prelude to drug redeployment and/or precision medicine approaches. Specifically, these data suggest that cancer related functions of NCOR1 are surprisingly most associated with sustained gene expression, and these targets, both in CML cell lines and patients, associate with sensitivity to clinically approved HDAC inhibitors. Citation Format: Mark D. Long, Patrick van den Berg, Moray J. Campbell, Prashant K. Singh, Sebastiano Battaglia. An integrative genomic pipeline to target the NCOR1 cistrome with precision. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2014-4186

Abstract 1717: Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

Abstract This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibirors (TKIs) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMTs) and a decrease in levels of microRNAs (miRNAs) miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune deficient mice that were treated by the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI-resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKIs. Citation Format: Takayuki Ikezoe, Chie Nishioka, Jing Yang, Akihito Yokoyama. Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2014-1717

Abstract 1456: MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia

Abstract Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells that is caused by the deregulated activity of the tyrosine kinase that is encoded by the chimeric bcr-abl oncogene. Given that CML cells are usually highly resistant to chemotherapy, novel and more efficacious strategies against this disease are urgently needed. MicroRNA (miRNA) is a set of newly discovered 20-22 nucleotides RNA molecules, and they broadly repress their cognate target genes at post-transcriptional/trans-lational level. To date, there are approximately 2,500 human miRNA transcripts have been characterized, which are estimated to modulate more than 30% human genes by being involved in many essential normal biological and pathological processes including differentiation, proliferation, apoptosis, and tumorigenesis as well. In this study, we studied let-7a, a miRNA with tumor suppressive signature, for its therapeutic potential in CML. When treating CML K562 and KU812 cell by imatinib, let-7a can be significantly induced. Overexpression of let-7a can sensitize K562 and KU812 cells to imatinib, while knock-down of let-7a can increase the resistance of these cells towards this drug. The functional studies found that let-7a was able to promote apoptosis of CML cells, inhibit the tumor cell proliferation, and lead to a cell cycle arrest in the G0/G1 phase. These results demonstrate that let-7a is a potential target for treating CML by imatinib and/or other anti-cancer drugs. The pro-apoptosis and anti-proliferation roles of let-7a in CML cells may provide novel insights into discovery and development of more efficacious drugs for treating this malignant disease. Citation Format: Bin Yi, Ruixia Ma, Xiangling Feng, Zhiwei Xing, Xiaoguo Zhang, Gary Piazza, Yaguang Xi. MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2014-1456

Linear antimicrobial peptides from Ectatomma quadridens ant venom.

Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105μg/g.

5-(2-Carboxyethenyl) isatin derivative induces G2/M cell cycle arrest and apoptosis in human leukemia K562 cells

Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC50) level of 3nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G2/M phase and accumulated subsequently in the sub-G1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G2/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated the expression of RKIP in chronic myelogenous leukemia (CML) K562 cells and the effects of RKIP on the characteristics of K562 cells. The recombinant plasmid pcDNA3.1-RKIP was established and transfected into K562 cells with the help of Lipofectamine 2000. At the same time, the RKIP-siRNA was transfected into K562 cells in another group. The expressions of RKIP in all groups were assayed by Western blot after 48 h. MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to analyze the cell viability. Flow cytometry (FCM) was used to examine the cell cycle and cell apoptosis. Colony forming unit (CFU) assay was used to analyze the effect of RKIP on the clonogenic growth of CML cells. Western blot or luciferase reporter assay was used to detect the effect of RKIP on the level of phospho-ERK1/2 or the transcriptional activity of NF-κB. Western blot analysis showed that the plasmid pcDNA3.1-RKIP or RKIP-siRNA significantly enhanced or decreased RKIP expression (p < 0.01), respectively. In addition, MTT, FCM, and CFU assay indicated that the overexpression of RKIP significantly lowered the cell viability, cell proliferation and the clonogenic growth (p < 0.05), but improved cell apoptosis (p < 0.01). Western blot analysis or luciferase reporter assay showed that the level of phospho-ERK1/2 or the transcriptional activity of NF-κB was strongly inhibited by overexpression of RKIP. All these results could bring us a new perspective for biological therapy in myelogenous leukemia in the future.

Bortezomib induces apoptosis by interacting with JAK/STAT pathway in K562 leukemic cells.

In the current study, we aimed to identify the cytotoxic and apoptotic effects of bortezomib (BOR) on human K562 chronic myelogenous leukemia cells and to evaluate the potential roles of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway members STAT3, STAT5, and JAK2 on BOR-induced cell death of leukemic cells. Cell viability was assessed via trypan blue dye exclusion test, and cytotoxicity of the BOR-treated cells was conducted by 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay. The relative messenger RNA (mRNA) expression levels of STAT3, STAT5A, STAT5B, and JAK2 were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). On the other hand, their protein expression levels were detected by western blot method. The obtained results indicated that BOR treatment reduced cell viability and induced leukemic cell apoptosis in a dose- and time-dependent manner as compared to untreated control cells. While mRNA expression levels of STAT5A, STAT5B, and STAT3 were significantly reduced following BOR treatment when compared to untreated controls, it had no effect upon JAK2 mRNA expression. As for protein levels, STAT expressions were downregulated after BOR treatment especially at 72nd and 96th hours. Our results pointed out that BOR treatment had a significant potential of being an anticancer agent for chronic myelogenous leukemia therapy, and this effect could be due to the expressional downregulations of JAK/STAT pathway members.

Ganglioside GM3 is required for caffeic acid phenethyl ester-induced megakaryocytic differentiation of human chronic myelogenous leukemia K562 cells.

The human chronic myelogenous cell line K562 has been used extensively as a model for the study of leukemia differentiation. We show here that treatment of K562 cells with caffeic acid phenethyl ester (CAPE) induced a majority of cells to differentiate towards the megakaryocytic lineage. Microscopy analysis showed that K562 cells treated with CAPE exhibited characteristic features of physiological megakaryocytic differentiation, including the presence of vacuoles and demarcation membranes. Differentiation of K562 cells treated with CAPE was also accompanied by a net increase in megakaryocytic markers. The transcriptional activity of lactosylceramide α-2,3-sialyltransferase (GM3 synthase) and synthesis of ganglioside GM3 were increased by CAPE treatment. The promoter analysis of GM3 synthase demonstrated that CAPE induced the expression of GM3 synthase mRNA via activation of the cAMP response element-binding protein (CREB), transcription factor in nucleus. Interestingly, the inhibition of ganglioside GM3 synthesis by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol (D-PDMP) and GM3 synthase-siRNA blocked the CAPE-induced expression of the megakaryocytic markers and differentiation of K562 cells. Taken together, these results suggest that CAPE induces ganglioside GM3-mediated megakaryocytic differentiation of human chronic myelogenous cells.

The anti-apoptotic form of tyrosine kinase Lyn that is generated by proteolysis is degraded by the N-end rule pathway.

The activation of apoptotic pathways results in the caspase cleavage of the Lyn tyrosine kinase to generate the N-terminal truncated LynΔN. This LynΔN fragment has been demonstrated to exert negative feedback on imatinib induced apoptosis in chronic myelogenous leukemia (CML) K562 cells. Our investigations focus on LynΔN stability and how reduced stability reduces imatinib resistance. As the proteolytical generated LynΔN has a leucine as an N-terminal amino acid, we hypothesized that LynΔN would be degraded by the N-end rule pathway. We demonstrated that LynΔN is unstable and that its stability is dependent on the identity of its N-terminus. Additionally we established that LynΔN degradation could be inhibited by inhibiting either the proteasome or knocking down the UBR1 and UBR2 ubiquitin E3 ligases. Importantly, we also demonstrate that LynΔN degradation by the N-end rule counters the imatinib resistance of K562 cells provided by LynΔN expression. Together our data suggest a possible mechanism for the N-end rule pathway having a link to imatinib resistance in CML. With LynΔN being an N-end rule substrate, it provides the first example that this pathway can also provide a pro-apoptotic function as previous reports have currently only demonstrated anti-apoptotic roles for the N-end rule pathway.

Methylation and decreased expression of SHP-1 are related to disease progression in chronic myelogenous leukemia

Despite the unprecedented success of tyrosine kinase inhibitors (TKIs) in treating chronic myelogenous leukemia (CML), some patients nevertheless progress to advanced stages of the disease. Thus far, the biological basis leading to CML progression remains poorly understood. SH2-containing tyrosine phosphatase 1 (SHP-1) is reported to bind to p210BCR‑ABL1 and to function as a tumor suppressor. Furthermore, its substrates have been found to be essential for p210BCR-ABL1 leukemogenesis or CML progression. In the present study, we found that SHP-1 mRNA and protein levels were markedly decreased in patients in the accelerated and blastic phases of CML (AP-CML and BP-CML) compared to those in the chronic phase (CP-CML). In vitro, we demonstrated that overexpression of SHP-1 reduced p210BCR-ABL1 protein expression and activity in the K562 CML cell line and negatively regulated the AKT, MAPK, MYC and JAK2/STAT5 signaling pathways. Moreover, using a methylation-specific polymerase chain reaction (MSP) assay, abnormal methylation of the SHP-1 gene promoter region was found both in K562 cells and bone marrow (BM) or peripheral blood (PB) cells from AP-CML and BP-CML patients. In conclusion, our findings suggest that decreased expression levels of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulating BCR-ABL1, AKT, MAPK, MYC and JAK2/STAT5 signaling.

Downregulation of miR‐217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibitors (TKI) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMT) and a decrease in levels of microRNA miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

Involvement of Mitochondrial and Reactive Oxygen Species in the Sonodynamic Toxicity of Chlorin e6 in Human Leukemia K562 Cells

It is well accepted that sonodynamic therapy (SDT) exerts cytotoxicity and anti-tumor activity in many human tumors through the induction of cell apoptosis. The aim of the work described here was to study the effect of chlorin e6 (Ce6)-mediated SDT on human chronic myelogenous leukemia K562 cells. Our results indicate that Ce6-mediated SDT can suppress the viability of K562 cells. SDT caused apoptosis as analyzed by annexin V-phycoerythrin/7-amino-actinomycin D staining as well as cleavage of caspase 3 and the polypeptide poly(ADP-ribose) polymerase. After SDT exposure, loss of mitochondrial membrane potential, translocation of Bax from cytoplasm to mitochondria and activation of caspase 9 indicated that the mitochondrial-related apoptotic pathway might be activated. This process was accompanied by rapid generation of reactive oxygen species (ROS). Scavenging of ROS significantly blocked caspase-3 expression and the killing effect of SDT on K562 cells. Stress-activated protein kinases c-jun NH2-terminal kinase (JNK) and the p38 mitogen-activated protein kinase were activated after SDT treatment. Together, these findings indicate that Ce6-mediated SDT triggers mitochondria- and caspase-dependent apoptosis; oxidative injury may play a vital role in apoptotic signaling cascades.

Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.

Olive (Olea europaea) leaf extract induces apoptosis and monocyte/macrophage differentiation in human chronic myelogenous leukemia K562 cells: insight into the underlying mechanism.

Differentiation therapy is an attractive approach aiming at reversing malignancy and reactivating endogenous differentiation programs in cancer cells. Olive leaf extract, known for its antioxidant activity, has been demonstrated to induce apoptosis in several cancer cells. However, its differentiation inducing properties and the mechanisms involved are still poorly understood. In this study, we investigated the effect of Chemlali Olive Leaf Extract (COLE) for its potential differentiation inducing effect on multipotent leukemia K562 cells. Results showed that COLE inhibits K562 cells proliferation and arrests the cell cycle at G0/G1, and then at G2/M phase over treatment time. Further analysis revealed that COLE induces apoptosis and differentiation of K562 cells toward the monocyte lineage. Microarray analysis was conducted to investigate the underlying mechanism of COLE differentiation inducing effect. The differentially expressed genes such as IFI16, EGR1, NFYA, FOXP1, CXCL2, CXCL3, and CXCL8 confirmed the commitment of K562 cells to the monocyte/macrophage lineage. Thus our results provide evidence that, in addition to apoptosis, induction of differentiation is one of the possible therapeutic effects of olive leaf in cancer cells.

Nucleostemin depletion induces post-g1 arrest apoptosis in chronic myelogenous leukemia k562 cells.

Despite significant improvements in treatment of chronic myelogenous leukemia (CML), the emergence of leukemic stem cell (LSC) concept questioned efficacy of current therapeutical protocols. Remaining issue on CML includes finding and targeting of the key genes responsible for self-renewal and proliferation of LSCs. Nucleostemin (NS) is a new protein localized in the nucleolus of most stem cells and tumor cells which regulates their self-renewal and cell cycle progression. The aim of this study was to investigate effects of NS knocking down in K562 cell line as an in vitro model of CML. NS gene silencing was performed using a specific small interfering RNA (NS-siRNA). The gene expression level of NS was evaluated by RT-PCR. The viability and growth rate of K562 cells were determined by trypan blue exclusion test. Cell cycle distribution of the cells was analyzed by flow cytometry. Our results showed that NS knocking down inhibited proliferation and viability of K562 cells in a time-dependent manner. Cell cycle studies revealed that NS depletion resulted in G(1) cell cycle arrest at short times of transfection (24 h) followed with apoptosis at longer times (48 and 72 h), suggest that post-G1 arrest apoptosis is occurred in K562 cells. Overall, these results point to essential role of NS in K562 cells, thus, this gene might be considered as a promising target for treatment of CML.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

VEGF depletion enhances bcr-abl-specific sensitivity of arsenic trioxide in chronic myelogenous leukemia

The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment.

Synthesis and Antiproliferative Activity of Quinolone Nucleosides Against the Human Myelogenous Leukemia K‐562 Cell Line

A set of 6-substituted quinolone nucleosides linked to aniline or phenol via N or O heteroatom-bridges presenting new compounds were synthesized by palladium-catalyzed Buchwald-Hartwig cross-coupling reactions. 6-Bromoquinolone nucleoside precursors, being protected by either benzoyl or TBDMS protecting groups on the ribose moiety, were subjected to different Buchwald-Hartwig conditions as the key step. Defined deprotection steps led, in good yields, to the final target compounds that carry, in position 3, either ester, acid, or amide functions. Thus, a series of novel quinolone nucleoside derivatives was obtained via a convergent synthesis route. Biological tests in human chronic myelogenous leukemia K562 cells exerted an efficient antiproliferative activity for two of them without induction of differentiation. These novel nucleosides deserve further experiments to determine their antiproliferative effects on other CML cell lines.