Chronic Myeloid Leukemia K562 Cells Research Articles

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.

Effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia K562 cells and its mechanism

Objective To explore the effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia cell line K562 and its mechanism. Methods K562 cells in logarithmic growth phase were treated with different concentrations of dehydrocostus lactone. Cell morphology was observed by Wright-Giemsa staining. Cell proliferation was detected by CCK-8 method. Cell cycle, apoptosis and the expressions of cell surface differentiation antigen CD14 and CD11b were detected by flow cytometry. JAK-STAT pathway and the expressions of apoptosis and cell cycle related proteins were detected by Western blot. Results Compared with the control group, the proliferation of K562 cells could be inhibited after treatment with different concentrations (4.0, 6.0, 8.0, 10.0 and 12.0 μmol/L) of dehydrocostus lactone for 24 h, and the difference was statistically significant (F = 109.510, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the apoptosis rates of K562 cells were (16.1±3.8)% and (29.6±4.3)%, which were higher than that of the control group [(3.1±0.5)%] (F = 83.255, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the proportion of G2/M phase cells in K562 cells were (17.0±3.2)% and (28.8±3.9)%, which were higher than that of the control group [(9.1±2.3)%] (F = 161.598, P < 0.05); the proportion of S phase cells in K562 cells were (48.1±3.9)% and (61.0±5.4)%, which were higher than that of the control group [(39.6±3.6)%] (F = 192.356, P < 0.05). After treatment with 2.5 and 5.0 μmol/L dehydrocostus lactone for 72 h, the expression rates of CD14 in K562 cells were (28.6±3.9)% and (41.1±4.4)%, which were higher than that in the control group [(3.1±0.5)%] (F = 132.811, P < 0.05); the expression rates of CD11b in K562 cells were (42.4±5.0)% and (61.2±5.7)%, which were higher than that in the control group [(4.2±1.1)%] (F = 179.553, P < 0.05). Dehydrocostus lactone could decrease the expressions of JAK2, STAT5, cyclin E, CDK2, cyclin A, CDC25C, cyclin B1, CDK1 and bcl-2 proteins, and up-regulate the expressions of p21 and bax proteins. Conclusion Dehydrocostus lactone can inhibit the proliferation of chronic myeloid leukemia K562 cells, which may be achieved by cell cycle arrest, induction of apoptosis and differentiation. Key words: Dehydrocostus lactone; Cell cycle; Apoptosis; Differentiation; Leukemia

TOPK is regulated by PP2A and BCR/ABL in leukemia and enhances cell proliferation.

Although treatment of chronic myeloid leukemia (CML) has improved with the development of tyrosine kinase inhibitors (TKIs), patients develop fatal blast crisis(BC) whilst receiving TKI treatment. Alternative treatments for cases resistant to TKIs are required. A serine/threonine protein kinase, T‑lymphokine‑activated killer cell‑originated protein kinase(TOPK), is highly expressed in various malignant tumors. Binding of peptides to human leukocyte antigen was assessed via mass spectrometry in K562 CML cells. TOPK expression was assessed in various CML cell lines and in clinical samples obtained from patients with CML using reverse transcription‑quantitative polymerase chain reaction and western blot assays. It was observed that TOPK was expressed abundantly in BCR/ABL‑positive cell lines and at significantly higher levels in CML clinical samples compared with healthy donor samples. Overexpression of BCR/ABL or the presence of its inhibitor imatinib upregulated and downregulated TOPK expression, respectively, indicating that TOPK may be a target of BCR/ABL. TOPK inhibitor OTS514 suppressed proliferation of BCR/ABL‑positive cell lines and colony formation of CD34‑positive cells from patients with CML compared with lymphoma patients without bone marrow involvement. Furthermore, phosphorylation of TOPK was increased by protein phosphatase2A(PP2A) inhibitor okadaic acid and was decreased in the presence of PP2A activator FTY720 compared with untreated samples. As constitutive BCR/ABL activity and inhibition of PP2A are key mechanisms of CML development, TOPK may be a crucial signaling molecule for this disease. Inhibition of TOPK may control disease status of CML, even in cases resistant to TKIs.

Tumor necrosis factor α knockout impaired tumorigenesis in chronic myeloid leukemia cells partly by metabolism modification and miRNA regulation.

PurposeHalf of the chronic myeloid leukemia (CML) patients with sustained deep molecular response suffer from relapse after discontinuation mainly because tyrosine kinase inhibitors (TKIs) cannot eradicate leukemia stem cells (LSCs). In addition, tumor necrosis factor α (TNF-α) is highly detected in CML patients. Our aim was to explore whether TNF-α is a potential target for LSC elimination.Materials and methodsWe applied a CRISPR/Cas9 gene editing technique, colony-forming cell assay, subcutaneous tumor models, miRNA-seq and liquid chromatography-mass spectroscopy (LC-MS) on metabonomics to explore the feasibility and mechanism of TNF-α as a new therapeutic target for CML.ResultsWe demonstrated that TNF-α knockout remarkably decreased the proliferative, colony-forming and in vivo tumorigenesis capacities of the CML K562 cell line. The apoptosis was increased when TNF-α knockout cells were cultured with imatinib. The mechanisms involved in the abovementioned phenomena were that TNF-α knockout inhibited the citrate cycle and increased starch, sucrose, amino sugar and nucleotide sugar metabolism. In addition, differentially expressed miRNAs between TNF-α knockout and control cells were involved in the cell cycle, CML, P13K-Akt and pathways in cancer.ConclusionWe identified that TNF-α may serve as a new target therapy for CML and described the metabolic pathways associated with TNF-α in CML cells for the first time.

MiR-378 promoted cell proliferation and inhibited apoptosis by enhanced stem cell properties in chronic myeloid leukemia K562 cells

Dysregulation of miR-378 has been found in diverse types of tumors as well as in leukemia. The role of miR-378 in chronic myeloid leukemia (CML) remains unclear. The aim of the study was to reveal the potential effects of miR-378 in the pathological process and progress in CML. Our results showed general level of miR-378 was significant higher in CML patients compared to controls. Overexpression of miR-378 dramatically promoted cell proliferation and drug-resistance. Additionally, apoptosis was inhibited in cells transfected with miR-378. More and bigger stem cell sphere formation was observed in miR-378 transfected cells. Furthermore, enhanced expression of miR-378 was associated with upregulation of stem-cell makers OCT4 and c-Myc. Further study validated that miR-378 inhibited the expression of FUS1. Our research demonstrated the oncogenic nature of miR-378 in CML, and might contribute to the progress of CML.

DNA-binding affinity, cytotoxicity, apoptosis, cell cycle inhibition and molecular docking studies of a new stilbene derivative

Stilbene derivatives have been found to possess promising anticancer activities against human cancer cell lines in vitro. In the present study, we have investigated cytotoxic, apoptosis induction and DNA binding activity of new stilbene derivative, (E)-1-(4-Chlorophenyl)-4,5-diphenyl-2-[4-(4-methoxystryl)phenyl]-1H-imidazol (STIM) on K562 chronic myeloid leukemia cell line. Via MTT assay STIM demonstrated cytotoxic activity against K562 cell line with IC50 value of 150 µM. Apoptosis, as the mechanism of cell death, was evaluated by morphological study and flow cytometric analysis. In vitro DNA binding property of STIM has been studied by vital spectroscopic techniques, which indicated that STIM interact with ctDNA through groove binding mode and binding constant (Kb) was estimated to be 6.9 × 104 M−1. Docking studies revealed that hydrophobic is the most important interaction in STIM-DNA complex, and that the ligand (STIM) interacts with DNA via groove binding mode and the bindiyspng energy was calculated as −13.37 kcal/mol. Taken together, the present study suggests that STIM exhibits anticancer effect on K562 cell line through the induction of apoptosis as well as cell cycle arrest at Sub-G1 phase and also can bind to double helix DNA in vitro.

BCR-Abl Silencing by siRNA: A Potent Approach to Sensitize Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitor Therapy.

Nonviral gene therapy with specific short interfering RNAs (siRNAs) against BCR-Abl can be an alternative and/or supportive therapy of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs), given the often observed resistance to TKIs in clinical setting. In this study, we explored the feasibility of BCR-Abl siRNA therapy in CML K562 cells in vitro by employing a cationic polymer derived from cholesterol (Chol) grafted low-molecular weight polyethyleneimine (PEI). The first generation TKI imatinib upregulated the expression of BCR-Abl in K562 cells as expected. Delivery of BCR-Abl siRNA in both drug-sensitive and drug-resistant K562 cells significantly downregulated the mRNA levels in both cell types. Similarly, the BCR-Abl siRNA treatment arrested the growth of both drug-sensitive and drug-resistant K562 cells with no obvious differences despite a large difference in drug responsiveness. The BCR-Abl gene silencing in combination with TKI treatments exhibited significant synergism in drug-resistant K562 cells in generating substantial antileukemic activity, where the TKIs on their own were not effective. The effect of BCR-Abl siRNA and TKIs on non-CML cells (Jurkat and primary fibroblast) was negligible, indicating the specificity of the proposed therapy. This strategy can significantly overcome TKI resistance in CML cells, suggesting a feasible and effective treatment model for CML patients suffering from clinical resistances.

Hetarylfuroxans: cytotoxic effect and induction of apoptosis in chronic myeloid leukemia K562 cells

Hetarylfuroxans (4-(2-methylpyridin-5-yloxy)-3-phenylfuroxan (1), bis(1,2,4-oxadiazol- 3-yl)furoxan (2), and 4-amino-3-(indenotriazin-3-yl)furoxan (3)) exhibit in vitro cytotoxic activity against chronic myeloid leukemia K562 cells. All studied furoxans induce apoptosis in K562 cells. The experiments with activation of caspases-3 and -7 showed that furoxan 3 possessed the highest apoptosis-inducing capacity. The studied hetarylfuroxans were found to be the promising drug candidates for the treatment of chronic myeloid leukemia K562.

VITAMIN E ACTIVATES EXPRESSION OF C/EBP ALPHA TRANSCRIPTION FACTOR AND G-CSF RECEPTOR IN LEUKEMIC K562 CELLS

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562cells. RNA extracted from K562cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). We have not found detectable expression of C/EBP alpha in K562cells. Upon 48-h culture with vitamin E at a dose of 100µM, K562cells expressed both C/EBP alpha and G-CSFR. VitaminE restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562cells. In this setting, G-CSFR expression in vitaminE treated K562cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

Role of Folate Receptor 3 in Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by Philadelphia chromosome and its molecular counterpart, the BCR-ABL1 fusion gene. Discontinuation of tyrosine kinase inhibitors (TKIs) therapy after achieving a persistent deep molecular response (DMR) is an urgently-needed treatment goal for patients and it was written in NCCN guideline Version 2.2017 for CML. Indeed, various studies have confirmed the feasibility of stopping TKIs therapy in many regions. However, increasing evidence demonstrated that TKIs are unable to eliminate quiescent leukemic stem cells (LSCs), which lead to treatment resistance or relapse after discontinuation of TKIs treatment. Multiple works have confirmed that approximately 50-60% of DMR patients stopping TKIs will lose their response and require retreatment. The patients who will lose the treatment-free remission (TFR) remains unknown currently.We selected bone marrow of 14 patients (matched 7 relapse and 7 non-relapse patients) who discontinued TKIs therapy in our center for transcriptome analysis to discover the differences between relapse and non-relapse patients. Bioinformatics indicated that folate receptor 3 (FOLR3) was highly expressed in non-relapse patients and only in the non-relapsers (Figure 1A). Further work found these samples all carried a common SNP mutation. Then we respectively transferred newly diagnosed chronic phase (CP) CML patients derived bone marrow CD34+ cells and CML K562 cells with lentiviral vectors containing FOLR3 SNP, FOLR3 SNP shRNA, and wild type FOLR3. The result indicated that FOLR3 SNP significantly promoted the clonogenicity of CD34+/K562 cells, while loss of FOLR3 SNP hindered cell differentiation (Figure 1B and D are data of CD34, Figure 1C and E are data of K562). K562 subcutaneous tumor formation in balb/c node mice confirmed that tumors weight and volume of FOLR3 SNP shRNA group were higher than control. While, small animal PET scanning showed that maximum standardized uptake value (SUVmax) of 18F-FDG of FOLR3 SNP+ group was higher than the rest. Experiments in vivo and vitro synergistically proved FOLR3 SNP promoted tumor cell differentiation, delayed tumor growth.To better understand how FOLR3 SNP promote tumor cell differentiation and delay tumor growth, we performed second transcriptome analysis. Consistently, both in FOLR3 SNP+ CD34+ and K562 cells, enrichment analysis revealed that differentially expressed genes were enriched in mitochondria associated Gene Ontology (GO) biological process (Figure F), in which mitochondria complex V matched genes were most significant, such as ATP5 family. In vitro, we demonstrated that ATP syntheses, maximal respiration and spare respiratory capacity of FOLR3 SNP+ CD34+/K562 cells were significantly higher than control and shFOLR3 counterpart through seahorse XF cell mito stress test (Figure G and H refer to CD34 and K562, separately). Electron microscope also exhibited an increase of mitochondrial in FOLR3 SNP+ cells.Mitochondria play an essential role in energy generation, cell signalling, differentiation, death and senescence in eukaryotic cells. We detected a series of genes related to aging, cell cycle and mitochondria unfolded protein response. The results showed that cell cycle kinase such as CDK4 decreased in FOLR3 SNP+ CD34+/K562 cells. On the other hand, senescence associated genes seemed increased. In conclusion, we highlighted the connection of FOLR3 and post-cessation relapse. FOLR3 SNP could be an indicator of TFR. Its internal mechanism might be the mitochondrion activation induced aging of residual leukemia cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites.

The functions of many long noncoding RNAs (lncRNAs) in the human genome remain unknown owing to the lack of scalable loss-of-function screening tools. We previously used pairs of CRISPR-Cas9 (refs. 1, 2, 3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs. Here we demonstrate genome-wide screening of lncRNA function using sgRNAs to target splice sites and achieve exon skipping or intron retention. Splice-site targeting outperformed a conventional CRISPR library in a negative selection screen targeting 79 ribosomal genes. Using a genome-scale library of splicing-targeting sgRNAs, we performed a screen covering 10,996 lncRNAs and identified 230 that are essential for cellular growth of chronic myeloid leukemia K562 cells. Screening GM12878 lymphoblastoid cells and HeLa cells with the same library identified cell-type-specific differences in lncRNA essentiality. Extensive validation confirmed the robustness of our approach.

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

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

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

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

Alteration of cellular and immune-related properties of bone marrow mesenchymal stem cells and macrophages by K562 chronic myeloid leukemia cell derived exosomes.

Leukemic cells can impact the bone marrow niche to create a tumor-favorable microenvironment using their secreted factors. Little knowledge is available about immunosuppressive and tumor-promoting properties of chronic myeloid leukemia derived exosomes in bone marrow stromal components. We report here that K562-derived exosomes can affect the gene expression, cytokine secretion, nitric oxide (NO) production, and redox potential of bone marrow mesenchymal stem cells (BM-MSCs) and macrophages. Human BM-MSCs and mouse macrophages were treated with K562-derived exosomes. Our results demonstrated that the expression of the genes involved in hematopoietic developmental pathways and immune responses, including C-X-C motif chemokine 12 (Cxcl12), Dickkopf-related protein 1 (DKK1), wnt5a, interleukin 6 (IL-6), transforming growth factor-beta, and tumor necrosis factor-alpha (TNF-alpha), changed with respect to time and exosome concentration in BM-MSCs. The TNF-alpha level was higher in exosome-treated BM-MSCs compared with the control. Exosome treatment of BM-MSCs led to an increased production ofNO and a decreased production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. We have shown that K562-derived exosomes induce overexpression of IL-10 and TNF-alpha and downregulation of iNOS transcript levels in macrophages. The enzyme-linked immunosorbent assay results showed that TNF-alpha and IL-10 secretions increased in macrophages. Treatment of macrophages with purified exosomes led to reduced NO and ROS levels. These results suggest that K562-derived exosomes may alter the local bone marrow niche toward a leukemia-reinforcing microenvironment. They can modulate the inflammatory molecules (TNF-alpha and NO) and the redox potential of BM-MSCs and macrophages and direct the polarization of macrophages toward tumor-associated macrophages.

Epigallocatechin-3-gallate and BIX-01294 have different impact on epigenetics and senescence modulation in acute and chronic myeloid leukemia cells

Myeloid leukemia treatment is quite successful nowadays; nevertheless the development of new therapies is still necessary. In the present study, we investigated the potential of epigenetic modulators EGCG (epigallocatechin-3-gallate) and BIX-01294 (N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine) to alter epigenetic state and cause cellular senescence in acute and chronic myeloid leukemia NB4 and K562 cells. We have shown that after leukemia cell treatment with EGCG and BIX-01294 the proliferation and survival were inhibited of both cell lines; however, only NB4 cells underwent apoptosis. Both epigenetic modulators caused cell cycle arrest in G0/G1 phase as assessed by RT-qPCR (p53, p21, Rb) and flow cytometry analysis. Increased levels of ATM, HMGA2, phosphorylated ATM, and SA-β-galactosidase staining indicated that EGCG caused cellular senescence, whereas BIX-01294 did not. Immunoblot analysis of epigenetic players DNMT1, HP1α, H3K9me3, EZH2, and SUZ12 demonstrated beneficial epigenetic modulation by both agents with exception of mainly no epigenetic changes caused in K562 cells by EGCG. Therefore, we suggest EGCG as a promising epigenetic modulator for acute promyelocytic leukemia therapy and as a potential cellular senescence inducer in both acute and chronic myeloid leukemia treatment, whereas BIX-01294 could be beneficial as an epigenetic modifier for both myeloid leukemias treatment.

Overexpression of miR-574-3p suppresses proliferation and induces apoptosis of chronic myeloid leukemia cells via targeting IL6/JAK/STAT3 pathway.

The present study aimed to elucidate the potential roles and regulatory mechanism of microRNA (miR)-574-3p in the development of chronic myeloid leukemia (CML). The expression of miR-574-3p in peripheral blood obtained from patients with CML was examined. Subsequently, miR-574-3p was overexpressed and suppressed in CML K562 cells to further investigate the effects of miR-574-3p on cell proliferation, and apoptosis. Furthermore, a luciferase reporter assay was performed to investigate whether interleukin-6 (IL-6) was a target of miR-574-3p. In addition, the regulatory association between miR-574-3p and the IL-6/Janus kinase (JNK)/signal transducer and activator of transcription-3 (STAT3) signaling pathway was explored. The expression of miR-574-3p in the peripheral blood obtained from patients with CML was significantly lower compared with that in healthy controls. Overexpression of miR-574-3p significantly inhibited the proliferation and induced the apoptosis of K562 cells, whereas suppression of miR-574-3p exhibited opposite effects. In addition, IL-6 was identified to be a direct target of miR-574-3p. Overexpression of IL-6 significantly promoted the proliferation and inhibited the apoptosis of K562 cells. Furthermore, overexpression of miR-574-3p inhibited the activation of the JAK/STAT3 signaling pathway, which was rescued by overexpression of IL-6. The results of the current study indicate that miR-574-3p overexpression may serve an important role in inhibiting proliferation and inducing apoptosis of K562 cells via suppression of IL-6/JAK/STAT3 signaling pathway activation. miR-574-3p may serve as a potential therapeutic target for CML.

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.

CDA gene silencing regulated the proliferation and apoptosis of chronic myeloid leukemia K562 cells

BackgroundAs a disease of hematopoietic stem cell, chronic myeloid leukemia (CML) possesses unique biological and clinical features. However, the biologic mechanism underlying its development remains poorly understood. Thus, the objective of the present study is to discuss the effect of cytidine deaminase (CDA) gene silencing on the apoptosis and proliferation of CML K562 cells.MethodsCDA mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzymatic activity of CDA was measured by a nuclide liquid scintillation method. RT-qPCR and Western blot analysis were used to detect CDA mRNA and protein expression. Cell proliferation, apoptosis and cell cycle were measured by CCK-8 assay and flow cytometry. The expression of proteins relevant to cell proliferation, apoptosis and cell cycle was measured by Western blot analysis. Tumor xenografts were implanted in nude mice to verify the effect of CDA silencing on tumor growth in vivo.ResultsCML and AL patients showed increased mRNA expression and enzymatic activity of CDA. Compared with the blank group, the mRNA and protein expression of CDA in the shRNA-1 and shRNA-2 groups decreased significantly. As a result, the proliferation of K562 cells was inhibited after CDA silencing and the cells were mainly arrested in S and G2 phases, while the apoptosis rate of these cells was increased. In addition, CDA gene silencing in K562 cells led to down-regulated p-ERK1/2, t-AKT, p-AKT and BCL-2 expression and up-regulated expression of P21, Bax, cleaved caspase-3/total caspase-3 and cleaved PARP/total PARP. Finally, CDA gene silencing inhibited tumor growth.ConclusionOur study demonstrated that CDA gene silencing could inhibit CML cell proliferation and induce cell apoptosis. Therefore, CDA gene silencing may become an effective target for the treatment of leukemia.

Decreased expression of microRNA-214 contributes to imatinib mesylate resistance of chronic myeloid leukemia patients by upregulating ABCB1 gene expression.

The aim of the present study was to determine the expression of adenosine triphosphate binding cassette subfamily B member 1 (ABCB1) gene and its protein P-glycoprotein (PGP) in bone marrow mononuclear cells from chronic myeloid leukemia (CML) patients with imatinib mesylate (IM) resistance, or IM-resistant CML K562 cells. In addition, the molecular mechanism of action of microRNA (miR)-214 on ABCB1 in IM resistance was investigated. A total of 26 CML patients with IM resistance were included in the present study. In addition, 31 CML patients who did not have IM resistance were included as the control group. Bone marrow was collected from all subjects. The K562R cell line, which is a K562 cell line with IM resistance, was used for cellular studies. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of ABCB1 mRNA and miR-214 in cells. Western blotting was employed to determine the expression of PGP. Dual luciferase reporter assay was carried out to identify interactions between ABCB1 mRNA and miR-214. MTT assay was used to determine the survival rate of cells. ABCB1 mRNA and PGP expression was upregulated in bone marrow mononuclear cells from CML patients with IM resistance. K562R cells had higher ABCB1 and PGP expression than K562 cells, potentially due to their different sensitivity to IM. Expression miR-214 was decreased in bone marrow mononuclear cells from patients with IM resistance and K562R cells. Notably, miR-214 was able to bind with the 3′-untranslated region, seed region of ABCB1 mRNA to regulate its expression. In addition, elevated expression of miR-214 restored IM sensitivity to K562R cells potentially by affecting ABCB1 expression. The present study demonstrated that upregulated expression of ABCB1 mRNA and PGP in bone marrow mononuclear cells from CML patients with IM resistance may be associated with the downregulation of miR-214. In addition, miR-214 may participate in the IM resistance of CML patients by regulating ABCB1 expression.

Abstract 5846: Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance

Abstract Background: Chronic myeloid leukemia (CML) is one of the most extensively studied hematopoietic disorders. Caused by reciprocal translocation t(9;22) and formation of the Philadelphia chromosome, it leads to constitutive expression of the BCR-ABL kinase and malignant transformation. Targeted therapy using tyrosine kinase inhibitors (TKI), such as imatinib or nilotinib, led to tremendous success in treatment. However, resistances are an uprising problem in patient cure. Besides BCR-ABL-dependent mechanisms (i.e. BCR-ABL amplification, mutations), cellular alterations of gene expression and its regulation are involved. Therefore, we performed an in vitro-study in K-562 CML cells to investigate changes of gene expression and epigenetics, as well as occurrence of mutations during the course of imatinib or nilotinib resistance. Methods: We analyzed mRNA expression, DNA-methylation and mutation status of treatment-naïve and TKI-resistant sublines (0.5 µM, 2 µM imatinib; 100 nM nilotinib) of the in vitro-cell line model (K-562). mRNA expression was analyzed using Affymetrix Human Gene 2.0 ST arrays and DNA-methylation by Infinium MethylationEPIC BeadChips (Illumina); exome sequencing was performed at GATC, Konstanz, Germany. Target verification was performed using qRT-PCR, pyrosequencing or Sanger sequencing. Effects on TKI-susceptibility were analyzed using transfection experiments with analyses of cell viability (WST-1 assay) and apoptosis (Caspase Glo 9 assay). Results: Cell resistant to low imatinib doses exhibited differentially expression of 485 mRNAs compared to treatment-naïve cells, whereas at high dose-resistance 166 mRNAs differed. 151 genes showed expression changes independent from the imatinib dose. In addition, we found 45 genes with expression changes during the course of imatinib resistance, i.e. the drug efflux transporter ABCG2, which was significantly upregulated at low and downregulated at high imatinib doses. A total of 254 genes was associated with nilotinib resistance, while 71 genes were differentially expressed in all resistant sublines. Regarding methylation and occurrence of mutations in imatinib resistance, we observed distinct variations pointing to genetic changes, especially at high-dose resistance, i.e. in the promoter region of microRNA-212, a proven regulator of ABCG2. In nilotinib resistance, we found 125 genes differentially methylated during development of resistance. Conclusions: Our data indicate that development of resistance to tyrosine kinase inhibitors is based on complex expression profile changes that can in part be explained by epigenetic alteration e.g. in drug efflux transporters. Besides, the mechanisms underlying resistances to imatinib or nilotinib seem to be dissimilar. After verification in clinical studies, these in vitro findings could be relevant in attempts to overcome TKI-resistances. Citation Format: Meike Kaehler, Inga Nagel, Henrike Bruckmueller, Ruwen Boehm, Ole Ammerpohl, Ingolf Cascorbi. Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5846.