Chronic Myeloid Leukemia K562 Cells Research Articles

The combination of venetoclax and quercetin exerts a cytotoxic effect on acute myeloid leukemia

Venetoclax is a BH3 mimetic that was recently approved for the treatment of acute myeloid leukemia (AML) treatment. However, the effect of venetoclax on AML remains limited, and a novel strategy is required. Here, we demonstrate for the first time that the cytotoxic effect of venetoclax drastically increased when by combined with the naturally occurring flavonoid quercetin. Combined treatment with venetoclax and quercetin caused most of AML KG-1 cells to exhibit a condensed morphology. Cell cycle analysis revealed that the combination strongly induced cell death. Caspase inhibitor blocked this cell death, and the combination induced poly (ADP-ribose) polymerase (PARP) cleavage, indicating that apoptosis was the primary mechanism. These effects were also observed in another AML cell line Kasumi-1 but not in chronic myeloid leukemia (CML) K562 cells. Public data analysis demonstrated that B-cell/CLL lymphoma 2 (Bcl-2) expression is increased in AML cells compared to other malignant tumors, and the survival and the growth of AML cell line depends on Bcl-2. We found that quercetin increased Bcl-2-associated X protein (Bax) expression in KG-1. Our study provides a novel function for quercetin and presents a promising strategy for AML treatment using venetoclax.

Vitamin K2 Protects Against SARS-CoV-2 Envelope Protein-Induced Cytotoxicity in Chronic Myeloid Leukemia Cells and Enhances Imatinib Activity.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by excessive proliferation of myeloid cells. The COVID-19 pandemic has raised concerns regarding the impact of SARS-CoV-2 on patients with malignancies, particularly those with CML. This study aimed to investigate the effects of SARS-CoV-2 proteins on CML cell viability and the protective role of vitamin K2 (VK2) in conjunction with imatinib. Experiments conducted on K562 CML cells demonstrated that the SARS-CoV-2 envelope protein induces cytotoxicity and activates caspase 3/7, which are key markers of apoptosis. VK2 mitigated these cytotoxic effects and decreased cytokine production while inhibiting colony formation. Furthermore, the combination of VK2 with imatinib significantly reduced cellular proliferation, diminished mitochondrial membrane potential, and markedly suppressed colony formation. These findings suggest that VK2 protects CML cells from SARS-CoV-2-induced cytotoxicity and enhances the therapeutic efficacy of imatinib, presenting a potential strategy to improve CML treatment during the COVID-19 pandemic.

Advancements in Chronic Myeloid Leukemia detection: Development and evaluation of a novel QCM aptasensor for use in clinical practice

Oncological diseases represent a significant global health challenge, with high mortality rates. Early detection is crucial for effective treatment, and aptamers, which demonstrate superior specificity and stability compared to antibodies, offer a promising avenue for diagnostic advancement. This study presents the design, development and evaluation of a quartz crystal microbalance (QCM) sensor functionalized with the T2-KK1B10 aptamer for the sensitive and specific detection of Chronic Myeloid Leukemia (CML) K562 cells. The research focuses on optimizing the biorecognition layer by adjusting the aptamer conditions, demonstrating the sensor's ability to detect these CML cells with high specificity and sensitivity. The aptamer-modified QCM sensor operates on the principle of mass change detection upon binding of target cells. By employing the Langmuir isotherm model, the performance of the sensor was optimized for the capture of CML cells from biological samples with LOD of 263 K562 cells. The sensor was also successfully regenerated multiple times without sensitivity loss. Validation of the sensor's performance was conducted under controlled laboratory settings, followed by extensive testing utilizing human lyophilized plasma and clinical samples from patients. The sensor exhibited high sensitivity and specificity in the detection of CML cells within clinical specimens, thereby illustrating its potential for practical clinical deployment. This research presents a novel approach to the early diagnosis of CML, facilitating timely intervention and enhanced patient outcomes. The developed aptasensor demonstrates potential for broader application in cancer diagnostics and personalized medicine.

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.

A Carbon 21 Steroidal Glycoside with Pregnane Skeleton from Cynanchum atratum Bunge Promotes Megakaryocytic and Erythroid Differentiation in Erythroleukemia HEL Cells through Regulating Platelet-Derived Growth Factor Receptor Beta and JAK2/STAT3 Pathway.

Erythroleukemia is a rare form of acute myeloid leukemia (AML). Its molecular pathogenesis remains vague, and this disease has no specific therapeutic treatments. Previously, our group isolated a series of Carbon 21 (C-21) steroidal glycosides with pregnane skeleton from the root of Cynanchum atratum Bunge. Among them, we found that a compound, named BW18, can induce S-phase cell cycle arrest and apoptosis via the mitogen-activated protein kinase (MAPK) pathway in human chronic myeloid leukemia K562 cells. However, its anti-tumor activity against erythroleukemia remains largely unknown. In this study, we aimed to investigate the anti-erythroleukemia activity of BW18 and the underlying molecular mechanisms. Our results demonstrated that BW18 exhibited a good anti-erythroleukemia activity in the human erythroleukemia cell line HEL and an in vivo xenograft mouse model. In addition, BW18 induced cell cycle arrest at the G2/M phase and promoted megakaryocytic and erythroid differentiation in HEL cells. Furthermore, RNA sequencing (RNA-seq) and rescue assay demonstrated that overexpression of platelet-derived growth factor receptor beta (PDGFRB) reversed BW18-induced megakaryocytic differentiation in HEL cells, but not erythroid differentiation. In addition, the network pharmacology analysis, the molecular docking and cellular thermal shift assay (CETSA) revealed that BW18 could inactivate Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway, which might mediate BW18-induced erythroid differentiation. Taken together, our findings elucidated a novel role of PDGFRB in regulating erythroleukemia differentiation and highlighted BW18 as an attractive lead compound for erythroleukemia treatment.

Synthesis, Biological Evaluation, Molecular Docking and ADME Profiling of Methylpiperidin‐4‐ylphenyl‐nicotinamide Derivatives

AbstractIn this paper, we designed, synthesized, and biologically screened a series of methyl‐piperidine‐phenyl‐nicotinamide derivatives with the aim of novel derivatives as effective anticancer agents. Compounds (9 a), (9 e) and (9 k) exhibited potent anticancer activity with an IC50 value of 5.9 μM, 5.9 μM and 5.0 μM, respectively using MV411 (Acute Myeloid Leukemia) and K562 (Chronic Myeloid Leukemia) cell line. Our docking studies show good binding affinity of these compounds for ABL1 kinase. Furthermore, these compounds exhibited a favourable in silico ADME profile with good solubility and low clearance in Human Liver Microsomes (HLM). These derivatives represent a promising approach to initiate the development of new anticancer drugs in oncology programs.

Abstract LB066: Acute systemic beta-adrenergic receptor activation to improve graft composition and outcomes in hematopoietic stem cell transplantation

Abstract Background: Graft-versus-host disease (GvHD) and relapse persist as significant hurdles in achieving successful outcomes following allogeneic hematopoietic cell transplantation (alloHCT). The composition of the graft plays a substantial role in influencing transplant outcomes. Higher numbers of naïve CD4 and naïve CD8 T cells correlate with increased GvHD risk, conversely, elevated numbers of NK cells are associated with enhanced graft-versus-leukemia (GvL) effects. Acute systemic β-adrenergic receptor activation triggers the mobilization of favorable effector lymphocytes into the bloodstream, leading to a significant alteration in the composition of circulating immune cells. Aim: This study investigated whether infusion with the non-selective beta-agonist isoproterenol (ISO), following granulocyte colony-stimulating factor (G-CSF) mobilization, would enhance human peripheral blood hematopoietic cell (PBHC) grafts and improve transplant outcomes in NSG mice. Methods: Ten healthy volunteers received ISO infusion (50 ng/kg/min) for 20 minutes after 5 days of G-CSF. Blood samples were collected before and during the last 5 minutes of ISO infusion. Results: ISO infusion increased the total number of G-CSF-mobilized CD34 cells (28±13 cell/µl to 32±13 cells/µl) and significantly improved G-CSF-mobilized PBHC composition, increasing favorable graft cell subsets—NK cells (9.5% to 27.9%) and γδ T cells (5.0% to 7.5%)—and reducing unfavorable graft components—B cells (12.6% to 7.7%), naïve CD4 (18.1% to 11.2%), and naïve CD8 (8.9% to 5.8%) T cells (all p&amp;lt;0.05). NSG mice receiving G-CSF+ISO mobilized grafts exhibited lower GvHD (P=0.012) and extended survival (median survival: G-CSF alone = 45 days, G-CSF+ISO = 78 days) compared to mice receiving grafts mobilized by G-CSF alone. No difference in human CD45 engraftment was observed between the groups, with ~50% engraftment in both groups of mice. Upon challenge with the K562 chronic myeloid leukemia cell line, 42% of NSG-Tg(Hu-IL15) mice receiving G-CSF+ISO mobilized grafts were alive with low tumor burden at day 40, compared to 15% of mice receiving G-CSF mobilized grafts. In vitro, PBHC mobilized by G-CSF+ISO demonstrated an 8-fold increase in cytolysis against K562 compared to PBHC mobilized by G-CSF only (P=0.0002). Conclusion: ISO infusion post-G-CSF treatment favorably enhances graft composition, mitigates GvHD, prolongs survival, and augments GvL effects. Our findings suggest that acute systemic beta-adrenergic receptor activation could be a valuable strategy to enhance outcomes in alloHCT. Citation Format: Helena Batatinha, Grace M. Niemiro, Nicole Pena, Giovannah Hoskin, Preteesh Mylabathula, Forrest Baker, Tiffany Zuniga, Kyle Smith, Douglass Diak, Michael Seckeler, Emmanuel Katsanis, Richard Simpson. Acute systemic beta-adrenergic receptor activation to improve graft composition and outcomes in hematopoietic stem cell transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB066.

Evaluation of multidrug resistance in tumors using 99mTc-doxorubicin

Doxorubicin (DOX) is a potent chemotherapeutic agent used as the first-choice drug in treating different types of cancer, such as leukemia, lymphoma, and breast cancer, among others. However, the development of multidrug resistance represents a significant obstacle to the successful treatment of tumors. In this study, we assessed whether labeled DOX could be uptaken by susceptible and multidrug-resistant cell lines and by tumors in vivo in animals. The efficiency of DOX labeling was maintained at around 90% in this work. To evaluate the effect of 99mTc-DOX on cells expressing active P-glycoprotein (Pgp), we utilized cultured human chronic myeloid leukemia K562 cells and the resistant counterpart Lucena 1 cells. Lucena 1 cell line showed overexpression of Pgp that was not observed in K562. Our results suggested that the resistance to labeled or unlabeled DOX can be reversed by Pgp inhibition and can be easily detected. In conclusion, administering 99mTc-DOX into mice enables the differentiation between multidrug-resistant tumors in a non-invasive way and the evaluation of tumor resistance and possible chemosensitizers.

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2–3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

An Active Compound from the Pyrazine Family Induces Apoptosis by Targeting the Bax/Bcl2 and Survivin Expression in Chronic Myeloid Leukemia K562 Cells.

It has been established that pyrazine derivatives, which have widespread bioactivities, can effectively treat cancer. In this study, we investigated the effects of 2-methoxy-5-(oxiran-2-ylmethyl) phenyl pyrazine-2- carboxylate (2-mOPP), a new pyrazine derivative, on proliferation, viability, and apoptosis induction in human leukemia K562 cells. For this purpose, the K562 cells were treated with various concentrations (20-120 μM) of the 2-mOPP for 24-72 hours. Cell viability was determined by MTT growth inhibition assay. Apoptotic activity of 2-mOPP was investigated morphologically by Hoechst staining, cell surface expression assay of phosphatidylserine by Annexin-V/PI technique, as well as DNA fragmentation assay. The effect of 2-mOPP on the K562 cell cycle was studied by flow cytometry. To determine the impact of 2-mOPP on the expression of intrinsic apoptosis-related genes, Bcl2 (anti-apoptotic), Bax (pro-apoptotic), and Survivin genes expression levels were evaluated before and after treatment with 2-mOPP through Real-Time PCR analysis. The results revealed that 2-mOPP inhibited viability with IC50 of 25μM in 72 h. Morphological changes assessment by fluorescence microscopy, Annexin V/PI double staining by flow cytometry, and DNA ladders formation upon cell treatment with the 2-mOPP showed that this compound induces apoptosis at IC50 value. Cell cycle arrest was observed in the G0/G1 phase, and the sub-G1 cell population (the sign of apoptosis) increased in a time-dependent manner. Low expression levels of Bcl2 and Survivin in K562 cells were observed 24-72 h after treatment. Along with the down-regulation of Survivin and Bcl2, the expression of Bax was increased after treatment with 2-mOPP. These findings demonstrate that the new pyrazine derivative plays a crucial role in blocking the proliferation of the leukemic cells by inducing cell cycle arrest and apoptosis.

Effect of TRIM59 Expression Interference on Daunorubicin Chemosensitivity of Chronic Myeloid Leukemia K562 Cells and Its Mechanism

To investigate the effect of tripipartite motif 59 (TRIM59) expression interference on the chemosensitivity of daunorubicin (DNR) in chronic myeloid leukemia (CML) K562 cells and the related molecular mechanism. The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR. Liposome-based transfection technology was used to transfect TRIM59-specific siRNA (si-TRIM59) into K562 cells which then were treated with DNR. The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry, respectively, and the expressions of apoptosis-related protein and Wnt/β-catenin signaling pathway-related protein were detected by Western blot. Compared with the bone marrow tissue of CML patients at the time of initial treatment, the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased (P <0.05). Compared with control group, the cell proliferation inhibition rate and apoptosis rate in si-TRIM59 group and DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased (P <0.05), while the expressions of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased (P <0.05). Compared with si-TRIM59 group and DNR group, the proliferation inhibition rate and apoptosis rate of si-TRIM59+DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased, while the expression of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased (P <0.05). TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR, and its mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.

SiRNA-mediated downregulation of BATF3 diminished proliferation and induced apoptosis through downregulating c-Myc expression in chronic myelogenous leukemia cells.

Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.

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.

NK3.3-Derived Extracellular Vesicles Penetrate and Selectively Kill Treatment-Resistant Tumor Cells.

Cancer treatments often become ineffective due to the development of tumor resistance, leading to metastasis and relapse. Treatments may also fail because of their inability to access cells deep within the tumor tissue. When this occurs, new therapeutic agents are needed. We previously reported that NK3.3EVs, extracellular vesicles (EVs) derived from the normal human natural killer (NK) cell line, NK3.3, have strong cytotoxic activity against leukemia and breast cancer cell lines, without harming normal cells. Here, we used a three-dimensional (3D) MCF7 breast cancer mammosphere model to reproduce a more physiological environment that NK3.3EVs would encounter in vivo. NK3.3EVs penetrated MCF7 mammospheres, inducing death by apoptosis. We generated an imatinib-resistant K562 chronic myeloid leukemia (CML) cell line to investigate whether NK3.3EVs were able to kill tumor cells resistant to front-line chemotherapy. NK3.3EVs were even more cytotoxic to imatinib-resistant cells than parental cells, inducing apoptosis via caspase-3/-7 activation. The small population of cancer stem cells (CSCs) within tumors also contributes to therapeutic resistance. NK3.3EVs reduced the CSC-like CD34+/CD38- subpopulation in imatinib-resistant and parental K562 cultures and decreased CSC-associated expression of tumor-promoting genes. Our results provide strong evidence that NK3.3EVs may be a potential new immunotherapeutic agent for difficult-to-treat cancers.

Biological evaluation of combinations of tyrosine kinase inhibitors with Inecalcitol as novel treatments for human chronic myeloid leukemia

BackgroundThe use of tyrosine kinase inhibitors (TKIs) as a treatment for chronic myeloid leukemia (CML) has improved the natural history of the disease and increased the duration of survival. Tyrosine kinase inhibitors represent the success of target therapies that work on molecular targets, although some patients still have therapy failure. Vitamin D has antiproliferative, pro-apoptotic, and anti-angiogenic effects on cells, therefore it can be considered as a potential cancer preventative and treatment agent. Inecalcitol (TX-522) is the 14-epi-analogue of Calcitriol (1,25(OH)2-vitamin D3), and inhibits cancer cell proliferation more effectively than Calcitriol. This study was conducted to evaluate the antiproliferative and synergistic effects of the anticancer drugs Imatinib and Dasatinib in combinations with Inecalcitol on human chronic myeloid leukemia K-562 cells. MethodThe growth inhibitory activities of Inecalcitol, Imatinib, Dasatinib, and different combinations of one of the two drugs (Imatinib and Dasatinib) with Inecalcitol, were determined in vitro using MTT assay against K-562 cell line. ResultsInecalcitol, Imatinib, and Dasatinib showed potent antiproliferative activities against K-562 cells with GI50 values of 5.6 µM, 0.327 µM, and 0.446 nM, respectively. Combinations of Imatinib or Dasatinib with different concentrations of Inecalcitol increased significantly the antiproliferative activities and potencies of both drugs (****p < 0.0001), with optimal GI50 values of 580 pM (Imatinib) and 0.51 pM (Dasatinib). Furthermore, the combination treatments showed synergistic interaction between the antileukemic drugs and Inecalcitol, with combination indices (CI) < 1. ConclusionThe study demonstrated that the human chronic myeloid leukemia K-562 cells were subjected to a synergistic growth inhibitory impact when antileukemic drugs (Imatinib or Dasatinib) were combined with Inecalcitol, therefore, it is recommended that these combinations be viewed as promising novel antileukemic medications and used in place of individual medications with lower dosages and negligible side effects in the treatment of CML.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

Apoptotic and antiproliferative effects of Urtica dioica L. extract on K562 chronic myeloid leukemia cell line

Apoptotic and antiproliferative effects of Urtica dioica L. extract on K562 chronic myeloid leukemia cell line

Cytotoxic and apoptotic effects of chemically synthesized silver nanoparticles loaded with recombinant Staphylococcus LukS-PV toxin.

Chronic myeloid leukemia (CML) accounts for approximately 15% of leukemias. LukS-PV, a Panton-Valentine leucocidin (PVL) component, is secreted by Staphylococcus aureus. Silver nanoparticles have increasingly been used for different purposes, most notably for drug delivery and anticancer agents. In this work, the cytotoxicity effect of recombinant LukS-PV protein, chemically synthesized AgNPs, and recombinant LukS-PV protein-loaded silver nanoparticles was investigated on human Chronic myeloid leukemia K562 cells and human normal embryonic kidney HEK293 cells. Cell apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles exhibited dose-dependent cytotoxicity and induced apoptosis in the K562 cells but had little effect on normal HEK293 cells. After 24h of exposure to recombinant LukS-PV protein-loaded silver nanoparticles (IC50 concentration), flow cytometry showed that 31.17% of K562 cells were apoptotic. These results indicate that recombinant LukS-PV protein-loaded silver nanoparticles maybe are a potential chemotherapeutic agent candidate against K562 cells. Hence, silver nanoparticles could be used as drug carriers for toxin release to cancer cells.

Amsacrine downregulates BCL2L1 expression and triggers apoptosis in human chronic myeloid leukemia cells through the SIDT2/NOX4/ERK/HuR pathway

Accumulating evidence indicates that the anticancer activity of acridine derivatives is mediated through the regulation of anti-apoptotic and pro-apoptotic BCL2 protein expression. Therefore, we investigated whether the cytotoxicity of amsacrine with an acridine structural scaffold in human chronic myeloid leukemia (CML) K562 cells was mediated by BCL2 family proteins. Amsacrine induced apoptosis, mitochondrial depolarization, and BCL2L1 (also known as BCL-XL) downregulation in K562 cells. BCL2L1 overexpression inhibited amsacrine-induced cell death and mitochondrial depolarization. Amsacrine treatment triggered SIDT2-mediated miR-25 downregulation, leading to increased NOX4-mediated ROS production. ROS-mediated inactivation of ERK triggered miR-22 expression, leading to increased HuR mRNA decay. As HuR is involved in stabilizing BCL2L1 mRNA, downregulation of BCL2L1 was noted in K562 cells after amsacrine treatment. In contrast, amsacrine-induced BCL2L1 downregulation was alleviated by restoring ERK phosphorylation and HuR expression. Altogether, the results of this study suggest that amsacrine triggers apoptosis in K562 cells by inhibiting BCL2L1 expression through the SIDT2/NOX4/ERK-mediated downregulation of HuR. Furthermore, a similar pathway also explains the cytotoxicity of amsacrine in CML MEG-01 and KU812 cells.

Thymoquinone Enhances Apoptosis of K562 Chronic Myeloid Leukemia Cells through Hypomethylation of SHP-1 and Inhibition of JAK/STAT Signaling Pathway.

The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML). SHP-1 functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement of SHP-1 expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent of Nigella sativa seeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression of SHP-1 through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status of SHP-1 was studied by pyrosequencing analysis. The expression of SHP-1, TET2, WT1, DNMT1, DNMT3A, and DNMT3B was determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated the DNMT1 gene, DNMT3A gene, and DNMT3B gene and upregulated the WT1 gene and TET2 gene. This led to hypomethylation and restoration of SHP-1 expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.