Human Leukemia Cell Line HL-60 Research Articles

CYTOTOXICITY OF ALOE VERA EXTRACT ON CHRONIC MYELOID LEUKEMIA CELLS; IN VITRO STUDY ON CYTOTOXICITY ON HL-60 CELLS

Aloe vera has been traditionally used for its various therapeutic properties, but its potential anticancer effects have not been thoroughly explored. Understanding its efficacy against leukemia could significantly impact care recommendations for leukemia patients. Objective: This in-vitro study aims to establish the anti-cancerous effects of Aloe vera on the Human Leukemia HL-60 cell line. Methods: Following approval from the Ethical Review Committee, this study involved culturing the HL-60 cell line and treating it with increasing concentrations of Aloe vera extract to determine the half-maximal inhibitory concentration (IC50). The MTT assay was employed to assess cell viability, and the absorbance was measured using an ELISA reader. Gene expression levels of pro-apoptotic biomarkers, Caspase-3 and Caspase-9, were also quantified to gauge apoptosis induction. Results: Aloe vera demonstrated a relatively low IC50 value of 13.1 µM in the HL-60 cell line, indicating potent cytotoxicity. Notably, pro-apoptotic biomarkers were highly expressed, with Caspase-3 and Caspase-9 recording gene expression levels of 11.1 and 13.7, respectively. Conclusion: Aloe vera exhibited significant pro-apoptotic effects against the leukemia HL-60 cell line, suggesting its potential as an anti-cancer agent. Further in-vivo and clinical studies are warranted to elucidate the specific pathways through which Aloe vera exerts these effects and to validate its use in clinical settings.

Anti-tumor withanolides as signal transducers and activators of transcription 3 (STAT3)-inhibition from Withania obtusifolia

The Solanaceae family and the Withania genus specifically are rich sources of medicinal plants. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) revealed a predominance of withanolides from an organic extract of Withania obtusifolia. A constructed molecular network uncovered the presence of potentially novel withanolides. A series of withanolides were then isolated and structurally characterized from the extract including two new withanolides (withafolia A and withafolia B) and seven previously reported metabolites. Of the isolated compounds, cytotoxicity of withanolide J, physaperuvin G, and a commercial STAT3 inhibitor (S3I-201) were assessed against a human leukemia HL-60 cell line resulting in IC50 values of 26, 29, and 120 μM, respectively. In silico molecular docking simulations indicate that withanolide J and physaperuvin G can bind as an inhibitor in the active site of STAT3 with docking scores comparable to the selective STAT3 inhibitor, S3I-201.

Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

Synthesis of New Chiral β-Carbonyl Selenides with Antioxidant and Anticancer Activity Evaluation-Part I.

A series of unsymmetrical phenyl β-carbonyl selenides with o-amido function substituted on the nitrogen atom with chiral alkyl groups was obtained. The compounds form a series of enantiomeric and diastereomeric pairs and present the first examples of this type of chiral Se derivatives. All obtained selenides were further evaluated as antioxidants and anticancer agents to define the influence of the particular stereochemistry of the attached functional groups on the bioactivity of the molecules. The highest H2O2 reduction potential was observed for N-(cis-2-hydroxy-1-indanyl)-2-((2-oxopropyl)selanyl)benzamide, and the best radical scavenging properties for N-(-1-hydroxy-2-butanyl)-2-((2-oxopropyl)selanyl)benzamide. Also, both enantiomers of the N-(1-hydroxy-2-butanyl) selenide expressed the highest cytotoxic potential towards human promyelocytic leukemia HL-60 cell line with similar IC50 values 14.4 ± 0.5 and 16.2 ± 1.1 µM, respectively. On the other hand, breast cancer cell line MCF-7 was most sensitive to N-((R)-(-)-1-hydroxy-2-butanyl)- 2-((2-oxopropyl)selanyl)benzamide (IC50 of 35.7 ± 0.6 µM). The structure-activity dependence of the obtained Se derivatives was discussed, and the most potent compounds were selected.

Cancer Cell Targeting Via Selective Transferrin Receptor Labeling Using Protein-Derived Carbon Dots.

Carbon dot (CD) nanoparticles offer tremendous advantages as fluorescent probes in bioimaging and biosensing; however, they lack specific affinity for biomolecules, limiting their practical applications in selective targeting. Nanoparticles with intrinsic affinity for a target have applications in imaging, cytometry, therapeutics, etc. Toward that end, we report the transferrin receptor (CD71) targeting CDs, synthesized for the first time. The formation of these particles is truly groundbreaking, as direct tuning of nanoparticle affinity was achieved by simple and careful precursor selection of a protein, which has the targeting characteristic of interest. We hypothesized that the retention of the original protein's peptides on the nanoparticle surface provides the CDs with some of the function of the precursor protein, enabling selective binding to the protein's receptor. This was confirmed with FTIR (Fourier transform infrared) data and subsequent affinity-based cell assays. These transferrin (Tf)-derived CDs have been shown to possess an affinity for CD71, a cancer biomarker that is ubiquitously expressed in nearly every cancer cell line due to its central role mediating the uptake of cellular iron. The CDs were tested using the human leukemia cell line HL60 and demonstrated the selective targeting of CD71 and specific triggering of transferrin-mediated endocytosis via clathrin-coated pits. The particle characterization results reflect a carbon-based nanoparticle with bright violet fluorescence and 7.9% quantum yield in aqueous solution. These unpresented CDs proved to retain the functional properties of the precursor protein. Indicating that this process can be repeated for other disease biomarkers for applications ranging from biosensing and diagnostic bioimaging to targeted therapeutics.

Circulating cell-free DNA fragmentation is a stepwise and conserved process linked to apoptosis

BackgroundCirculating cell-free DNA (cfDNA) is a pool of short DNA fragments mainly released from apoptotic hematopoietic cells. Nevertheless, the precise physiological process governing the DNA fragmentation and molecular profile of cfDNA remains obscure. To dissect the DNA fragmentation process, we use a human leukemia cell line HL60 undergoing apoptosis to analyze the size distribution of DNA fragments by shallow whole-genome sequencing (sWGS). Meanwhile, we also scrutinize the size profile of plasma cfDNA in 901 healthy human subjects and 38 dogs, as well as 438 patients with six common cancer types by sWGS.ResultsDistinct size distribution profiles were observed in the HL60 cell pellet and supernatant, suggesting fragmentation is a stepwise process. Meanwhile, C-end preference was seen in both intracellular and extracellular cfDNA fragments. Moreover, the cfDNA profiles are characteristic and conserved across mammals. Compared with healthy subjects, distinct cfDNA profiles with a higher proportion of short fragments and lower C-end preference were found in cancer patients.ConclusionsOur study provides new insight into fragmentomics of circulating cfDNA processing, which will be useful for early diagnosis of cancer and surveillance during cancer progression.

Dasatinib and Hesperidin Loaded Nano Formulation and Preclinical Evaluation for Anticancer Activity

In this study, we aimed to develop Dasatinib/hesperidin-loaded-SLNs for chronic myeloid leukemia (CML). Utilizing a high-shear homogenizer for the synthesis, “central composite design (CCD)” was used to enhance the dasatinib/hesperidin loaded-SLNs. The optimized SLNs had PDI, particle size, and average entrapment efficiency of 0.12%, 162.3 nm, and 93%, respectively. Therefore, by enhancing the total amount of Compritol as well as sonication time the polydispersity was increased. Poloxamer 188 content had a significant influence in decreasing the polydispersity index and the entrapment efficiency (EE) of the SLN was found to be 93%. Through TEM, SEM, FTIR, DSC, and HPLC analysis, SLNs were characterized, and their anticancer efficiency was assessed in both in-vitro and in-vitro cell viability tests (MTT). SLNs containing dasatinib and hesperidin have rounded and spherical shape having a diameter of 200 nm. DSC and FTIR tests showed compatibility between the drugs and excipients. The drug release from optimized SLN formulation was under observation for 48 hours. It was found that the medication released its drug components over a protracted period of time, with 30% of the drug being released in the first four hours and the remainder 76% in the next 48 hours. According to the IC50 values determined by an independent study, dasatinib, hesperidin, and SLN were 33.97, 5158, and 4.03 μg/mL, respectively. A comparison of SLN and free drugs revealed that SLN was more effective at cytotoxicity. In this study, dasatinib and hesperidin-loaded SLNs for chronic myeloid leukemia (CML) against HL60 human leukemia cell lines were prepared and evaluated using a novel formulation approach free of toxic excipients.

The Peptide Salamandrin-I Modulates Components Involved in Pyroptosis and Induces Cell Death in Human Leukemia Cell Line HL-60.

Amphibian secretions have been extensively investigated for the production of bioactive molecules. Salamandrin-I is an antioxidant peptide, isolated from the skin secretion of the fire salamander, that has induced no toxicity in microglia or erythrocytes. Importantly, the administration of antioxidants may constitute an adequate therapeutic approach to cancer treatment. Here, with the purpose of better characterizing the therapeutic potential of salamandrin-I, we investigated whether this antioxidant peptide also exerts anticancer activity, using the human leukemia cell line HL-60 as a cancer model. Salamandrin-I treatment induced a significant reduction in HL-60 proliferation, which was accompanied by cell cycle arrest. Furthermore, the peptide-induced cell death showed a significant increase in the LDH release in HL-60 cells. The cellular toxicity exerted by salamandrin-I is possibly related to pyroptosis, since the HL-60 cells showed loss of mitochondrial membrane potential and hyperexpression of inflammasome components following the peptide treatment. This is the first demonstration of the anticancer potential of the salamandrin-I peptide. Such results are important, as they offer relevant insights into the field of cancer therapy and allow the design of future bioactive molecules using salamandrin-I as a template.

Cytotoxicity and Induction of G1 Phase Cell Cycle Arrest in human acute promyelocytic leukemia HL60 cells by indole alkaloids isolated from Dialium corbisieri seeds.

The phytochemical investigation of Dialium corbisieri seeds led to the isolation of five monoterpenoid indole alkaloids along with a phytoserotonin, 1-6 and among the known compounds, the spectroscopic data of (5S)-methoxy-akuammiline (1) was reported for the first time. The structures were elucidated based on NMR spectroscopic techniques such as UV, IR, HRESITOFMS, and ECD spectrum calculations. The isolated compounds were evaluated for their cytotoxicity and cell progression in the human acute promyelocytic leukemia HL60 cell line.

Novel indole retinoid derivative induces apoptosis and cell cycle arrest and modulates AKT and ERK signaling in HL-60 cells.

Chemotherapy with targeted drugs is the first line therapy option for acute and chronic myeloid leukemia. However, hematopoietic stem cell transplantation may be used in high-risk patients or patients with failed responses to chemo drugs. Discovery and development of more effective new agents with lower side effects is the main aim of leukemia treatment. In this study, a novel retinoid compound with tetrahydronaphthalene ring was synthesized and evaluated for anticancer activity in human chronic and acute myeloid leukemia cell lines K562 and HL-60. Novel N-(1H-indol-1-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide was synthesized based on molecular hybridization of the two different bioactive structures retinoid head and indole. The effects of the synthesized carboxamide compound, which was referred to as compound 5, were determined in K562 chronic myeloid leukemia and HL-60 acute myeloid leukemia cell lines and L929 fibroblast cell line, which served as a control. Colorimetric MTT and caspase3 activity tests, flow cytometry, western blot, and microscopic examinations were used to evaluate biological activity. Compound 5 more effectively induced cell death in HL60 cells in comparison to K562 cells and L929 fibroblast cells. Therefore, further mechanism of cell death was investigated in HL60 cell line. It was found that compound 5 induced remarkable cytotoxicity, caspase3 activation, and PARP fragmentation in HL60 cells. Flow cytometric staining showed that the percentage of cells arrested in G0/G1 was also increased with compound 5 treatment. Important modulator proteins of cell proliferation p-ERK, p-AKT, and p-m-TOR were also found to be inhibited with compound 5 treatment. Collectively, our results reveal compound 5, which is a novel indole retinoid compound as a potential active agent for the treatment of acute promyelocytic leukemia.

Synthesis and characterization of new metal complexes containing azo-indole moiety and anti-leukemia human (HL-60) study of its palladium (II) complex

The research included formation of (E)-2-(1H-indol-3yl)diazenyl)thiazole (HIDAT) ligand resulting from the reaction diazonium salt of 2-aminothiazole compound with indole compound. Azo dye ligand (HIDAT) and its complexes of the transition elements (cobalt, nickel, copper and palladium) were prepared and characterized by different technique such as elemental analysis (C.H.N.S), mass spectroscopy, 1H NMR, FT-IR, UV–Visible and magnetic measurements as well as molar conductivity. The HIDAT complexes were synthesized under molar ratio (1:2) of metal to ligand excepting Pd(II) complex prepared under mole ratio equal to 1:1. The HIDAT ligand presented significant ability to know pH for aqueous solutions. According to the infrared data, HIDAT dye functions as a bidentate ligand. The spectral studies were designated to octahedral geometry for the synthesized complexes excepting Pd complex was square planar. The cytotoxicity effect of palladium complex was demonstrated in human leukemia cell line HL-60 and HdFn healthy cells and the MTT results against HL-60 cells were more susceptible to the palladium complex's cytotoxicity than HdFn cells. The docking (MOE) study of the palladium complex against leukemia cancer protein (3s7s) has promising inhibition activity.

Green synthesis of copper oxide spindle like nanostructure using Hibiscus cannabinus flower extract for antibacterial and anticancer activity applications

In this study, CuO nanospindle synthesized using petal extract of Hibiscus cannabinus (H. cannabinus) was attempted to have better antibacterial activity against Gram-positive and Gram-negative bacteria. Cytotoxic activity was found to be 70 % against human acute myeloid leukemia HL60 cell line at 500 µg/ml. Morphological features of CuO was analyzed by scanning electron microscopy (SEM). Structural properties were analyzed by X-ray diffraction (XRD) pattern and the Fourier transform infrared (FTIR) spectrum of CuO provides additional evidence for the formation of monoclinic crystal structured CuO. Energy dispersive X-ray spectroscopy (EDAX) analysis exposed the pristine properties of CuO nanospindles.

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n=49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Antiproliferative Activity and Apoptotic Potential of Curcumin on Human Acute Myeloid Leukemia Cell Line (HL-60)

Curcuma Longa or also known as turmeric is a prominent natural remedy in Asia that has been utilized to heal diseases for decades. It has been demonstrated to have substantial biological activities, including anticarcinogenic, antimutagenic, antioxidant, anti-inflammatory, anti-infectious, hypocholesterolemic, and chemopreventive properties. Curcumin is a therapeutic substance extracted from Curcuma Longa that is thought to have anti-cancer properties against a variety of cancer types. However, its anti-cancer potential on acute myeloid leukemia (AML) remains uncertain [1,2]. &#x0D; &#x0D; The objective of this intervention study was to examine the anti-proliferative and apoptotic effects of curcumin on the HL-60 cell line of acute myeloid leukemia. A stock solution of pure curcumin compound was obtained by dissolving it in dimethyl sulfide (DMSO). HL-60 cells were treated with six different concentrations which were 100, 50, 25, 12.5, 6.25, 3.125 µg/ml and incubated at three distinct incubation period (24, 48 and 72 hours). The in vitro cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and quantified using a microplate reader at 570 nm. Acridine orange (AO) and propidium iodide (PI) staining were employed to evaluate morphological changes. &#x0D; &#x0D; MTT assays results show that curcumin has a high cytotoxicity effect on HL-60 cells. The maximal half inhibitory concentration (IC50) value of curcumin was consistently decreased when the incubation time increased. The IC50 of curcumin on HL-60 cells were reported to be 13, 8, and 3.5 µg/ml at 24, 48 and 72 hours of incubation, respectively. There was a significantly different IC50 value (p&lt;0.05) of curcumin between the incubation periods (Figure 1). The morphological alteration was shown in AO/PI staining by the appearance of early apoptosis, late apoptosis, and necrotic cells.&#x0D; These findings illustrated that curcumin revealed an effective anti-cancer action on human acute myeloid leukemia HL-60 cell line. Further research is required to determine the mechanism of cell death in HL-60 cells treated with pure curcumin compound.&#x0D; &#x0D; In conclusion, the study revealed the role of putative targeted genes in the biological mechanism in gout pathogenesis has provides insight of the potential biomarkers in developing the personalized medicine better treatment for gout patient in ensuring better patient healthcare.

Anti-Leukemic Activity of Brassica-Derived Bioactive Compounds in HL-60 Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells mainly treated with chemotherapy for cancer remission, but this non-selective treatment also induces numerous side effects. Investigations with bioactive compounds from plant-derived foods against cancer have increased in the last years because there is an urgent need to search for new anti-leukemic agents possessing higher efficacy and selectivity for AML cells and fewer negative side effects. In this study, we analyzed the anti-leukemic activity of several phytochemicals that are representative of the major classes of compounds present in cruciferous foods (glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols, and anthocyanins) in the human acute myeloid leukemia cell line HL-60. Our results revealed that among the different Brassica-derived compounds assayed, sulforaphane (SFN) (an aliphatic isothiocyanate) showed the most potent anti-leukemic activity with an IC50 value of 6 µM in dose-response MTT assays after 48 h of treatment. On the other hand, chlorogenic acid (a hydroxycinnamic acid) and cyanidin-3-glucoside (an anthocyanin) also displayed anti-leukemic potential, with IC50 values of 7 µM and 17 µM after 48 h of incubation, respectively. Importantly, these compounds did not show significant cell toxicity in macrophages-like differentiated cells at 10 and 25 µM, indicating that their cytotoxic effects were specific to AML cancer cells. Finally, we found that these three compounds were able to induce the NRF2/KEAP1 signaling pathway in a dose-dependent manner, highlighting SFN as the most potent NRF2 activator. Overall, the present evidence shed light on the potential for using foods and ingredients rich in anticancer bioactive phytochemicals from Brassica spp.

Ultraviolet Radiation Promoted Hypoxia-Induced Apoptosis in HL-60 Human Promyelocytic Leukemia Cell Line.

Minimal residual disease (MRD) is an important reason for the failure of autologous hematopoietic stem cell transplantation (auto-HSCT). Reducing MRD in grafts is particularly important to improve the efficacy of auto-HSCT. Previously, we reported that ultraviolet light-emitting diode (UV LED) suppressed the expression of Bcl-2 to induce apoptosis in HL-60 cells. Leukemia can lead to severe hypoxia of the bone marrow. Therefore, this study aimed to investigate the effect of UV LED on leukemia cells under hypoxia. HL-60 cells were irradiated with a UV LED (30 J/m2) and simulated under hypoxia with cobalt chloride. We found that UV LED irradiation or CoCl2 inhibited proliferation, induced apoptosis, decreased the Bcl-2/Bax ratio, and increased the levels of caspase 3, cleaved-caspase 3, and caspase 9 in HL-60 cells. In particular, the combined application of UV and CoCl2 significantly enhanced the apoptosis of HL-60 cells. In conclusion, UV LED in hypoxia exacerbated the inhibition of proliferation and induction of apoptosis and necrosis in HL-60 cells via the regulation of caspase 3/9 and the Bcl-2/Bax ratio-dependent pathway. The application of UV LEDs in hypoxia conditions may be a promising approach to kill residual drug-resistant leukemia cells in autologous grafts.

Antitumor activity against human promyelocytic leukemia and in silico studies of some benzoxazines

Cancer is one of the deadliest diseases in the world today, and the incidence of cancer is increasing. Leukemia is a type of blood cancer defined as the uncontrolled proliferation of abnormal leukocytes in the blood and bone marrow. The HL-60 (human promyelocytic leukemia) cell line, derived from a single patient with acute promyelocytic leukemia, provides a unique in vitro model system for studying the cellular and molecular events involved in the proliferation and differentiation of leukemic cells. In this study, antitumor activities on the HL-60 of some of the resynthesized benzoxazine derivatives (BXN-01 and BXN-02) were investigated. The results of in vitro studies obtained were compared a standard drug, etoposide. In vitro results showed that BXN-01 and BXN-02 were found to be extremely effective compared to etoposide (IC50 value: 10 µM) with IC50 values of 5 nM and 25 nM, respectively. Furthermore, molecular docking studies were carried out for preliminary prediction of possible interaction modes between compounds and the active site of the target macromolecules, hTopo IIα, HDAC2, and RXRA. Then, in silico ADME/Tox studies were performed to predict drug-likeness and pharmacokinetic properties of BXN-01 and BXN-02. Communicated by Ramaswamy H. Sarma

Carboxyl Group-Modified Myoglobin Induces TNF-α-Mediated Apoptosis in Leukemia Cells.

Previous studies have shown that chemical modification may increase the activity of proteins or confer novel activity to proteins. Some studies have indicated that myoglobin (Mb) is cytotoxic; however, the underlying mechanisms remain unclear. In this study, we investigated whether chemical modification of the carboxyl group by semicarbazide could promote the Mb cytotoxicity in human leukemia U937 cells and the underlying mechanism of semicarbazide-modified myoglobin (SEM-Mb)-induced U937 cell death. The semicarbazide-modified Mb (SEM-Mb) induced U937 cell apoptosis via the production of cleaved caspase-8 and t-Bid, while silencing of FADD abolished this effect. These findings suggest that SEM-Mb can induce U937 cell death by activating the death receptor-mediated pathway. The SEM-Mb inhibited miR-99a expression, leading to increased NOX4 mRNA and protein expression, which promoted SIRT3 degradation, and, in turn, induced ROS-mediated p38 MAPK phosphorylation. Activated p38 MAPK stimulated miR-29a-dependent tristetraprolin (TTP) mRNA decay. Downregulation of TTP slowed TNF-α mRNA turnover, thereby increasing TNF-α protein expression. The SEM-Mb-induced decrease in cell viability and TNF-α upregulation were alleviated by abrogating the NOX4/SIRT3/ROS/p38 MAPK axis or ectopic expression of TTP. Taken together, our results demonstrated that the NOX4/SIRT3/p38 MAPK/TTP axis induces TNF-α-mediated apoptosis in U937 cells following SEM-Mb treatment. A pathway regulating p38 MAPK-mediated TNF-α expression also explains the cytotoxicity of SEM-Mb in the human leukemia cell lines HL-60, THP-1, K562, Jurkat, and ABT-199-resistant U937. Furthermore, these findings suggest that the carboxyl group-modified Mb is a potential structural template for the generation of tumoricidal proteins.

Ethylenediaminetetraacetic acid (EDTA) enhances cAMP production in human TDAG8-expressing cells

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds tightly to metal ions. We found that cAMP response element (CRE)-driven promoter activity by protons was enhanced by EDTA in human T-cell death-associated gene 8 (TDAG8)-overexpressed HEK293T cells. The enhancing action by EDTA was also detected by proton-induced cAMP production that is located upstream from the CRE-driven promoter activity even at physiological proton concentration pH7.4. The proton-induced CRE-driven promoter activity was not enhanced by other chelating agents, ethylene glycol tetraacetic acid (EGTA) and sodium citrate. The enhanced CRE-driven promoter activity by EDTA was not attenuated by increasing the extracellular calcium ion concentration. These results indicate that the EDTA-enhancing action may not be due to its chelating action but might rather be another EDTA-specific effect. Enhanced cAMP production by EDTA was also detected in a human leukemia cell line HL-60, in which TDAG8 and OGR1 (ovarian cancer G-protein-coupled receptor 1) were endogenously expressed, suggesting that the medical use of EDTA would influence the physiological and pathophysiological functions of hematopoietic cells.

Effects of SIDT2 on the miR-25/NOX4/HuR axis and SIRT3 mRNA stability lead to ROS-mediated TNF-α expression in hydroquinone-treated leukemia cells.

Our previous studies indicated that the benzene metabolite hydroquinone (HQ) evokes the ROS/p38 MAPK/protein phosphatase 2A/tristetraprolin axis, leading to increased TNF-α expression in human acute myeloid leukemia cell lines U937 and HL-60. In this study, we aimed to identify the upstream pathway involved in ROS-mediated TNF-α expression. HQ treatment increased SIDT2 expression, which subsequently decreased miR-25 and SIRT3 expression in U937 cells. Notably, miR-25 downregulation promoted SIDT2 expression in HQ-treated U937 cells. SIDT2 induced lysosomal degradation of SIRT3 mRNA, but inhibited miR-25 expression through a lysosome-independent pathway. MiR-25 inhibition reduced NOX4 mRNA turnover, resulting in increased NOX4 protein levels. NOX4 induces mitochondrial ROS production and HuR downregulation. Restoration of HuR expression increased SIRT3 expression, suggesting that NOX4-mediated HuR downregulation promotes SIDT2-mediated degradation of SIRT3 mRNA. Inhibition of NOX4 or SIRT3 overexpression abolished HQ-induced ROS production, thereby abolishing TNF-α upregulation. Overall, these results indicate that SIDT2 regulates the miR-25/NOX4/HuR axis and SIRT3 mRNA destabilization, leading to ROS-mediated TNF-α upregulation in HQ-treated U937 cells. HQ-induced increase in TNF-α expression in HL-60 cells was also mediated through a similar pathway.