Functional characterization of native Piezo1 as calcium and magnesium influx pathway in human myeloid leukemia cells.

Piezo1 is a Ca2+-permeable mechanically activated ion channel that is involved in various physiological processes and cellular responses to mechanical stimuli. The study of biophysical characteristics of Piezo1 is important for understanding the mechanisms of its function and regulation. Stretch activation, a routine approach that is applied to stimulate Piezo1 activity in the plasma membrane, has a number of significant limitations that complicate precise single-channel analysis. Here, we aimed to determine pore properties of native Piezo1, specifically to examine permeation for physiologically relevant signaling divalent ions (calcium and magnesium) in human myeloid leukemia K562 cells using Piezo1-specific chemical agonist, Yoda1. Using a combination of low-noise single-current patch-clamp recordings of Piezo1 activity in response to Yoda1, we have determined single-channel characteristics of native Piezo1 under various ionic conditions. Whole-cell assay allowed us to directly measure Piezo1 single currents carried by Ca2+ or Mg2+ ions in the absence of other permeable cations in the extracellular solutions; unitary conductance values estimated at various concentrations of Mg2+ revealed strong saturation effect. Patch clamp data complemented with fluorescent imaging clearly evidenced Ca2+ and Mg2+ entry via native Piezo1 channel in human leukemia K562 cells. Mg2+ influx via Piezo1 was detected under quasi-physiological conditions, thus showing that Piezo1 channels could potentially provide the physiological relevant pathway for Mg2+ ion transport and contribute to the regulation of Mg2+-dependent intracellular signaling.

CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.

c-Jun targets miR-451a to regulate HQ-induced inhibition of erythroid differentiation via the BATF/SETD5/ARHGEF3 axis.

Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50 µM HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells.

Objective: Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. Methods: In this study, we treated K562 cells with 40 µmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. Results: One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Conclusion: Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

A Crucial Role of Proteolysis in the Formation of Intracellular Dinitrosyl Iron Complexes.

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings.

Saracatinib prompts hemin-induced K562 erythroid differentiation but suppresses erythropoiesis of hematopoietic stem cells.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

"Combination treatments of imatinib with astaxanthin and crocin efficiently ameliorate antioxidant status, inflammation and cell death progression in imatinib-resistant chronic myeloid leukemia cells".

BACKGROUND: Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. AIMS: This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. METHODS AND RESULTS: After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48 h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). CONCLUSION: Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC.

Altered regulatory T cell-mediated Natural Killer cells suppression may lead to generalized vitiligo.

Generalized vitiligo (GV) is characterized by white patches due to autoimmune loss of melanocytes. Regulatory T cells (Tregs) maintain immune homeostasis, while NK cells eliminate pathogens and tumors. Increased NK cell frequency and reduced Treg frequency and suppressive capacity are observed in vitiligo patients. However, studies assessing Treg-mediated suppression of NK cell functions in GV are lacking. Therefore, our study aimed to assess in vitro Treg-mediated suppression of NK cells function over K562 and SK-Mel-28 cells in 31 GV patients and 30 controls using the BrdU-cell proliferation assay. We found decreased Treg-mediated suppression of NK cell function in GV patients (p = 0.0289). Moreover, increased NK cell-mediated K562 and SK-Mel-28 cells' suppression was observed in GV patients (p = 0.0207,p = 0.0419). Disease activity-based analysis, suggested reduced Treg-mediated suppression of NK cell function and increased NK cell function in active vitiligo patients (p = 0.03,p = 0.0436). Interestingly, age-based analysis suggested decreased Treg-mediated suppression of NK cell function in 1-20 and 21-40 years age groups compared to 41-60 years age group of GV patients (p = 0.005,p = 0.0380). Overall, our study, for the first time, suggests that decreased Treg-mediated suppression of NK cells may lead to increased destruction of melanocytes in GV, and this knowledge may help in developing effective therapeutics based on Tregs and NK cells for GV.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies.

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

[Periplaneta americana extract Câ ¡-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway].

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 µg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated ß-galactosidase stain kit(SA-ß-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 µg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 µg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-ß-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

Regulation of erythroid differentiation in K562 cells by the EPAS1-IRS2 axis under hypoxic conditions.

Red blood cells (RBCs) produced in vitro have the potential to alleviate the worldwide demand for blood transfusion. Hematopoietic cell differentiation and proliferation are triggered by numerous cellular physiological processes, including low oxygen concentration (<5%). In addition, hypoxia inducible factor 2α (HIF-2α) and insulin receptor substrate 2 (IRS2) were found to be involved in the progression of erythroid differentiation. However, the function of the HIF-2α-IRS2 axis in the progression of erythropoiesis is not yet fully understood. Therefore, we used an in vitro model of erythropoiesis generated from K562 cells transduced with shEPAS1 at 5% O2 in the presence or absence of the IRS2 inhibitor NT157. We observed that erythroid differentiation was accelerated in K562 cells by hypoxia. Conversely, knockdown of EPAS1 expression reduced IRS2 expression and erythroid differentiation. Intriguingly, inhibition of IRS2 could impair the progression of hypoxia-induced erythropoiesis without affecting EPAS1 expression. These findings indicated that the EPAS1-IRS2 axis may be a crucial pathway that regulates erythropoiesis and that drugs targeting this pathway may become promising agents for promoting erythroid differentiation.

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca2+ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca2+ imaging showed that both SK and IK channel inhibitors affect Ca2+ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane.

Utilization of Dairy By-Products as a Source of Functional and Health Compounds-The Role of Ovine Colostrum and Milk Whey on Chronic Myeloid Leukemia Cells.

Nowadays, the search for food products that promote consumers' health has gained interest, and dairy by-products, due to their biological quality, could have a prominent position among products with health benefits. However, little is known about their activity on cancer cells. This study aimed to provide evidence about the effect of ovine colostrum and milk whey on K562 cells, a model of the human chronic myeloid leukemia cell line. The exposure of K562 cells to a single administration of sheep by-products at different concentrations for three days and three treatments for three days was carried out. Using a flow cytometric approach, we found that CD235a expression remained stable in the cells exposed to ovine whey (milk and colostrum) at concentrations ranging from 1 ng/mL to 100 µg/mL, after three days from one or three administrations, respectively. A significant reduction in fluorescent cells was observed in the populations exposed to 1 mg/mL of both milk and colostrum at the same time points. In these conditions, the size and granularity of the leukemic cells also changed, with a substantial reduction in the number of actively dividing cells in the S phase of the cell cycle. This phenomenon was highlighted by the Annexin V/PI cytofluorimetric test, which is able to provide quantitative results regarding the population of cells in early or late apoptosis or necrotic cells after exposure to a single dose or three doses of colostrum or sheep whey for three days, respectively. This report showed that both colostrum and milk whey were able to modify the phenotypic profile and cell cycle of the K562 cell line, inducing apoptosis at the highest concentration.

Development of a Simple and Stable NanoESI Spray System Using Suction Wind from the MS Inlet.

Improving the reproducibility of proteome analysis systems presents a challenge; therefore, in this study, we developed a new insertion spray ionization (InSpIon) system wherein an InSpIon tube was designed with a spray tip inserted into the tube. This system stabilized the spray and subsequently improved the reproducibility of the analysis.

Anti-Tumor Effect of Protoscolex Hydatid Cyst Somatic Antigen on Inhibition Cell Growth of K562.

BACKGROUND AND OBJECTIVE: Today, cancer is one of the most important causes of death in the world, and so far, many treatment methods have been used in this field. Immunotherapy is considered one of the newest developments in this science, and it is still being investigated in some forms in different cancers and with a variety of antigens as well. One of the subsets of cancer immunotherapy is its treatment using parasitic antigens. The present study evaluated the effect of using somatic antigens of protoscoleces of Echinococcus granulosus on K562 cancer cells. METHODS: In this study, hydatid cysts' protoscolex antigens were extracted, purified, and added to K562 cancer cells at three concentrations (0.1, 1, and 2 mg/ml) and on three times (24, 48, and 72 h). The number of apoptotic cells was compared to the control flask. The antigen concentration of 2 mg/ml was used as a control sample to investigate its cytotoxic effect on the growth of healthy HFF3 cells. Annexin V and PI tests were also performed to differentiate apoptosis from necrosis. RESULTS: In flasks treated with hydatid cyst protoscolex antigen, all three concentrations significantly reduced the growth of cancer cells compared with the control flask, and concentration 2 of crude antigen significantly caused the death of cancer cells. Furthermore, more cancer cells underwent apoptosis by increasing the time of exposure to the antigen. On the other hand, flow cytometry results also showed that the amount of apoptosis has increased compared to the control group. In fact, Protoscolex hydatid cyst somatic antigens induce programmed cell death in K562 cancer cells while not having a cytotoxic effect on normal cells. CONCLUSION: Therefore, it is suggested to do more research on the anti-cancer and therapeutic properties of the antigens of this parasite.

Impact of P-glycoprotein on intracellular drug concentration in peripheral blood mononuclear cells and K562 cells.

P-glycoprotein (P-gp) expression in lymphocytes is variable and 2-fold higher in rheumatoid arthritis (RA) patients with treatment resistance than in healthy subjects. To date the information on P-gp-mediated drug interaction in lymphocyte is limited. We analyzed the importance on P-gp in lymphocytes using peripheral blood mononuclear cells (PBMCs) together with K562, K562/Adr, and K562/Vin cells, which have various P-gp levels, as cell models, and dexamethasone, nintedanib and apafant as weak to good P-gp substrates. P-gp levels in K562, K562/Adr, and K562/Vin cells were 0.3-, 20-, and 106-fold of healthy PBMCs, respectively. While cell accumulation of apafant and nintedanib decreased in all cells with increasing P-gp levels, dexamethasone accumulation in K562/Adr was comparable to that in healthy PBMCs and K562 cells. Cell accumulations of substrates in cells with low P-gp expression were not significantly changed by the P-gp inhibitors at therapeutic concentrations. However, accumulation increased to 1.4-fold at highest in K562/Adr cells with higher P-gp expression than in PBMCs of the RA patients. These results suggest P-gp controls the cellular concentration of P-gp substrates in PBMCs or K562 cells but cellular concentration of a weak P-gp substrate would not be apparently affected even in cells with a sufficient P-gp expression.

[Effects of miR-144-3p on Proliferation, Cell Cycle and Apoptosis of K562 Cells].

OBJECTIVE: To investigate the effects of miR-144-3p on cell proliferation, cell cycle and apoptosis of blast phase chronic myelogenous leukemia (CML) K562 cells. METHODS: K562 cells were cultured in vitro and mimics negative control, hsa-miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor were respectively transfected into K562 cells with transfection reagents. The cells were divided into five groups including blank control, mimics negative control, miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor. After transfection, the cell proliferation activity was detected by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometry. RESULTS: Compared with the blank control and mimics negative control groups, the proliferation rate of miR-144-3p mimics group was significantly decreased (P<0.05), the proportion of S phase cells was markedly increased (P<0.05), while the proportion of G1 phase cells was obviously decreased (P<0.05), and the apoptosis rate was significantly increased (P<0.05). Compared with the blank control and inhibitor negative control groups, the proliferation rate of miR-144-3p inhibitor group was obviously increased (P<0.05), the proportion of S phase cells was markedly decreased (P<0.05), while the proportion of G1 phase cells was obviously increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05). CONCLUSION: miR-144-3p can inhibit the proliferation and promote apoptosis of K562 cells, affect the cell cycle, and block K562 cells in S phase, which indicates that miR-144-3p is involved in the cell cycle activity of CML during blastic phase.