Human K562 Cells Research Articles

Establishing capsaicin's anti-cancer intricacies in chronic myelogenous leukemia care: insights from human K562 cells

Objective: We aimed to establish capsaicin as a potent natural chemotherapeutic agent in chronic myeloid leukemia by unveiling anti-cancer mechanisms, concentrating at pro-apoptotic and anti-proliferative effects on K562 cells. Methodology: After REC approval, this research proceeded with culturing K562 cells and treatment with serial concentrations of capsaicin for calculation of subsequent 50% Inhibitory Concentration (IC50) values via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, expression analysis of gene, comparative analysis of relative gene fold (intrinsic biomarkers caspase-3, caspase-9) as apoptosis mediator biomarkers followed by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). Anti-proliferative activity was confirmed via Nitric Oxide (NO) releasing Assay. Results: For K562 cells, the IC50 of capsaicin (12.1 µM) showed high gene expression of intrinsic pro-apoptotic biomarkers caspase-3 and caspase-9 with the relative gene fold of 12.1 and 13.9 respectively. Persistent peaks of NO levels confirmed the anti-proliferative potential of the compounds. Strongest growth inhibitory activity was seen at the peak time of 100-120 min. Conclusion: Capsaicin proved to be a strong pro-apoptotic and anti-proliferative phytochemical agent leading to therapeutic effects against K562 cells. Further studies would help in identifying key molecular intricacies by which capsaicin exhibits diversified anti-cancer potential. Abbreviations: CML - chronic myelogenous leukemia; NO - Nitric oxide; ROS - Reactive Oxygen Species; ROS - Reactive Oxygen Species Keywords: Capsaicin, Pro-apoptotic Effects, Anti-Cancer Mechanisms, K562 cells, Natural Chemotherapeutics Citation: Devi D, Nangdev P, Khaliq HMH, Anique M, Moqaddas A, Aziz O, Javed W. Establishing capsaicin's anti-cancer intricacies in chronic myelogenous leukemia care: insights from human K562 cells. Anaesth. pain intensive care 2024;28(5):830−835; DOI: 10.35975/apic.v28i5.2494 Received: June 26, 2024; Reviewed: August 10, 2024; Accepted: August 16, 2024

Abstract P181: Class 1 human leukocyte antigens exhibit marked variability in capacity to present isolevuglandin adducts

Introduction: Isolevuglandins (isoLGs) are lipid peroxidation products that covalently modify lysine residues on self-proteins. These modified self-proteins are processed to peptides that are presented in the context of class I major histocompatibility complexes (MHC-I). In hypertension, this modification enhances CD8 + T cell activation, contributing to inflammation and elevated blood pressure. In mice we found that IsoLG-adduct presentation is highly dependent on class I MHC structure. We sought to determine if human class I MHC (HLAs) exhibit similar variability in their ability to present these adducts by screening alleles representing 90% of the population. Hypothesis: We hypothesize that HLA subtypes exhibit variable capacity for isoLG presentation. Methods: HLA alleles representing 90% demographic frequency were identified from the US National Merit Donor Program Allele Frequency Database. HLA-null human K562 cells were transduced with these alleles, expanded, and enriched for HLA expression by flow cytometry to produce single HLA allele lines. To induce isoLG adduct presentation, cells were treated with 1mM tert-Butyl hydroperoxide (tBHP). After 24 hours, we employed Forster resonance energy transfer (FRET) between HLAs and isoLG-adducts using antibodies specific for both, which we have shown is indicative of IsoLG-adduct presentation (Fig 1A-C). Results: Substantial variability for isoLG-adduct presentation was observed between HLA subtypes A, B and C (Figure 1A-1C). Scikit-learn’s KMeans clustering revealed four patterns of isoLG-adduct presentation among HLAs: Low baseline/low activation, low baseline/high activation, intermediate baseline/intermediate activation, and high baseline/high activation alleles (Fig 1D). Conclusions: These findings identify specific HLA alleles as high isoLG presenters, potentially associated with increased immune activation risk. Combining these findings, HLA alleles, and diagnosis code databases could aid in identifying high-risk haplotypes for immune activation in human hypertension.

Decoding the genomic landscape of chromatin-associated biomolecular condensates

Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Exploring the Composition and Bioactivity of Hedychium flavum Leaf and Stem Essential Oil: In Vitro Assessment of Antibacterial, Antioxidant, Cytotoxicity, and Enzyme Inhibitory Activities

Hedychium flavum is an edible, medicinal, and aromatic plant widely cultivated for its essential oil. So far, little studies have been done on its phytoconstituents and bioactivities. Hence, the research aimed to determine the chemical constituents of leaf essential oil (L-EO) and stem essential oil (S-EO) of H. flavum and first estimate their antibacterial, antioxidant, cytotoxic, and enzyme inhibitory activities. According to gas chromatography-flame ionization detector/mass spectrometer (GC-FID/MS) assay, L-EO was mainly composed of β-pinene (33.4%), α-pinene (10.4%), humulene (6.8%), β-caryophyllene (6.0%), eucalyptol (6.0%), caryophyllene oxide (5.5%), endo-borneol (3.8%), humulene epoxide II (3.7%), and D-limonene (3.2%). The predominant components of S-EO were β-pinene (17.2%), eucalyptol (9.7%), nerolidol (7.8%), α-phellandrene (6.7%), α-pinene (5.8%), β-caryophyllene (5.4%), terpinen-4-ol (4.5%), D-limonene (4.4%), p-cymene (3.7%), endo-borneol (3.5%), and α-terpineol (3.5%). For the bioactivities, L-EO and S-EO showed strong antibacterial activity against Escherichia coli, Enterococcus faecalis, and Bacillus subtilis with the minimum inhibitory concentration (MIC) of 9.77–625.00 µg/mL and the minimum bactericidal concentration (MBC) of 9.77–1250.00 µg/mL. Both L-EO and S-EO exhibited moderate antioxidant activity in the DPPH (14.52 ± 0.93 mg/mL and 4.73 ± 0.15 mg/mL, respectively) and ABTS (4.05 ± 0.75 mg/mL and 2.38 ± 0.14 mg/mL, respectively) free radical scavenging capacity. S-EO showed selective cytotoxicity on human leukemic K562 cell line (IC50 = 93.94 ± 0.91 µg/mL), which was nearly three times that of noncancer L929 cell line (IC50 = 294.49 ± 9.40 µg/mL). In enzyme inhibitory properties, L-EO exerted a potent inhibition on α-glucosidase (IC50 = 1.03 ± 0.02 mg/mL), and both essential oils had weak inhibition against cholinesterase and tyrosinase. Thus, H. flavum L-EO and S-EO possess antibacterial, antioxidant, cytotoxic, and enzyme inhibitory activities with potential for exploitation in the food, cosmetic, and pharmaceutical industries.

Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

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.

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.

Reversal Effect of Arctigenin on the Drug Resistance in Leukemia K562/A02 Cells and Its Mechanism

To study the effect of arctigenin(ARG) on adriamycin(ADM) resistance of leukemia cell line K562/A02 and the underlying mechanism. Human leukemia cell line K562 and ADM-resistant cell line K562/A02 were cultured and treated with 2.5-50 µmol/L ADM. Cell proliferation was measured using CCK-8 method, and half maximal inhibitory concentration (IC50) was calculated. K562/A02 cells were treated with different concentrations of ARG (1, 2, 4, 8, 16 mmol/L) to detect the effect of ARG on K562/A02 cells, and a suitable concentration (2 mmol/L) was selected for subsequent experiments. K562/A02 cells were treated with 2 mmol/L ARG and 5 µmol/L ADM, and cell apoptosis was detected by flow cytometry, the expression of P-gp, MRP, cleaved caspase-3, Bax, Bcl-2 proteins and the TLR4/NF-κB signaling pathway-related proteins were measured by Western blot. TLR4 overexpression plasmid was transfected into K562/A02 cells which were co-treated with ARG and ADM, then drug sensitivity and cell apoptosis were measured. The IC50 value of ADM on K562/A02 cells was 36.57 µmol/L, which was significantly higher than that on K562 cells (1.30 µmol/L). ARG with a concentration of ≤2 mmol/L did not have a significant effect on K562/A02 cells. 2 mmol/L ARG significantly reduced the IC50 of ADM on K562/A02 cells. In 5 µmol/L ADM-treated K562/A02 cells, compared with the control group, the apoptosis rate of K562/A02 cells in the ARG group was significantly increased, the expressions of cleaved caspase-3, Bax proteins were significantly upregulated, the expressions of P-gp, MRP, Bcl-2, TLR4, MyD88, and p-NF-κB proteins were significantly downregulated, and the differences were statistically significant (P < 0.05). After transfection with TLR4 overexpression plasmid, the sensitivity of ARG-treated K562/A02 cells to ADM was reduced (P < 0.05), the cell apoptosis was decreased, and the expressions of P-gp, MRP, Bcl-2 and TLR4/NF-κB signaling pathway-related proteins were significantly elevated, while the expressions of cleaved caspase-3 and Bax proteins were significantly decreased (all P < 0.05). ARG may reverse the resistance of human leukemia cell line K562/A02 to ADM by inhibiting TLR4/NF-κB signaling pathway.

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.

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis.

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model.The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Х3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Х3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

Curcumin prevents cadmium or H2O2-induced oxidative stress via Nrf2/ARE signaling and autophagy in myeloid cells

The evidence linking high levels of environmental pollutants to chronic degenerative diseases is alarming, with heavy metals (HM) identified as a key factor. Research suggests that certain phytochemicals in the diet can reduce HM levels and mitigate their adverse health effects.Curcumin (Curc), a natural polyphenol, is particularly effective in protecting against Cadmium (Cd) toxicity. The present study demonstrates that preincubation with low doses of Curc (1 μM) in differentiated HL-60 and K-562 human myeloid cells can significantly protect against cytotoxicity induced by Cd and or hydrogen peroxide (H2O2). Curc reduced the increased levels of Reactive Oxygen Species (ROS) generated by Cd or H2O2 by inducing a protective form of autophagy, a relevant process for the cellular homeostasis. Curc activated mild oxidative stress that triggers the expression of Nrf2-dependent transcripts, such as Nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent transcripts, such as human NADPH Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase (HO1). The potential chemopreventive effects of Curc against redox stress have been strengthened by the observation that free and unmetabolized Curc is detectable inside the cells after 5 min of treatment, and its presence parallels with increased levels of intracellular Glutathione (GSH). These findings suggest that supplementation with Curc in the form of nutraceuticals may represent a promising way to protect people living in highly polluted areas against the adverse effects of HM contaminants.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from β-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia.

Knockouts of TNFRSF1A and TNFRSF1B Genes in K562 Cell Line Lead to Diverse Long-Lasting Responses to TNF-α.

This research delves into the intricate landscape of tumor necrosis factor-alpha (TNF-α) signaling, a multi-functional cytokine known for its diverse cellular effects. Specifically, we investigate the roles of two TNF receptors, TNFR1 and TNFR2, in mediating TNF-α-induced transcriptional responses. Using human K562 cell lines with TNFR1 and TNFR2 knockouts, we explore changes in gene expression patterns following TNF-α stimulation. Our findings reveal distinct transcriptional profiles in TNFR1 and TNFR2 knockout cells, shedding light on the unique contributions of these receptors to TNF-α signaling. Notably, several key pathways associated with inflammation, apoptosis, and cell proliferation exhibit altered regulation in the absence of TNFR1 or TNFR2. This study provides valuable insights into the intricate mechanisms governing TNF-α signaling and its diverse cellular effects, with potential implications for targeted therapeutic strategies.

Discovery of Transfer Factors in Plant-Derived Proteins and an In Vitro Assessment of Their Immunological Activities.

Repeated exposure to pathogens leads to evolutionary selection of adaptive traits. Many species transfer immunological memory to their offspring to counteract future immune challenges. Transfer factors such as those found in the colostrum are among the many mechanisms where transfer of immunologic memory from one generation to the next can be achieved for an enhanced immune response. Here, a library of 100 plants with high protein contents was screened to find plant-based proteins that behave like a transfer factor moiety to boost human immunity. Aqueous extracts from candidate plants were tested in a human peripheral blood mononuclear cell (PBMC) cytotoxicity assay using human cancerous lymphoblast cells-with K562 cells as a target and natural killer cells as an effector. Plant extracts that caused PBMCs to exhibit enhanced killing beyond the capability of the colostrum-based transfer factor were considered hits. Primary screening yielded an 11% hit rate. The protein contents of these hits were tested via a Bradford assay and Coomassie-stained SDS-PAGE, where three extracts were confirmed to have high protein contents. Plants with high protein contents underwent C18 column fractionation using methanol gradients followed by membrane ultrafiltration to isolate protein fractions with molecular weights of <3 kDa, 3-30 kDa, and >30 kDa. It was found that the 3-30 kDa and >30 kDa fractions had high activity in the PBMC cytotoxicity assay. The 3-30 kDa ultrafiltrates from the top two hits, seeds from Raphanus sativus and Brassica juncea, were then selected for protein identification by mass spectrometry. The majority of the proteins in the fractions were found to be seed storage proteins, with a low abundance of proteins involved in plant defense and stress response. These findings suggest that Raphanus sativus or Brassica juncea extracts could be considered for further characterization and immune functional exploration with a possibility of supplemental use to bolster recipients' immune response.

A Cytochrome c Variant Enhances Megakaryocytic Maturation

Mutations in genes which play a role in the processes of megakaryopoiesis and platelet production cause thrombocytopenia. Five pathogenic variants in the gene encoding cytochrome c ( CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. The role of cytochrome c in megakaryopoiesis and platelet formation is unknown, and it is unclear how mutations in cytochrome c cause thrombocytopenia. The goal of this project is to understand how CYCS mutations cause thrombocytopenia. We are using the human leukemic K562 cell line as a model system to study the impact of CYCS mutations on megakaryocyte differentiation and mitochondrial function. One of the CYCS pathogenic variants (c.145T&amp;gt;C; p.Tyr49His) was introduced into K562 cells using the CRISPR-Cas9 base-editing method. K562 cells have three copies of CYCS-containing chromosome 7 and we isolated CYCS WT/WT/WT, CYCS WT/WT/Y49H, CYCS WT/Y49H/Y49H, CYCS Y49H/Y49H/Y49H clonal lines, confirmed by sequencing and mass spectrometry. K562 cells undergo megakaryocyte differentiation in response to PMA. We monitored the expression of megakaryocyte markers CD61 and CD41a by flow cytometry in response to PMA in all clonal lines. The K562- CYCS Y49H/Y49H/Y49H line showed faster maturation compared to all other clonal lines and the parental K562 cells. We assessed mitochondrial function using the Seahorse XFe96 Analyzer. Our results show that the mutation does not impact mitochondrial respiratory function. Finally, we have introduced the Y49H mutation into WTC11 iPSCs. Investigations of the impact of the Y49H variant on apoptosis showed that there is no significant differences in apoptosis between WT and the clonal line carrying the Y49H variant. Our results suggest that the pathogenic c.145T&amp;gt;C; p.Tyr49His CYCS variant enhances megakaryocyte maturation.

CHEX-seq detects single-cell genomic single-stranded DNA with catalytical potential

Genomic DNA (gDNA) undergoes structural interconversion between single- and double-stranded states during transcription, DNA repair and replication, which is critical for cellular homeostasis. We describe “CHEX-seq” which identifies the single-stranded DNA (ssDNA) in situ in individual cells. CHEX-seq uses 3’-terminal blocked, light-activatable probes to prime the copying of ssDNA into complementary DNA that is sequenced, thereby reporting the genome-wide single-stranded chromatin landscape. CHEX-seq is benchmarked in human K562 cells, and its utilities are demonstrated in cultures of mouse and human brain cells as well as immunostained spatially localized neurons in brain sections. The amount of ssDNA is dynamically regulated in response to perturbation. CHEX-seq also identifies single-stranded regions of mitochondrial DNA in single cells. Surprisingly, CHEX-seq identifies single-stranded loci in mouse and human gDNA that catalyze porphyrin metalation in vitro, suggesting a catalytic activity for genomic ssDNA. We posit that endogenous DNA enzymatic activity is a function of genomic ssDNA.

Synthesis of new Cu (II) and Zn (II) complexes derived from isatin‐3‐thiosemicarbazone: X‐ray, anticancer activity, molecular docking, ADMET study, and quantum chemical calculations

The current work aimed to synthesize new mononuclear Cu (II) and Zn (II) complexes derived from isatin‐based thiosemicarbazide, (Z)‐N‐methyl‐2‐(2‐oxoindolin‐3‐ylidene)hydrazinecarbothioamide ligand (L1) using the reflux conventional method. Structural characterization for the synthesized compounds was achieved via elemental analyses, spectroscopic analysis, magnetic susceptibility, molar conductivity, and thermal analysis. The compounds were further explored for their prospective anticancer properties with the aid of in silico methodology. Complexation of metal centers with L1 was observed using the shifting of the v(C=N) peak to lower frequencies and the emergence of new peaks in the range of 454–512 cm−1 assigned to v(M‐N) and v(M‐O). The non‐electrolytic character of Cu2+ and Zn2+ compounds was displayed by their low molar conductivities. Quantum chemical calculations were employed to investigate the electronic and molecular properties of the complexes. The frontier molecular orbitals were performed by density functional theory (DFT) calculations to provide valuable information regarding their molecular orbitals, reactivity indices, and electronic structures, assisting in understanding their structure–activity relationships and potential mechanisms of action. Additionally, molecular docking was carried out to comprehend the interaction of the compounds with the selected enzyme and determine their potential binding mode and energy. Meanwhile, the ADMET study exhibited that all of the compounds possess good oral bioavailability suggesting potent and suitable anticancer drug candidates. All the compounds were tested against human leukemia K562 cancer cell line and human fibroblasts Hs27 cell line. Overall, this comprehensive study highlights the synthesis, characterization, anticancer activity, molecular docking, ADMET evaluation, and quantum chemical calculations of new Cu (II) and Zn (II) complexes derived from isatin‐3‐thiosemicarbazone.