Migration Of K562 Cells Research Articles

Effect of Csn-B Combined with Imatinib on Apoptosis of Chronic Myeloid Leukemia Cells through Nur77/Pim-1/Drp1 Pathway

To investigate the anti- chronic myelogenous leukemia (CML) activity of Nur77-specific agonist Csn-B combined with imatinib by promoting Nur77 expression, and explore the potential role of its signaling pathway. Firstly, CCK-8 and Transwell assay were used to detect the inhibitory effects of Csn-B, imatinib, and their combination on the proliferation and migration of K562 cells. Furthermore, the apoptosis rate of K562 cells treated with Csn-B, imatinib, and their combination was detected by flow cytometry. The expression levels of Nur77, Pim-1, Drp1, p-Drp1 S616, Bcl-2 and Bax in K562 cells were detected by Western blot. Finally, the expression levels of reactive oxygen species (ROS) in K562 cells treated with Csn-B, imatinib and their combination were detected by immunofluorescence assay. The level of Nur77 in CML patients decreased significantly compared with normal population in dataset of GSE43754 (P < 0.001). Csn-B combined with imatinib could significantly inhibit the proliferation and migration of K562 cells (both P < 0.001), and induce apoptosis (P < 0.001). Csn-B promoted Nur77 expression in K562 cells, and synergistically enhanced imatinib sensitivity when combined with imatinib. Csn-B combined with imatinib could significantly enhanced ROS levels in K562 cells and mitochondria compared with single-drug treatment (both P < 0.001). Csn-B combined with imatinib can enhance ROS expression and induce apoptosis of K562 cells through Nur77/Pim-1/Drp1 pathway.

Effect and molecular mechanism of hesperadin-induced ferroptosis in chronic myeloid leukemia K562 cells

Objective: To investigate the effect and molecular mechanism of hesperadin in inducing ferroptosis in chronic myeloid leukemia cell line K562 cells. Methods: The effects of hesperadin on the viability, proliferation, and migration of K562 cells were detected though CCK8, EDU-594, and Transwell assays, and the apoptotic rate of K562 cells was detected by flow cytometry. In addition, C11-BODIPY and FerroOrange were utilized to detect intracellular lipid peroxidation and Fe(2+) levels. Meanwhile, the expression levels of ferroptosis-associated protein solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in cells were detected through Western blot. Lipid peroxidation and Fe(2+) levels were also detected after transfection of cells with SLC7A11 overexpression plasmid. Results: Hesperadin decreased cell viability in a dose-dependent manner with IC(50) of 0.544 μmol/L. Hesperadin concentrations of 0.4 and 0.8 μmol/L were selected for follow-up experiments. EDU-594, Transwell, and flow cytometry showed significantly decreased proliferation and migration rate of K562 cells after 0.4 and 0.8 μmol/L hesperadin treatment for 24 h, and the apoptosis rate was significantly increased compared with the control group (P<0.05). Western blot indicated a downregulated expression of the antiapoptotic protein Bcl-2 and an elevated expression of proapoptotic proteins Bax and Caspase-3. Moreover, hesperadin increased intracellular lipid peroxidation and Fe(2+) levels compared with the control treatment (P<0.05). The combination of ferroptosis inhibitor (Fer-1) and hesperadin could reverse the effect of hesperadin on K562 cells. The mRNA and protein levels of ferroptosis-related genes SLC7A11 and GPX4 were significantly decreased in the 0.8 μmol/L hesperadin-treated group (P<0.05). SLC7A11 overexpression can inhibit hesperadin effect and alleviate ferroptosis. Conclusion: Hesperadin can promote ferroptosis in K562 cells by regulating the SLC7A11/GPX4 axis.

HnRNPA0 promotes MYB expression by interacting with enhancer lncRNA MY34UE-AS in human leukemia cells

MYB is a key regulator of hematopoiesis and erythropoiesis, and dysregulation of MYB is closely involved in the development of leukemia, however the mechanism of MYB regulation remains still unclear so far. Our previous study identified a long noncoding RNA (lncRNA) derived from the −34 kb enhancer of the MYB locus, which can promote MYB expression, the proliferation and migration of human leukemia cells, and is therefore termed MY34UE-AS. Then the interacting partner proteins of MY34UE-AS were identified and studied in the present study. hnRNPA0 was identified as a binding partner of MY34UE-AS through RNA pulldown assay, which was further validated through RNA immunoprecipitation (RIP). hnRNPA0 interacted with MY34UE-AS mainly through its RRM2 domain. hnRNPA0 overexpression upregulated MYB and increased the proliferation and migration of K562 cells, whereas hnRNPA0 knockdown showed opposite effects. Rescue experiments showed MY34UE-AS was required for above mentioned functions of hnRNPA0. These results reveal that hnRNPA0 is involved in leukemia through upregulating MYB expression by interacting with MY34UE-AS, suggesting that the hnRNPA0/MY34UE-AS axis could serve as a potential target for leukemia treatment.

Effect of CPEB4 on Migration and Cycle of Chronic Myeloid Leukemia Cell

To investigate the effects of CPEB4 on the migration and cycle of K562 cells and the changes of protein molecules that may be involved in the regulatory mechanism. Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4, silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells by electroporation so as to change CPEB4. The transfection efficiency was detected by Western blot. Finally, the migration and cycle of different cells were detected by Transwell chamber and flow cytometry.Western blot was used to detect the expression changes of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins. Compared with normal white blood cells, the expression of CPEB4 protein in K562 cells was significantly enhanced (P＜0.01); Compared with the control group, CPEB4-silenced K562 cells showed that the cell migration ability was significantly enhanced (P＜0.01); G0/G1 phase cell ratio reduced, G2/M phase cell ratio increased, and cell cycle progression accelerated（P＜0.01）, The expression levels of MMP2 (P＜0.05), MMP9 (P＜0.05), CDK4 (P＜0.01), CyclinD1 (P＜0.01) proteins increased significantly. The expression level of P21 protein significantly decreased (P＜0.01). The migration ability of K562 cells after CPEB4 overexpression was decreased (P＜0.01), the cell ratio of G0/G1 phase in the cell cycle increased, the cell proportion of S phase decreased and the cell cycle progression was arrested at G0/G1 phase (P＜0.01). The expression of P21 protein increased, MMP2 , MMP9, CDK4, CyclinD1 protein expression decreased significantly（P＜0.05-0.01）. CPEB4 can inhibit the migration of K562 cells and arrest cell cycle progression at G0/G1 phase. Its mechanism may be related with regulating the exprossion of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins.

MicroRNA-141-5p Acts as a Tumor Suppressor via Targeting RAB32 in Chronic Myeloid Leukemia

MicroRNA-141-5p (miR-141-5p), an important member of the miR-200 family, has been reported to be involved in cellular proliferation, migration, invasion, and drug resistance in different kinds of human malignant tumors. However, the role and function of miR-141-5p in chronic myeloid leukemia (CML) are unclear. In this current study, we found that the level of miR-141-5p was significantly decreased in peripheral blood cells from CML patients compared with normal blood cells and human leukemic cell line (K562 cells) compared with normal CD34+ cells, but was remarkably elevated in patients after treatment with nilotinib or imatinib. Suppression of miR-141-5p promoted K562 cell proliferation and migration in vitro. As expected, overexpression of miR-141-5p weakened K562 cell proliferation, migration, and promoted cell apoptosis. A xenograft model in nude mice showed that overexpression of miR-141-5p markedly suppressed tumor growth in vivo. Mechanistic studies suggested that RAB32 was the potential target of miR-141-5p, and silencing of RAB32 suppressed the proliferation and migration of K562 cells and promoted cell apoptosis. Taken together, our study demonstrates that miR-141-5p plays an important role in the activation of K562 cells in vitro and may act as a tumor suppressor via targeting RAB32 in the development of CML.

Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment

AbstractAbstract 4426 Background/Aims:Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes.Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA.We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562.Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD. Results:We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration.Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied. Conclusions:IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression. Disclosures:No relevant conflicts of interest to declare.

Effects of SHIP Over Expression On the Invasion and Migration of K562 Cells and Its Mechanism.

Abstract 5051 IntroductionThe phosphatidylinositol-3 kinase (PI3K) pathway plays a central role in regulating numerous biologic processes, including survival, adhesion, migration, metabolic activity, proliferation, differentiation, and end cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3)). The SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) is known to play an important role in the negative regulation by PI3K-dependent signaling cascades activated by several tyrosine-kinase coupled cytokine receptors. SHIP knockout mice display myeloproliferation and hyper-responsiveness to growth factor stimulation and have short life span with myeloid cell infiltration into vital organs. We have proved SHIP inhibits growth of leukemia cells, and it may have an important role in apoptosis of leukemia cells. However, to date the role of anti-migrated effect of SHIP and its mechanism is unclear. MethodsBased on the results of our previous studies, with different human leukemia cell lines K562 as study samples, including K562 transfected with wtSHIP (Group A), K562 transfected with muSHIPC1076T (Group B) and K562 transfected with empty vector (Group C); Expression of SHIP at mRNA and protein level was detected by real-time PCR and Western blot, respectively; By Transwell chamber assay was used to count the numbers of Group A and Group B cells that penetrated the matrigel to the back of PVPF membrane after transfection; Primary migration associated factor FAK, p-FAK and NFκB were screened by Western blot. The expression of MMP-2 and MMP-9 was examined by zymography. ResultsCompared with Group C and Group B, the migration of Group A cells was decreased (32±6 vs 78±13 and 83±16)(P<0.01); Over expression of wild-type SHIP does not affect the MMP2 secretion in K562, but site-directed mutant in C terminus of SHIP can significantly block MMP-9 secretion in K562 cells, By Western blot analysis, the expression of p-FAK and NFκB protein in Group A cells were down-regulated to 44% and 63% of the Group C, respectively. ConclusionsThe results confirmed SHIP as a negative regulator for cell migration and invasion in bcr/abl transformed cells through decreasing the MMP-9 expression, which may be induced by reduced phopha-FAK and NFαB expression, and implied that it may function through its PXXP domain. DisclosuresNo relevant conflicts of interest to declare.