Viability Of K562 Cells Research Articles

Anti-cancer activity of brucine loaded Cu(OH)2/ZnO nanoparticles against chronic myeloid leukemia cell line (K562) via apoptotic signaling pathway

Background and objectiveChronic myeloid leukemia (CML) is marked by abnormal blood cell production and traditional treatments often cause severe side effects and resistance. This study improves cancer therapy by using copper (II) hydroxide (Cu(OH)₂) and zinc oxide (ZnO) nanoparticles combined with brucine (Bru) to enhance targeted drug delivery and efficacy. Materials and methodsBru-Cu(OH)₂-ZnO NPs were synthesized and characterized using various analytical techniques. Their cytotoxicity was evaluated on K562 myeloid leukemia cells through cell viability assays, dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and measurement of oxidative stress indicators such as nitric oxide and lipid peroxidation levels. Antioxidant activity was assessed by glutathione (GSH) levels, while apoptosis was investigated via Caspase 3 and 9 activity and gene expression analysis (Bax, p53, and Bcl-2) using real-time polymerase chain reaction (RT-PCR). ResultsThe novel Bru-Cu(OH)₂-ZnO NPs demonstrated significant anti-cancer activity by markedly reducing K562 cell viability and enhancing cytotoxicity in a dose-dependent manner. These nanoparticles effectively scavenged free radicals, increased lipid peroxidation and nitric oxide levels, and improved caspase activity. Notably, gene expression analysis revealed that Bru-Cu(OH)₂-ZnO NPs led to overexpression of pro-apoptotic genes (Bax and p53) and downregulation of the anti-apoptotic gene (Bcl-2), highlighting their potential as effective cancer therapeutics. ConclusionThe synthesized Bru-Cu(OH)2-ZnO NPs shown excellent anti-cancer efficacy against the K562 cell line. This study underscores the potential of combining bioactive compounds with metal oxide nanoparticles to enhance targeted drug delivery and therapeutic effectiveness, offering a promising approach for advancing cancer treatment.

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.

Anti-Leukemic Effects of Small Molecule Inhibitor of c-Myc (10058-F4) on Chronic Myeloid Leukemia Cells.

As one of the main molecules in BCR-ABL signaling, c-Myc acts as a pivotal key in disease progression and disruption of long-term remission in patients with CML. To clarify the effects of c-Myc inhibition in CML, we examined the anti-tumor property of a well-known small molecule inhibitor of c-Myc 10058-F4 on K562 cell line. This experimental study was conducted in K562 cell line for evaluation of cytotoxic activity of 10058-F4 using Trypan blue and MTT assays. Flow cytometry and Quantitative RT-PCR analysis were also conducted to determine its mechanism of action. Additionally, Annexin/PI staining was performed for apoptosis assessment. The results of Trypan blue and MTT assay demonstrated that inhibition of c-Myc, as shown by suppression of c-Myc expression and its associated genes PP2A, CIP2A, and hTERT, could decrease viability and metabolic activity of K562 cells, respectively. Moreover, a robust elevation in cell population in G1-phase coupled with up-regulation of p21 and p27 expression shows that 10058-F4 could hamper cell proliferation, at least partly, through induction of G1 arrest. Accordingly, we found that 10058-F4 induced apoptosis via increasing Bax and Bad; In contrast, no significant alterations were observed NF-KB pathway-targeted anti-apoptotic genes in the mRNA levels. Notably, disruption of the NF-κB pathway with bortezomib as a common proteasome inhibitor sensitized K562 cells to the cytotoxic effect of 10058-F4, substantiating the fact that the NF-κB axis functions probably attenuate the K562 cells sensitivity to c-Myc inhibition. It can be concluded from the results of this study that inhibition of c-Myc induces anti-neoplastic effects on CML-derived K562 cells as well as increases the efficacy of imatinib. For further insight into the safety and effectiveness of 10058-F4 in CML, in vivo studies will be required.

Unlocking the therapeutic potential: Green synthesized zinc oxide/silver nanoparticles from Sophora pachycarpa for anticancer activity, gene expression analysis, and antibacterial applications

Hematologic malignancies and bloodstream infections rank among the most lethal medical conditions. Research on new treatments for these diseases is crucial. In this study, we investigated the anticancer properties and molecular mechanisms of zinc oxide nanoparticles doped with silver synthesized using a green approach involving Sophora pachycarpa (S. pachycarpa) plant seed extract (SPS@ZnO/Ag NPs). Additionally, we explored the antibacterial effects of these nanoparticles and S. pachycarpa extract. The SPS@ZnO/Ag NPs were characterized using XRD, FTIR, zeta potential, EDS, FESEM, and TEM analyses. Subsequently, we assessed the viability of K562 cells in the presence of different nanoparticles and extract. Molecular mechanisms underlying cell death were examined through flow cytometry analysis, Hoechst staining, and relative expression of pro-apoptotic genes (Apaf-1, Cytochrome c, Caspases 3, 6, 9) relative to the gene B-actin. Also, BAX/BCL2 ratio was determined. Antibacterial effects were evaluated against Gram-negative and Gram-positive bacteria. The results confirmed successful synthesis of spherical SPS@ZnO/Ag NPs with positive surface charge, purity, and size ranging from 50 to 65 nm. SPS@ZnO/Ag NPs significantly reduced K562 cell viability compared to S. pachycarpa extract and chemical nanoparticles, with the 1:1 ratio of zinc oxide and silver nitrate exhibiting the highest cell death. The findings from flow cytometry analysis, Hoechst staining, and molecular pathway analysis indicate that SPS@ZnO/Ag NPs induce cancer cell death through apoptosis. Evaluation of antibacterial properties demonstrated the destruction of all studied strains by SPS@ZnO/Ag NPs. Overall, our study demonstrates that green synthesis, in comparison to chemical synthesis, exerts a notable impact on the anticancer properties of zinc oxide nanoparticles doped with silver. Moreover, SPS@ZnO/Ag NPs exhibit targeted induction of cancer cell apoptosis, showcasing their potential application in biomedical fields.

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 h. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2 A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Discovery of (−)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (−)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages.

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.

Abstract 4404: EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2

Abstract The polycomb protein EZH2 is a commonly deregulated or mutated transcriptional regulator in various cancers. In Chronic Myeloid Leukemia (CML) it has been reported to be upregulated and associated with transcriptional reprograming, rendering CML leukemic stem cells (LSCs) sensitive to EZH2 inhibitor treatment. Mechanistically this seems to be linked to the silencing of apoptotic target genes by EZH2-mediated histone H3 lysine 27 tri-methylation (H3K27me3). We hypothesized that the regulation of apoptotic genes is not the only pathway responsible for its oncogenic function, and that EZH2 might also act as a modulator of the mRNA splicing landscape. To test this, we treated the CML cell line K562 with the EZH2 inhibitors GSK126 and EPZ-6438 and performed RNA-seq followed by splicing analysis. Using two different bioinformatics tools, we detected differential splicing of several hundreds of splicing events. However, differentially spliced genes were not associated with increased EZH2 or H3K27me3 signals, suggesting an indirect mechanism of splicing regulation. Indeed, we found 11 splicing factors transcriptionally upregulated upon EZH2 inhibitor treatment. CELF2 was identified as top candidate to mediate part of the EZH2 inhibitor induced phenotype. Upon overexpression, we observed 1) reduced cell growth, viability, and colony formation of K562 cells, 2) a change in the splicing landscape, significantly overlapping with EZH2 inhibitor induced changes, 3) the downregulation of cMYC signaling, also found upon EZH2 inhibitor treatment. To test if CELF2 is indeed acting as EZH2-regulated tumor suppressor in CML, we extended our study to CML patient samples: we performed RNA-seq followed by splicing and gene expression analysis in seven CML patient-derived CD34+ samples and eight healthy donor-derived CD34+ samples, as well as three CML patient samples transduced with CELF2-containing expression constructs. While we found Celf2 mRNA downregulated in CML derived samples, overexpression led to decreased colony formation and cMYC signaling, validating our findings from K562 cells. We also performed an analysis of CML associated splicing changes and found hundreds of deregulated splicing events, of which a significant portion overlapped with CELF2 overexpression mediated changes, suggesting direct regulation by CELF2. Based on this data we propose a novel role of EZH2, where it exerts part of its oncogenic function in CML through the transcriptional repression of splicing factors, in particular CELF2, leading to splicing deregulation, increased cMYC signaling, and enhanced cell growth and viability. Finally, data from other publicly available EZH2 inhibitor treated cancer models suggests that splicing modulation by EZH2 might not be restricted to CML. Citation Format: Reinhard Brunmeir, Ying Li, Baohong Lin, Leong Qiao Zheng, Henry Yang, Wee Joo Chng. EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4404.

The Effects of the Steroids 5-Androstenediol and Dehydroepiandrosterone and Their Synthetic Derivatives on the Viability of K562, HeLa, and Wi-38 Cells and the Luminol-Stimulated Chemiluminescence of Peripheral Blood Mononuclear Cells from Healthy Volunteers.

In order to evaluate the role of substituents at 3-C and 17-C in the cytotoxic and cytoprotective actions of DHEA and 5-AED molecules, their derivatives were synthesized by esterification using the corresponding acid anhydrides or acid chlorides. As a result, seven compounds were obtained: four DHEA derivatives (DHEA 3-propionate, DHEA 3-butanoate, DHEA 3-acetate, DHEA 3-methylsulfonate) and three 5-AED derivatives (5-AED 3-butanoate, 5-AED 3,17-dipropionate, 5-AED 3,17-dibutanoate). All of these compounds showed micromolar cytotoxic activity toward HeLa and K562 human cancer cells. The maximum cytostatic effect during long-term incubation for five days with HeLa and K562 cells was demonstrated by the propionic esters of the steroids: DHEA 3-propionate and 5-AED 3,17-dipropionate. These compounds stimulated the growth of normal Wi-38 cells by 30-50%, which indicates their cytoprotective properties toward noncancerous cells. The synthesized steroid derivatives exhibited antioxidant activity by reducing the production of reactive oxygen species (ROS) by peripheral blood mononuclear cells from healthy volunteers, as demonstrated in a luminol-stimulated chemiluminescence assay. The highest antioxidant effects were shown for the propionate ester of the steroid DHEA. DHEA 3-propionate inhibited luminol-stimulated chemiluminescence by 73% compared to the control, DHEA, which inhibited it only by 15%. These data show the promise of propionic substituents at 3-C and 17-C in steroid molecules for the creation of immunostimulatory and cytoprotective substances with antioxidant properties.

Sorafenib combination with imatinib enhances apoptotic and cell cycle arrest effects with no effect on viability and BCR-ABL expression of K-562 cells

Chronic myeloid leukemia (CML) is one of the myeloproliferative disorders that affect hematopoiesis. Historically, many therapies have been used for the management of this disease with moderate response. The discovery of tyrosine kinase inhibitors (TKIs), marked by the introduction of imatinib, revolutionized the therapy of CML. Resistance to imatinib, however, marks CML cells in refractory disease. Additionally, advanced stages of CML may still be refractory to available therapies. Sorafenib is an inhibitor of multiple intracellular and receptor protein kinases that is approved by the FDA for the treatment of advanced hepatocellular carcinoma patients and renal cell cancer. Other TKIs, such as erlotinib and sunitinib, are under study for the treatment of CML patients. In this study, the synergistic effect of the combination of imatinib and sorafenib on the K-562 CML cell line was evaluated. We used viability assay, cell cycle analysis, apoptosis assay, reverse transcription quantitative real-time PCR detect the expression of Breakpoint Cluster Region – abelson fusion oncogene and to evaluate the interaction of the combination. The study showed that the addition of sorafenib to imatinib did not produce a significant synergistic effect on the viability of cells. The combination, however, had a significant effect on cell cycle arrest and induction of apoptosis with no effect on the reduction of BCR-ABL mRNA expression on K-562 cells.

Artemisia vulgaris Induces Tumor-Selective Ferroptosis and Necroptosis via Lysosomal Ca2+ Signaling.

To evaluate the chemical composition and effects of Artemisia vulgaris (AV) hydroalcoholic extract (HEAV) on breast cancer cells (MCF-7 and SKBR-3), chronic myeloid leukemia (K562) and NIH/3T3 fibroblasts. Phytochemical analysis of HEAV was done by high-performance liquid chromatography-mass (HPLC) spectrometry. Viability and cell death studies were performed using trypan blue and Annexin/FITC-7AAD, respectively. Ferrostatin-1 (Fer-1) and necrostatin-1 (Nec-1) were used to assess the mode of HEAV-induced cell death and acetoxymethylester (BAPTA-AM) was used to verify the involvement of cytosolic calcium in this event. Cytosolic calcium measurements were made using Fura-2-AM. HEAV decreased the viability of MCF-7, SKBR-3 and K562 cells (P<0.05). The viability of HEAV-treated K562 cells was reduced compared to HEAV-exposed fibroblasts (P<0.05). Treatment of K562 cells with HEAV induced cell death primarily by late apoptosis and necrosis in assays using annexin V-FITC/7-AAD (P<0.05). The use of Nec-1 and Fer-1 increased the viability of K562 cells treated with HEAV relative to cells exposed to HEAV alone (P<0.01). HEAV-induced Ca2+ release mainly from lysosomes in K562 cells (P<0.01). Furthermore, BAPTA-AM, an intracellular Ca2+ chelator, decreased the number of non-viable cells treated with HEAV (P<0.05). HEAV is cytotoxic and activates several modalities of cell death, which are partially dependent on lysosomal release of Ca2+. These effects may be related to artemisinin and caffeoylquinic acids, the main compounds identified in HEAV.

Co-treatment of Naringenin and Ketoprofen-RGD Suppresses Cell Proliferation via Calmodulin/PDE/cAMP/PKA Axis Pathway in Leukemia and Ovarian Cancer Cells.

Naringenin (Nar) has anti-inflammatory and anticarcinogenic properties. Arginine-glycine- aspartate (RGD) is a tripeptidic sequence used as an integrin ligand and targeting system for delivering chemotherapeutic agents to cancer cells. In this study, the inhibitory effects of Nar and ketoprofen-RGD on leukemia and ovarian cancer cells (K562 and SKOV3) were explored for the first time, focusing on their proliferation activity and their anti-inflammatory capacity. Analyses were conducted on the calmodulin (CaM)-dependent phosphodiesterase 1 (PDE1) activation by ketoprofen-RGD, Nar, and their combination. These drugs' effects on protein kinase A (PKA) activation, intracellular cyclic adenosine monophosphate (cAMP) level, and PDE1 inhibition were identified. Later, it was also evaluated if ketoprofen-RGD alone or in combination with Nar had anti-inflammatory effects. Nar improved the antagonizing consequences of ketoprofen-RGD on the CaM protein, which hinders PDE1, improving PKA activity and cAMP levels. A mixture of ketoprofen-RGD and Nar and ketoprofen-RGD alone diminished K562 and SKOV3 cell viability through the cAMP/PKA pathway by inhibiting PDE1 and CaM. These two compounds showed anti-inflammatory effects on both cell lines. This study indicated for the first time that combining ketoprofen-RGD and Nar can be a promising anti-inflammatory therapeutic regimen for treating leukemia and ovarian cancer.

KOENZİM Q0 İNSAN KRONİK MYELOİD LÖSEMİ K562 HÜCRELERİNİN PROLİFERASYONUNU ENGELLER VE MAPK VE AKT SİNYAL YOLAKLARINI MODÜLE EDER

Objective: This study evaluated the antiproliferative and pro-apoptotic effects of coenzyme Q0 (CoQ0) in human chronic myeloid leukemia K562 cell line. Material and Method: The cytotoxic effect of CoQ0 on human chronic myeloid leukemia cell line, K562 was determined by MTT test. The activity of caspase-3, expression of proteins involved in apoptosis, MAPK and AKT signaling pathways were determined with enzymatic assay and western blot analysis, respectively. Result and Discussion: Results showed that CoQ0 inhibited cell viability of K562 cells at 5 μM and higher concentrations and Bax protein expression was significantly decreased at 12.5 μM concentration of CoQ0. However, CoQ0 did not significantly affect caspase 3 activity and Bcl-2 protein expression. p-c-Raf (Ser259) protein expression was significantly decreased at 12.5 μM of CoQ0. Treatment with 10 μM of CoQ0 induced significantly phosphorylation of p38 MAPK and 12.5 μM CoQ0 caused a nonsignificant decrease in p-ERK1/2 protein expression in K562 cell line. Interestingly, in K562 cells, phosphorylation of Akt (Ser473) was diminished at 12.5 μM of CoQ0, with no change observed in p-Akt (Thr308) protein expression among groups. In conclusion, CoQ0 inhibited cell proliferation and suppressed phosphorylation of c-Raf (Ser259), Akt (Ser473), but not ERK1/2 in K562 cells. There is still a need for new insights into the anticancer mechanisms of CoQ0 and develop treatment strategies for chronic myeloid leukemia.

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.

Anti-Cancer Effects of Oxygen-Atom-Modified Derivatives of Wasabi Components on Human Leukemia Cells.

1-Isothiocyanato-6-(methylsulfinyl)-hexanate (6-MITC) is a natural compound found in Wasabia japonica. The synthetic derivatives 1-Isothiocyanato-6-(methylsulfenyl)-hexane (I7447) and 1-Isothiocyanato-6-(methylsulfonyl)-hexane (I7557) were obtained from 6-MITC by deleting and adding an oxygen atom to the sulfone group, respectively. We previously demonstrated that extensive mitotic arrest, spindle multipolarity, and cytoplasmic vacuole accumulation were induced by 6-MITC and inhibited the viability of human chronic myelogenous leukemia K562 cells. In this study, we examined the anti-cancer effects of 6-MITC derivatives on human chronic myelogenous leukemia (CML) cells. Autophagy was identified as the formation of autophagosomes with double-layered membranes using transmission electron microscopy. Cell cycle and differentiation were analyzed using flow cytometry. Apoptosis was detected by annexin V staining. After treatment with I7447 and I7557, the G2/M phase of cell cycle arrest was revealed. Cell death can be induced by a distinct mechanism (the simultaneous occurrence of autophagy and aberrant mitosis). The expression levels of acridine orange were significantly affected by lysosomal inhibitors. The natural wasabi component, 6-MITC, and its synthetic derivatives have similar effects on human chronic myelogenous leukemia cells and may be developed as novel therapeutic agents against leukemia.

Investigating the Activity of Indole-2-on Derivative Src Kinase Inhibitors Against Chronic Myeloid Leukemia Cells.

Src family tyrosine kinases play a potential role in Bcr-Abl-induced leukemogenesis. Src kinase inhibitors are reported as selective inhibitors of chronic myeloid leukemia. Since Src kinase inhibitors have an inhibitive effect on chronic myeloid leukemia, indole derivatives (C-1, C-2, C-3) previously found as potent inhibitors of Src kinase were tested against chronic myeloid leukemia in this study. Cell viability of K562 and R/K562 cells, antiproliferative and antioxidant effects, and inhibition profiles of Bcr-Abl kinase of indole derivatives were determined compared to dasatinib and imatinib. The results showed that compounds affected cell proliferation and decreased the levels of Bcr/Abl. These results confirmed that the antileukemic activity of compounds was related to Bcr/Abl expression. Docking studies also presented that compounds are inhibitors of both Src and Abl kinases. Calculation of drug-like properties showed that compounds could be potential drug candidates. Among indole-2-on derivatives, previously identified as Src kinase inhibitors, C-2 has been discovered to be a strong anticancer drug that is active against susceptible and resistant K562 cell lines and induces apoptosis.

X-ray-irradiated K562 feeder cells for expansion of functional CAR-T cells

Immunotherapy, particularly CAR-T therapy has recently emerged as an innovator for cancer treatment. Gamma-irradiated K562 cells is a common and effective method to stimulated CAR-T cells prior to treatment. However, high cost and limited equipment of gamma-irradiation is drawback of this method. This requires the establishment of CAR-T-expanding alternatives, such as X-ray-irradiated K562 cells.X-ray irradiation was used to deactivate K562 cells. The post-irradiative cell survival was investigated by counting of the number of cells, staining with Trypan Blue and PI. FACS analysis was applied to detect the expression of cell surface markers. The production of CD19-CAR-T cells were executed from fresh blood donor by CD19-CAR-plasmid transfection, followed by the stimulation with X-ray-irradiated K562 feeder cells. The function of produced CAR-T cells was checked by their ability to kill Daudi cells.X-ray-irradiation inhibited the propagation and viability of K562 cells in a dose- and time-dependent manner. Interestingly, CAR-T-stimulating effectors were remained on the surface of X-ray-irradiated K562 cells. CD-19-CAR-T cells were produced successfully, suggested by number of CAR-positive cells in transfected and stimulated population, compared to un-transfected group. Lastly, our data showed that engineered CAR-T cells effectively killed Daudi cells.Our data demonstrated the efficacy of X-ray on deactivation K562 feeder cells which subsequently stimulated and expanded functional CAR-T cells. Thus, X-ray can be used as an alternative to inactivate K562 cells prior to using as a feeder of CAR-T cells.

Development of the Novel Cytosine Analog Aza-T-Dcyd (AB1) As a Fetal Hemoglobin Inducer to Treat Sickle Cell Disease.

Introduction: Induction of fetal hemoglobin (HbF) expression in sickle cell disease (SCD) patients using small molecules is considered an effective therapeutic strategy to ameliorate severe clinical phenotypes. With this objective in mind, several drug molecules have been tested for HbF induction. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. Studies have reported that DNA methyltransferase inhibitors such as decitabine stimulate γ-globin gene expression through DNA hypomethylation. However, decitabine has cytotoxic side effects and is most effective by intravenous administration. Thus, it is critical to develop alternative safe and orally active hypomethylating agents for inducing HbF. Our lab in collaboration with AkiraBio, conducted preclinical in vitro studies to assess a novel cytosine analog Aza-T-dCyd (AB1), for its ability to induce HbF in human K562 cells and erythroid progenitor cells derived from SCD patients. Methods: K562 cells were cultured in Iscove's Modified Dulbecco's Eagle medium, 10% fetal bovine serum and penicillin/streptomycin. The human erythroid progenitors were generated from peripheral blood mononuclear cells isolated from two SCD patients under IRB exempt protocol as previously established in our lab (Li et al., Haematologica. 2018). The cultured K562 cells were treated either with AB1 alone at gradually increasing concentrations from 3nm to 200nm, decitabine (0.2µM), hydroxyurea (HU100µM) or combined AB1 with HU for 48 hours. The cultured sickle erythroid progenitors were treated with AB1 on day 8 and then harvested on day 10. The harvested cells were analyzed for cell viability and flow cytometry to measure HbF positive cells (F-cells). The γ-globin and β-globin mRNA levels were quantified by qRT-PCR and the protein levels of HbF and DNMT-1 were detected by Western blot technique. Results: Initial studies to evaluate drug toxicity were completed. We observed higher cell viability of K562 cells treated with AB1 10nM and 30nM compared to untreated and HU 100µM (p=0.005) treated cells. Hence, subsequent experiments were conducted with the lower AB1 concentrations. By flow cytometry analysis, AB1 treatment significantly increased F-cells from 13.95% to 20.11% and 22.46% in 10nM and 30nM treated cells (p=0.0002) respectively. Moreover, mean fluorescence intensity analysis showed significant HbF expression in AB1 treated cells compared to untreated cells (p=0.00001). At the transcription level, AB1 increased γ-globin mRNA levels 1.4-fold compared to untreated cells. K562 cells treated with a combination of AB1 and HU at their maximum response concentrations (AB1 10 and 30nM; HU 100 µM) did not show additive effects on F-cells or γ-globin mRNA expression. Other combination concentrations were not assessed. In subsequent studies we investigated the HbF inducing capacity of AB1 in human erythroid progenitors generated from SCD patients (N=2). Normal cell viability was observed in sickle progenitors at 3nm, 6nM and 10nm AB1 treatment. The F-cell level increased from 16.21% to 27.68% at 10nm AB1 (p=0.02). By qRT-PCR assay, a significant 2-fold increase in γ-globin mRNA level was observed in AB1 treated erythroid cells, when normalized with total γ-globin and β-globin mRNA; β globin mRNA levels were unchanged. Finally, when we analyze the effects of AB1 on protein levels of HbF and DNMT-1 by Western blot analysis, 10nM and 30nM AB1 induced HbF and inhibited DNMT-1 expression the sickle erythroid progenitors. Conclusion: Our study findings demonstrate that AB1 induced γ-globin transcription in K562 cells and human sickle erythroid progenitors. Studies to test the effects of AB1 in the Townes SCD mouse model are in progress.

EVI1 upregulates PTGS1 (COX1) and decreases the action of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia cells.

Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic myelogenous leukemia (CML). However, primary and acquired drug resistance to TKIs have been reported. In this study, we used RNA sequencing followed by RQ-PCR to show that the proto-oncogene EVI1 targets the drug-metabolizing gene PTGS1 in CML. The PTGS1 promoter element had an EVI1 binding site, and CHIP assay confirmed its presence. Data from a publicly available CML microarray dataset and an independent set of CML samples showed a significant positive correlation between EVI1 and PTGS1 expression in CML. Downregulation of EVI1 in K562 cells and subsequent treatment with TKIs resulted in a lower IC50 in the control cells. Furthermore, combined inhibition of BCR-ABL with imatinib and PTGS1 with FR122047 (PTGS1 inhibitor) synergistically reduced the viability of imatinib-resistant K562 cells. We conclude that elevated EVI1 expression contributes to TKIs resistance and that combined inhibition of PTGS1 and BCR-ABL may represent a novel therapeutic approach.

Effect of pre-low-dose irradiation on anticancer activities of gallic acid in leukemic K562 and K562/Dox cells: cell viability and cellular energetic state studies.

The aim of this study was to determine the effects of pre-low-dose irradiation followed by gallic acid (GA) on cell viability and cellular energetic state of leukemic K562 and K562/Dox cells. The cells were irradiated with 0.02, 0.05, and 0.1Gy of X-rays. For determining cell viability, pre-low-dose irradiation was followed by 10 or 100µM GA at 24h post-irradiation, and the cell viability was then determined at 48h post-irradiation. For cellular energetic state, pre-low-dose irradiation was followed by 10 or 100µM GA at 1.5h post-irradiation and the mitochondrial activity, mitochondrial membrane potential (ΔΨm), and ATP level were determined at 3h post-irradiation. The % cell viability was significantly decreased in both cells that were irradiated with X-rays followed by treatment with 10 or 100µM GA at 24h post-irradiation, when compared with control group. However, this did not happen when compared with GA alone without any pre-low-dose irradiation. The mitochondrial activity had significantly decreased in 10µM GA-treated K562 cells and the mitochondrial activity, ΔΨm, and ATP levels had significantly decreased in 10µM GA-treated K562/Dox cells after irradiation to X-rays when compared with GA alone group. In addition, the ΔΨm and ATP levels was significantly decreased in only 100µM GA-treated K562/Dox cells, but was not decreased in 100µM GA-treated K562 cells after exposure to X-rays. These findings suggest that pre-low-dose irradiation followed by GA could not kill K562 and K562/Dox cells, but could improve cellular energetic damage of GA effects possibly through mitochondrial impairment.