Myeloid Leukemia HL-60 Research Articles

Moojecin: The first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

Disintegrins are a class of peptides found in snake venom that inhibit the activity of integrins, which are essential cell adhesion receptors in tumor progression and development. In this work, moojecin, a RGD disintegrin, was isolated from Bothrops moojeni snake venom, and its antitumor potential in acute myeloid leukemia (AML) HL-60 and THP-1 cells was characterized. The isolation was performed using a C18 reverse-phase column in two chromatographic steps, and its molecular mass is 7417.84 Da. N-terminal and de novo sequencing was performed to identify moojecin. Moojecin did not show cytotoxic or antiproliferative activity in THP-1 and HL-60 at tested concentrations, but it exhibited significant antimigratory activity in both cell lines, as well as inhibition of angiogenesis in the tube formation assay on Matrigel in a dose-dependent manner. A stronger interaction with integrin αVβ3 was shown in integrin interaction assays compared to α5β1, and the platelet aggregation assay indicated an IC50 of 5.039 μg/mL. Preliminary evaluation of disintegrin toxicity revealed no incidence of hemolysis or cytotoxic effects on peripheral blood mononuclear cells (PBMCs) across the tested concentrations. Thus, this is the first study to report the isolation, functional and structural characterization of a disintegrin from B. moojeni venom and bring a new perspective to assist in AML treatment.

Baicalein Promotes Acute Myeloid Leukemia Cell Autophagy via miR-424 and the PTEN/PI3K/AKT/mTOR Pathway

Objective: To explore the autophagic effect of baicalein on acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and miR-424, which regulates the baicalein effect on HL-60 and THP-1 in which autophagy was observed. Methods: The cell counting kit-8 (CCK-8) assay was used to detect the optimal concentration of baicalein in the HL-60 and THP-1 cell lines. miR-424 was detected by qPCR. The influence of baicalein on the autophagy of the HL-60 and THP-1 cells was demonstrated by detecting the expression of Beclin-1, LC3- I, and LC3-II using western blot. The phosphatase and tensin homolog (PTEN)/PI3K/AKt/mTOR pathways were determined by western blot. Results: The optimum concentration of baicalein used and the time of treatment in the HL-60 and THP-1 cell lines were 40 μM and 48 hours, respectively. The expression of miR-424 in the baicalein-treated cells was lower than that in the blank group both in the HL-60 cells and THP-1 cells. The expression of PTEN was promoted by baicalein. However, baicalein inhibited PI3K expression, mTOR phosphorylation, and AKT phosphorylation in the two cell lines. LC3-Ⅰ/Ⅱ, which is the biomarker for autophagy, increased after the cells were treated with baicalein. The baseline expression also increased after the cells were treated with baicalein. Conclusion: Baicalein could promote the autophagy of the HL-60 and THP-1 cells via miR-424 and the PTEN/ PI3K/AKT/mTOR pathway.

Expression and secretion of galectin-12 in the context of neutrophilic differentiation of human promyeloblastic HL-60 cells.

Galectin-12 is a tissue-specific galectin that has been largely defined by its role in the regulation of adipocyte differentiation and lipogenesis. This study aimed to evaluate the role of galectin-12 in the differentiation and polarization of neutrophils within a model of acute myeloid leukemia HL-60 cells. All-trans retinoic acid and dimethyl sulfoxide were used to induce differentiation of HL-60 cells which led to the generation of two phenotypes of neutrophil-like cells with opposite changes in galectin-12 gene (LGALS12) expression and different functional responses to N-formyl- l-methionyl- l-leucyl- l-phenylalanine. These phenotypes showed significant differences of differentially expressed genes on a global scale based on bioinformatics analysis of available Gene Expression Omnibus (GEO) data sets. We also demonstrated that HL-60 cells could secrete and accumulate galectin-12 in cell culture medium under normal growth conditions. This secretion was found to be entirely inhibited upon neutrophilic differentiation and was accompanied by an increase in intracellular lipid droplet content and significant enrichment of 22 lipid gene ontology terms related to lipid metabolism in differentiated cells. These findings suggest that galectin-12 could serve as a marker of neutrophilic plasticity or polarization into different phenotypes and that galectin-12 secretion may be influenced by lipid droplet biogenesis.

Cytotoxic depsidones and xanthones from Garcinia esculenta Y. H. Li

Six new compounds, including two depsidones garciculendepsidones A and B (1 and 2), one prenylated xanthone garciculenxanthone (3) and three dimeric xanthones bigarciculenxanthones A–C (4–6), were isolated from the twigs and leaves of Garcinia esculenta Y. H. Li. Their structures were elucidated based on comprehensive analyses of spectral data, including HRESIMS, 1D and 2D NMR, and ECD calculation. All the isolates were tested for their cytotoxicity against five human cancer cell lines (myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480), among them, compounds 3–5 displayed cytotoxic potential, especially garciculenxanthone (3) had the lowest IC50 value of 8.2 μm for lung cancer A-549 cells.

In silico and In vitro Determination of Antiproliferative Activity of Series N-Pyrrolyl Hydrazide-Hydrazones and Evaluation of their Effects on Isolated Rat Mycrosomes and Hepatocytes.

The significant increase in patients suffering from different types of cancer, guides scientists to take prompt measures in the development of novel and effective antiproliferative agents, where the intercalation of heterocyclic fragments in the designed molecules has proven to be a useful practice. The newly synthesized compounds were obtained from the corresponding 1,4-dicarbonyl derivative through multicomponent reactions to produce biologically active target molecules and assessed by in silico and in vitro assays for their possible antitumor activity. The pharmacological bioassay was conducted in the panel of human tumor cell lines (i) SKW-3 (ACC 53) - human T-cell leukemia and (ii) HL-60 (ACC 3) - human acute myeloid leukemia (AML). The statistical processing of MTT data included the paired Student's t-test with p ≤ 0.05 set as significance level. All evaluated structures displayed a higher cytotoxic effect against the acute myeloid leukemia HL-60 with 11o and 11p as the most active compared to the activity against SKW-3 cell line. Throughout the cytotoxicity screening two molecules, 11l and 12o, displayed comparable chemosensitivity on both cell lines. The corresponding hepatotoxicity on isolated rat hepatocytes and microsomes was also established, identifying 11, 12 and 12a as the least toxic and 11x, 11d, 12x and 12d as the most toxic derivatives. As the most promising compound is underlined ethyl 1-(2-(2-((1-acetyl-1H-indol-3-yl)methylene)hydrazinyl)-2- oxoethyl)-5-(4-bromophenyl)-2-methyl-1H-pyrrole-3-carboxylate (11l) demonstrating highest activity on both evaluated tumor cell lines, decreased hepatotoxicity on all evaluated parameters and docking score of -7.517 kcal/mol.

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.

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.

Anti-leukemic activity of Cyperus rotundus L. on human acute myeloid leukemia HL-60 cells in vitro

Context: Acute myeloid leukemia (AML) is the most common form of acute leukemia. Currently, many people use medicinal herbs for the treatment of cancers. Nut grass (Cyperus rotundus L.) is a medicinal plant widely used in conventional medicine due to its role as an anti-cancer. Aims: To evaluate the effect of Cyperus rotundus tuber (CRT) ethanolic extract on cell proliferation, differentiation, cell cycle, and apoptotic of HL-60 cells. Methods: HL-60 cells line as a model for AML was cultured under the influence of CRT extract concentrations (35.4, 354, and 3540 µg/mL) for 48 h. Furthermore, the cell was subjected to carboxyfluorescein succinimidyl ester staining for proliferation, using a CD117 marker for differentiation. Annexin V-FITC and PI for cell cycle and apoptosis. The effect of this herb was studied by flow cytometry. The data were statistically analyzed with one-way ANOVA (p≤0.05) and the Tukey test using SPSS version 16 for Windows. Results: The results showed that the CRT inhibited proliferation activity, differentiation, cell cycle arrest, and apoptosis. Conclusions: The data clearly showed the potential anti-cancer activity of CRT on HL-60 cells and suggested that it could help develop promising therapeutic agents for AML treatments.

Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells

A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.

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]. 
 
 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. 
 
 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<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.
 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.
 
 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.

Overexpressed fibroblast growth factor receptors increase 1,25-dihydroxyvitamin D-dependent differentiation of acute myeloid leukemia cells

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2–FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1–3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1–3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D-induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation.

Gallium (III) Complexes with 5-Bromosalicylaldehyde Benzoylhydrazones: In Silico Studies and In Vitro Cytotoxic Activity.

Gallium (III) complexes with the ligands 5-bromosalicylaldehyde-4-hydroxybenzoylhydrazone and 5-bromosalicylaldehyde isonicotinoylhydrazone were synthesized to receive compounds with improved antiproliferative action. Compounds were characterized by elemental analysis, IR, and NMR spectroscopy. Density functional theory calculations with Becke’s 3-parameter hybrid functional and 6-31+G(d,p) basis set were carried out to investigate the structural features of the ligands and Ga(III) complexes. Cytotoxic screening by MTT-dye reduction assay was carried out using cisplatin and melphalan as reference cytotoxic agents. A general formula [Ga(HL)2]NO3 for the complexes obtained was suggested. The complexes are mononuclear with the Ga(III) ions being surrounded by two ligands. The ligands acted as monoanionic tridentate (ONO) donor molecules. The analysis revealed coordination binding through deprotonated phenolic-oxygen, azomethine-nitrogen, and amide-oxygen atoms. The bioassay demonstrated that all compounds exhibited concentration-dependent antiproliferative activity at low micromolar concentrations against the acute myeloid leukemia HL-60 and T-cell leukemia SKW-3 cell lines. IC50 values of 5-bromo-derivative ligands and gallium (III) complexes are lower than those of cisplatin and much lower than these of melphalan. The coordination to gallium (III) additionally increased the cytotoxicity compared to the metal-free hydrazones.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term “Myc active pathway” according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed “MYC_TARGETS_V2” with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as “TNFA_SIGNALING_VIA_NFKB”, “APOPTOSIS”, and “TGF_BETA_SIGNALING” were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

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.

Molecular Mechanism Investigation on Monomer Kaempferol of the Traditional Medicine Dingqing Tablet in Promoting Apoptosis of Acute Myeloid Leukemia HL-60 Cells.

The traditional medicine Dingqing Tablet produces effective efficacy in treating acute myeloid leukemia, but its specific mechanism remains to be investigated. Dingqing Tablet consists of Codonopsis, Indigo Naturalis, Cortex Moutan, Radix Notoginseng, Citrus Reticulata, and Eolite. The active components of Dingqing Tablets were screened by the TCMSP database. Meanwhile, the SwissTargetPrediction database was utilized to predict the corresponding targets. Relevant disease targets of acute myeloid leukemia were obtained from GeneCards. The obtained targets of Dingqing Tablets and genes of acute myeloid leukemia were used, and the overlapped genes were presented in the Venn diagram. A drug-component-target network was constructed via Cytoscape 3.6.0 software. Molecular docking methodology was also used with AutoDock Vina 1.1.2. Furthermore, the effects of kaempferol on the proliferation and apoptosis of HL-60 cells were identified using 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), 5-Ethynyl-2′-deoxyuridine (EDU), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assays. The combination of kaempferol and AKT1 was verified using an immunoprecipitation (IP) experiment and the effects of Kaempferol on HL-60 cell apoptosis by western blot (WB) and qPCR. The key component kaempferol and the core target gene AKT1 were sorted out using a drug-component target network diagram. Molecular docking results revealed that the binding energy between kaempferol and AKT1 was lower than -5 kcal/mol. MTT and EDU assays indicated that kaempferol markedly inhibited the proliferation of HL-60 cells. Flow cytometry and TUNEL assays suggested that kaempferol substantially promoted HL-60 cell apoptosis. IP assay results testified that kaempferol could bind to AKT1, thereby reducing the level of P-AKT and promoting HL-60 cell apoptosis. The monomer kaempferol of Dingqing Tablet could promote apoptosis of HL-60 cells, and the mechanism might correlate with the combination of kaempferol and AKT1, reducing the level of P-AKT and promoting the expression of the apoptotic signaling pathway.

Grape seed proanthocyanidin extract induces apoptosis of HL-60/ADR cells via the Bax/Bcl-2 caspase-3/9 signaling pathway.

BackgroundOur previous study detailed the direct induction of apoptosis by grape seed proanthocyanidin extract (GSPE) in a multidrug resistant human acute myeloid leukemia (AML) HL-60/adriamycin (HL-60/ADR) cell line, although the mechanism of this effect was not detailed. This study aims to elucidate the mechanism underlying GSPE-induced cell apoptosis in HL-60/ADR cells.MethodsHL-60/ADR cells were studied to evaluate effects of GSPE (0–100 µg/mL); a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to identify the cytotoxic effect of varying GSPE concentrations. Trypan blue staining was used to observe changes in cell viability; flow cytometry assays were used to verify apoptosis. Expression of Bax and Bcl-2 mRNA was analyzed using real-time polymerase chain reaction (PCR). Activity of caspase-3 and caspase-9 was also detected.ResultsHere, GSPE was found to inhibit HL-60/ADR cell growth and induce cell apoptosis in a dose-dependent manner. Real-time PCR findings revealed that GSPE concentrations above 75 µg/mL significantly increase expression of Bax mRNA (P<0.001). GSPE concentrations above 25 µg/mL were found to significantly decrease expression of Bcl-2 mRNA (P<0.01), while concentrations above 50 µg/mL were found to significantly increase caspase-3 activity after 6, 12 and 24 h (P<0.01). However, only 100 µg/mL GSPE was found to significantly increase caspase-9 activity (P<0.001 at 6 and 12 h; P<0.05 at 24 h).ConclusionsGSPE inhibits the proliferation of HL-60/ADR cells by the induction of apoptosis in a dose-dependent manner via the Bax/Bcl-2 caspase-3/9 signaling pathway.

Tingenone and 22-hydroxytingenone target oxidative stress through downregulation of thioredoxin, leading to DNA double-strand break and JNK/p38-mediated apoptosis in acute myeloid leukemia HL-60 cells

Acute myeloid leukemia (AML) is the most lethal form of leukemia. Standard anti-AML treatment remains almost unchanged for decades. Tingenone (TG) and 22-hydroxytingenone (22-HTG) are quinonemethide triterpenes found in the Amazonian plant Salacia impressifolia (Celastraceae), with cytotoxic properties in different histological types of cancer cells. In the present work, we investigated the anti-AML action mechanism of TG and 22-HTG in the AML HL-60 cell line. Both compounds exhibited potent cytotoxicity in a panel of cancer cell lines. Mechanistic studies found that TG and 22-HTG reduced cell growth and caused the externalization of phosphatidylserine, the fragmentation of internucleosomal DNA and the loss of mitochondrial transmembrane potential in HL-60 cells. In addition, pre-incubation with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, prevented TG- and 22-HTG-induced apoptosis, indicating cell death by apoptosis via a caspase-dependent pathway. The analysis of the RNA transcripts of several genes indicated the interruption of the cellular antioxidant system, including the downregulation of thioredoxin, as a target for TG and 22-HTG. The application of N-acetyl-cysteine, an antioxidant, completely prevented apoptosis induced by TG and 22-HTG, indicating activation of the apoptosis pathway mediated by oxidative stress. Moreover, TG and 22-HTG induced DNA double-strand break and phosphorylation of JNK2 (T183/Y185) and p38α (T180/Y182), and co-incubation with SP 600125 (JNK/SAPK inhibitor) and PD 169316 (p38 MAPK inhibitor) partially prevented apoptosis induced by TG and 22-HTG. Together, these data indicate that TG and 22-HTG are new candidate for anti-AML therapy targeting thioredoxin.

ZEB2 Knock-down Induces Apoptosis in Human Myeloid Leukemia HL-60 Cells.

Acute myeloid leukemia (AML) is the most prevalent type of cancer in the adult hematopoietic system. Conventional therapies are associated with unfavorable side effects in individuals diagnosed with AML. These after-effects with partial remission reflect the urgent need for novel therapeutic approaches for inducing apoptosis, specifically in malignant cells, without affecting other cells. As a transcription factor (TF), ZEB2 (Zinc Finger E-Box Binding Homeobox 2) regulates the expression of specific genes in normal conditions. However, increased expression of ZEB2 is reported in various cancers, especially in AML, which is related to a higher degree of apoptosis inhibition of malignant cells. In this work, the role of ZEB2 in apoptosis inhibition is surveyed through ZEB2 specific knocking-down in human myeloid leukemia HL-60 cells. Transfection of HL-60 cells was conducted using ZEB2-siRNA at concentrations of 20, 40, 60, and 80 pmol within 24, 48, and 72 h. After determining the optimum dose and time, flow cytometry was used to measure the apoptosis rate. The MTT assay was also utilized to evaluate the cytotoxic impact of transfection on the cells. The expression of candidate genes was measured before and after transfection using qRT-PCR. According to obtained results, suppression of ZEB2 expression through siRNA was associated with the induction of apoptosis, increased pro-apoptotic, and decreased anti-apoptotic gene expression. Transfection of ZEB2-siRNA was also associated with reduced cell proliferation and viability. Our study results suggest that ZEB2 suppression in myeloid leukemia cells through apoptosis induction could be a proper therapeutic method.

Xanthohumol Induces ROS through NADPH Oxidase, Causes Cell Cycle Arrest and Apoptosis.

Reactive oxygen species (ROS) are either toxic in excess or essential for redox signalling at the physiological level, which is closely related to the site of generation. Xanthohumol (XN) is an important natural product of hops (Humulus lupulus L.) and was reported to induce ROS in mitochondria. While in the present study, our data indicate that NADPH oxidase (NOX) is another site. In human acute myeloid leukemia HL-60 cells, we first identified that cell proliferation was inhibited by XN without affecting viability, and this could be alleviated by the antioxidant N-acetyl-L-cysteine (NAC); cell cycles were blocked at G1 phase, apoptosis was induced in a dose-dependent manner, and malondialdehyde (MDA) content was upregulated. XN-induced ROS generation was detected by flow cytometry, which can be inhibited by diphenyleneiodonium chloride (DPI, a NOX inhibitor), while not by NG-methyl-L-arginine acetate (L-NMMA, a nitric oxide synthase inhibitor). The involvement of NOX in XN-induced ROS generation was further evaluated: immunofluorescence assay indicated subunits assembled in the membrane, and gp91phox knockdown with siRNA decreased XN-induced ROS. Human red blood cells (with NOX, without mitochondria) were further selected as a cell model, and the XN-induced ROS and DPI inhibiting effects were found again. In conclusion, our results indicate that XN exhibits antiproliferation effects through ROS-related mechanisms, and NOX is a source of XN-induced ROS. As NOX-sourced ROS are critical for phagocytosis, our findings may contribute to the anti-infection and anti-inflammatory effect of XN.

L, D-Polydeoxyribonucleotides to provide an essential inhibitory effect on DNA polymerase β of human myeloid leukemia HL60 cells

The inhibitory effect of D and L-polynucleotides of a given length (40-50n) on the catalytic activity of DNA polymerase β isolated from chromatin cells of acute myeloid leukemia HL-60 was evaluated. The synthesized L enantiomer was found to have a higher inhibitory activity than the synthesized and isolated D enantiomers of polynucleotides. The work also proposes a biophysical model that describes this effect.