Imatinib-sensitive K562 Cells Research Articles

Bioinformatic analysis of the expression profile and identification of RhoGDI2 as a biomarker in imatinib-resistant K562 cells

ABSTRACTObjectivesChronic myeloid leukemia (CML) is an aggressive malignancy originating from hematopoietic stem cells. Imatinib (IM), the first-generation tyrosine kinase inhibitor, has greatly improved theliving quality of CML patients. However, owing to the recurrence and treatment failure coming from tyrosine kinase inhibitor (TKIs) resistance, some CML patients still bear poor prognosis. Therefore, we aimed to seek potential signaling pathways and specific biomarkers for imatinib resistance.MethodsWe performed mRNA and miRNA expression profiling in imatinib-sensitive K562 cells (IS-K562) and imatinib-resistant K562 cells (IR-K562). Differentially expressed genes (DEGs) were identified and pathway enrichment analyses were performed to explore the potential mechanism. The protein–protein interaction (PPI) network and miRNA-mRNA regulatory network were constructed to explore potential relationships among these genes. RT-qPCR, western blot and CCK8 were used for further experiments.ResultsA total of 623 DEGs and 61 differentially expressed miRNAs were identified. GO revealed that DEGs were mainly involved in cell adhesion, cell migration, differentiation, and inflammatory response. KEGG revealed that DEGs were typically enriched in the Rap1 signaling pathway, focal adhesion, proteoglycans and transcriptional misregulation in cancer, signaling pathways regulating pluripotency of stem cells and some immune-related pathways. The protein–protein interaction (PPI) network and miRNA-mRNA regulatory network revealed a web of diverse connections among genes. Finally, we proved that RHoGDI2 played a critical role in imatinib resistance.ConclusionThe dynamic interplay between genes and signaling pathways is associated with TKIs resistance and RHoGDI2 is identified as a biomarker in IR-K562.

MiR-145-5p and miR-203a-5p overcome imatinib resistance in myelogenous leukemic cells through metabolic reprogramming

Imatinib is the most effective therapy for chronic myeloid leukemia (CML), but many patients eventually develop resistance to it after an initial satisfactory response. This study investigated the potential of three miRNAs (miR-106b-5p, miR-145-5p, miR-203a-5p) in overcoming imatinib resistance in leukemic cells. The imatinib-resistant K562 (IR-K562) cells were developed and transfected with one of the three miRNAs to evaluate their potency in overcoming imatinib resistance. The changes in the metabolic profile were studied using flux balance analysis (FBA) and the data was validated using qRT-PCR.Among the three miRNAs, the ectopic expression of either miR-145-5p or miR-203a-5p was able to sensitize the IR-K562 cells to imatinib. The concentration of key oncometabolites; glucose, lactate, and glutamine, in the culture media of the miR-transfected IR-K562 cells, reverted to the same levels as seen in imatinib-sensitive K562 cells. In addition, the FBA analysis revealed that the metabolism of lipid, fatty acids, and electron transport chain were significantly altered in resistant cells. The FBA data was also validated at the molecular level. Interestingly, the imatinib treatment coupled with the transfection of miR-145-5p or miR-203a-5p cells could reverse the metabolic flux of IR-K562 to the levels seen in imatinib-sensitive K562 cells. This study highlights the key metabolic changes that occur during development of imatinib resistance. It also identifies the specific miRNAs which can be targeted to overcome imatinib resistance in CML.

Antileukemic Activity of hsa-miR-203a-5p by Limiting Glutathione Metabolism in Imatinib-Resistant K562 Cells.

Imatinib has been the first and most successful tyrosine kinase inhibitor (TKI) for chronic myeloid leukemia (CML), but many patients develop resistance to it after a satisfactory response. Glutathione (GSH) metabolism is thought to be one of the factors causing the emergence of imatinib resistance. Since hsa-miR-203a-5p was found to downregulate Bcr-Abl1 oncogene and also a link between this oncogene and GSH metabolism is reported, the present study aimed to investigate whether hsa-miR-203a-5p could overcome imatinib resistance by targeting GSH metabolism in imatinib-resistant CML cells. After the development of imatinib-resistant K562 (IR-K562) cells by gradually exposing K562 (C) cells to increasing doses of imatinib, resistant cells were transfected with hsa-miR-203a-5p (R+203). Thereafter, cell lysates from various K562 cell sets (imatinib-sensitive, imatinib-resistant, and miR-transfected imatinib-resistant K562 cells) were used for GC-MS-based metabolic profiling. L-alanine, 5-oxoproline (also known as pyroglutamic acid), L-glutamic acid, glycine, and phosphoric acid (Pi)-five metabolites from our data, matched with the enumerated 28 metabolites of the MetaboAnalyst 5.0 for the GSH metabolism. All of these metabolites were present in higher concentrations in IR-K562 cells, but intriguingly, they were all reduced in R+203 and equated to imatinib-sensitive K562 cells (C). Concludingly, the identified metabolites associated with GSH metabolism could be used as diagnostic markers.

Restoration of miR-23a expression by chidamide sensitizes CML cells to imatinib treatment with concomitant downregulation of CRYAB

ABSTRACT MicroRNAs (miRNAs) are involved in various processes from the initiation and development of cancers, including chronic myeloid leukemia (CML). In this report, we aimed to investigate the roles of miR-23a in the regulation of imatinib mesylate (IM) sensitivity in CML cells and the possible mechanisms involved in this process. We demonstrated that the expression of miR-23a was markedly low in bone marrow mononuclear cells from patients in whom IM treatment had failed and imatinib-resistant K562/G01 cells when compared to patients with optimal responses and imatinib-sensitive K562 cells, respectively. Overexpression of miR-23a was shown to induce apoptosis of K562/G01 cells and sensitize these cells to imatinib treatment. With the aid of bioinformatics analysis, we revealed that CRYAB could be a potential downstream effector of miR-23a, contributing to miR-23a-mediated IM resistance. We also observed that the expression of CRYAB was inversely correlated with miR-23a expression in CML cell lines and patient samples. Importantly, chidamide upregulated miR-23a expression and reversed the IM resistance of CML cells. Together, these findings strongly suggest that miR-23a acts as a tumor suppressor by downregulating CRYAB expression. Restoration of miR-23a by chidamide may therefore have a therapeutic effect in controlling the sensitivity of CML cells to imatinib.

RanBP3 Regulates Proliferation, Apoptosis and Chemosensitivity of Chronic Myeloid Leukemia Cells via Mediating SMAD2/3 and ERK1/2 Nuclear Transport.

Abnormal subcellular localization of proteins is an important cause of tumorigenesis and drug resistance. Chromosome region maintenance 1 (CRM1), the nuclear export regulator of most proteins, has been confirmed to be over-expressed in various malignancies and is regarded as an efficient target. But the potential role of the CRM1 cofactor RanBP3 (Ran Binding Protein 3) is left unrevealed in chronic myeloid leukemia (CML). Here, we first detected the level of RanBP3 in CML and found an elevated RanBP3 expression in CML compared with control. Then we used shRNA lentivirus to down-regulated RanBP3 in imatinib sensitive K562 cells and resistant K562/G01 cells and found RanBP3 silencing inhibited cell proliferation by up-regulating p21, induced caspase3-related cell apoptosis, and enhanced the drug sensitivity of IM in vitro. Notably, we observed that RanBP3 silencing restored imatinib sensitivity of K562 cells in NOD/SCID mice. Mechanistically, the nuclear aggregation of SMAD2/3 revealed that tumor suppressor axis (TGF-β)-SMAD2/3-p21 was the anti-proliferation program related to RanBP3 knockdown, and the decrease of cytoplasmic ERK1/2 caused by RanBP3 interference leaded to the down-regulation of anti-apoptosis protein p(Ser112)-BAD, which was the mechanism of increased cell apoptosis and enhanced chemosensitivity to imatinib in CML. In summary, this study revealed the expression and potential role of RanBP3 in CML, suggesting that targeting RanBP3 alone or combined with TKIs could improve the clinical response of CML.

EPS8 regulates proliferation, apoptosis and chemosensitivity in BCR-ABL positive cells via the BCR-ABL/PI3K/AKT/mTOR pathway.

Although the introduction of tyrosine kinase inhibitors greatly improved the survival of patients with chronic myeloid leukemia (CML), drug resistance remains a problem. Thus, mechanism-based novel therapeutic targets warrant exploration. Recently, epidermal growth factor receptor kinase substrate 8 (EPS8), which has been identified as an oncogene and plays an important role in a broad spectrum of solid tumours, was reported to be related to poor prognosis or chemoresistance in acute leukemia patients. However, its role in CML remains unclear. In the present study, using q-RT-PCR, we demonstrated that CML patients expressed a higher level of EPS8 mRNA in bone marrow mononuclear cells than healthy controls. Then, to determine the effect of EPS8 on the biological functions of CML cells, EPS8 expression was knocked down in the human CML cell line K562. Reduced proliferation, increased apoptosis, impaired adhesion and migration were observed in K562 cells after EPS8 silencing. Notably, attenuation of EPS8 increased chemosensitivity both in imatinib-sensitive K562 cells and in the imatinib-resistant murine BCR-ABL+ 32D-p210BCR/ABL-T315I cells. Mechanistically, knockdown of EPS8 downregulated p-BCR/ABL and its downstream AKT/mTOR signalling pathway. Finally, knockdown of EPS8 attenuated K562 cell proliferation in BALB/c nude mice. These data indicated that EPS8 regulated the proliferation, apoptosis and chemosensitivity in BCR-ABL positive cells via the BCR-ABL/PI3K/AKT/mTOR pathway. Targeting EPS8 alone or combined with a tyrosine kinase inhibitor may be a promising alternative therapeutic strategy.

Persistent STAT5-mediated ROS production and involvement of aberrant p53 apoptotic signaling in the resistance of chronic myeloid leukemia to imatinib.

The persistent activation of signal transducer and activator of transcription5 (STAT5) may principally be attributed to breakpoint cluster region (BCR)-Abelson murine leukemia viral oncogene homolog1 (ABL1), and have multi-faceted effects in the development of chronic myeloid leukemia(CML). The p53 protein network regulates important mechanisms in DNA damage repair, cell cycle regulation/checkpoints, and cell senescence and apoptosis, as demonstrated by its ability to positively regulate the expression of various pro-apoptotic genes, including B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated Xprotein(Bax). In the present study, it was observed that the mRNA levels of STAT5A and STAT5B were upregulated in patients with imatinib-resistant CML and in the imatinib-resistant K562/G CML cell line. In addition, increased expression of STAT5 was observed in the BCR-ABL1 mutation group, compared with that in the non-BCR-ABL1 mutation group, regardless of patient imatinib resistance state. Elevated levels of reactive oxygen species(ROS) and DNA double-strand breaks were identified in K562/G cells using flow cytometric and phosphorylated H2AX(γ-H2AX) foci immunofluorescence assays, respectively, compared with the imatinib-sensitive K562 cells. The levels of intracellular ROS and γ-H2AX were decreased by the ROS scavenger (N-acetylcysteine), and ROS levels were also markedly reduced by STAT5 inhibitor (SH-4-54). In addition, imatinib significantly inhibited the proliferation of K562 and K562/G cells, with half maximal inhibitory concentration values of 0.17±0.07 and 14.78±0.43µM, respectively, and the levels of apoptosis were significantly different between K562 and K562/G cells following treatment with imatinib. The mRNA and protein levels of STAT5 and mouse double minute2 homolog(MDM2) were upregulated, whereas those of Bax were downregulated in K562/G cells, as determined using western blot analysis. Additionally, although the two cell lines exhibited relatively low protein expression levels of p53, lower levels of p53 and TPp53BP1 transcripts were detected in the K562/G cells. Taken together, these findings suggest that the resistance of CML to the tyrosine kinase inhibitor, imatinib, may be associated with persistent STAT5-mediated ROS production, and the abnormality of the p53 pathway.

Downregulation of miR-224 and let-7i contribute to cell survival and chemoresistance in chronic myeloid leukemia cells by regulating ST3GAL IV expression

Acquired resistance to imatinib is frequently associated with poor clinical outcome of chronic myeloid leukemia (CML) patient. To date, evidence indicates that protein glycosylation and its upstream regulators might be implicated in tumorigenesis and chemoresistance occurrence. In current study we initially explored N-glycan profiles on the surface of CML cell lines and bone marrow mononuclear cells (BMMC) of CML patients by using mass spectrometry (MS) analysis. An elevated sialylation was detected in K562R cells (CML cells with imatinib resistance phenotype) compare to K562 cells. By quantitative real time-PCR (qRT-PCR) and western blotting analysis we observed that imatinib resistant K562R cells exhibited marked high levels of CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase (ST3Gal IV) as compared to imatinib sensitive K562 cells. Further studies revealed that manipulated expression of ST3GAL IV led to the significant alterations of cell cycle distribution, apoptotic signal, cell proliferation and the effectiveness of imatinib treatment. Using microRNA array, miRNA database searching and luciferase reporter assay, we identified that miR-224 and let-7i directly regulate the expression of ST3GAL IV gene. Moreover, engineered expression of miR-224 and let-7i in K562 and K562R cells could significantly affect ST6Gal IV-induced proliferation rate and drug-resistance. Thus we propose that miR-224 and let-7i regulate the proliferation and chemosensitivity of CML cells probably via targeting ST3GAL IV.

A molecular and biophysical comparison of macromolecular changes in imatinib-sensitive and imatinib-resistant K562 cells exposed to ponatinib.

Chronic myeloid leukemia (CML) is a type of hematological malignancy that is characterized by the generation of Philadelphia chromosome encoding BCR/ABL oncoprotein. Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, and dasatinib, are used for the frontline therapy of CML. Development of resistance against these TKIs in the patients bearing T315I mutation is a major obstacle in CML therapy. Ponatinib, the third-generation TKI, is novel drug that is effective even in CML patients with T315I mutation. The exact mechanism of ponatinib in CML has been still unknown. In this study, we aimed to determine the potential mechanisms and structural metabolic changes activated by ponatinib treatment in imatinib-sensitive K562 human CML cell lines and 3μM-imatinib-resistant K562/IMA3 CML cell lines generated at our lab. Apoptotic and antiproliferative effects of ponatinib on imatinib-sensitive and 3μM-imatinib-resistant K562/IMA3 CML cells were determined by proliferation and apoptosis assays. Additionally, the effects of ponatinib on macromolecules and lipid profiles were also analyzed using Fourier transform infrared spectroscopy (FTIR). Our results revealed that ponatinib inhibited cell proliferation and induced apoptosis as determined by loss of mitochondrial membrane potential, increased caspase-3 enzyme activity, and transfer of phosphatidylserine to the plasma membrane in both K562 and K562/IMA-3 cells. Furthermore, cell cycle analyses revealed that ponatinib arrested K562 and K562/IMA-3 cells at G1 phase. Moreover, ponatinib treatment created a more ordered nucleic acid structure in the resistant cells. Although the lipid to protein ratio increased in imatinib-sensitive K562 cells with a little decrease in the K562/IMA-3 cells, ponatinib treatment indicated significant changes in the lipid composition such as a significant increase in the cellular cholesterol amounts much more in the K562/IMA-3 cells than the sensitive counterparts. Unsaturation in lipids was higher in the resistant cells; however, increases in lipids without phosphate and the number of acyl chains were much higher in the K562 cells. Taken together, all these results showed powerful antiproliferative and apoptotic effects of ponatinib in both imatinib-sensitive and imatinib-resistant CML cells in a dose-dependent manner, and hence, the use of ponatinib for the treatment of TKI-resistant CML patients may be an effective treatment approach in the clinic. More importantly, these results showed that FTIR spectroscopy can detect drug-induced physiological changes in cancer drug resistance.

Suppression of STAT5A increases chemotherapeutic sensitivity in imatinib-resistant and imatinib-sensitive K562 cells

STAT proteins are cytoplasmic transcription factors that are involved in the regulation of numerous cellular activities such as cell growth, differentiation, and survival. In this study, we aimed to identify the expression pattern of STAT genes in imatinib-sensitive and -resistant K562 cells, and further, to reveal the effects of STAT5A siRNA knockdown on cell growth and apoptosis induction. The XTT cell proliferation assay showed that both sensitive and resistant K562 cells were sensitized to imatinib upon transfection with STAT5A siRNA. Caspase-3 enzyme activity was increased significantly in both cells. These results may open up new opportunities to overcome chemotherapeutic resistance in leukemia.

In Vitro and In Vivo Study of Synergistic Effect of Imatinib and Simvastatin in Chronic Myelogenous Leukemia.

Abstract 2202Poster Board II-179 Background:Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods:Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results:Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion:We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures:No relevant conflicts of interest to declare.

Imatinib Inhibits Proteasomal Activity in CML Cells.

Imatinib mesylate (Gleevec) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of CML it has become indispensable and shows few side effects. Nevertheless, imatinib has been described to influence the function and differentiation of APCs, inhibit the effector function of T lymphocytes and decrease the immunogenicity of CML cells. In the present study, we analyzed the possible effects of imatinib on proteasomal activity in CML cells. Proteasomal activity was determined using a biotinylated active site-directed probe which, depending on their activity, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5. We show that Imatinib treatment leads to a decreased activity of the proteasome in BCR-ABL positive cells. Imatinib sensitive K-562 cells as well as HLA-A2 transfected K-562 cells displayed a profound downregulation of proteasome activity at sub-therapeutic concentrations of imatinib. This effect was not due to downregulation of the protein expression of proteasome subunits or the induction of apoptosis. In imatinib resistant K-562R cells a higher concentration of imatinib was required for a detectable inhibition of proteasomal activity corresponding to a higher expression of BCR-ABL in these cells. Interestingly, the use of specific siRNA against the BCR-ABL fusion site b3a2 to downregulate the expression of the protein in K-562 and K-562R cells did not influence the activity of the proteasome indicating that this effect is independent of the expression and activity of BCR-ABL. In line with these results transfection of BaF/3 cells with BCR-ABL had no effect on proteasomal activity. To further analyze the mechanisms involved in proteasome inhibition by imatinib we performed activity assays with isolated 20S and 26S proteasomes. Incubation of both proteasome subtypes with imatinib resulted in a reduced acitivity in a concentration dependent manner as shown by reduced proteolytic cleavage of fluorogenic substrates. As the reduction in activity was seen in the ATP-independent 20S proteasome as well as the ATP-dependent 26S proteasome, a competition of imatinib with ATP similar to the mechanism of BCR-ABL inhibition is not responsible for this effect. In order to expand these studies, we additionally examined HLA class I expression on the cell surface to determine the influence of imatinib and subsequent proteasome inhibition on antigen presentation. Correlating with the inhibition of proteasome activity, HLA class I expression was strongly reduced on HLA-A2 transfected K-562 cells after imatinib treatment. Taken together these findings show that imatinib directly affects proteasomal activity in vitro. This effect is more pronounced in BCR-ABL positive cells, which might be due to BCR-ABL mediated deregulation of cellular pathways, but seems to be, however, independent of BCR-ABL.