Human HL-60 Research Articles

Structures and Biologic Activity of Chitonoidosides I, J, K, K1 and L-Triterpene Di-, Tri- and Tetrasulfated Hexaosides from the Sea Cucumber Psolus chitonoides.

Five new triterpene di-, tri- and tetrasulfated hexaosides (chitonoidosides I (1), J (2), K (3), K1 (4) and L (5)) were isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from a depth of 100–150 m. The structural variability of the glycosides concerned both the aglycones (with 7(8)- or 9(11)-double bonds) and carbohydrate chains differing from each other by the third sugar residue (Xyl or sulfated by C-6 Glc) and/or by the fourth—terminal in the bottom semi-chain—residue (Glc or sulfated by C-6 MeGlc) as well as by the positions of a sulfate group at C-4 or C-6 in the sixth—terminal in the upper semi-chain—residue (MeGlc). Hemolytic activities of these compounds 1–5 against human erythrocytes as well as cytotoxicity against human cancer cell lines, HeLa, DLD-1 and HL-60, were studied. The hexaosides, chitonoidosides K (3) and L (5) with four sulfate groups, were the most active against tumor cells in all the tests. Noticeably, the sulfate group at C-4 of MeGlc6 did not decrease the membranolytic effect of 5 as compared with 3, having the sulfate group at C-6 of MeGlc6. Erythrocytes were, as usual, more sensitive to the action of the studied glycosides than cancer cells, although the sensitivity of leukemia promyeloblast HL-60 cells was higher than that of other tumor cells. The glycosides 1 and 2 demonstrated some weaker action in relation to DLD-1 cells than against other tumor cell lines. Chitonoidoside K1 (4) with a hydroxyl at C 25 of the aglycone was not active in all the tests. The metabolic network formed by the carbohydrate chains of all the glycosides isolated from P. chitonoides as well as the aglycones biosynthetic transformations during their biosynthesis are discussed and illustrated with schemes.

Formulation of a novel anti-leukemia drug and evaluation of its therapeutic effects in comparison with Cytarabine

In the recent research, we investigated the application of gold nanoparticles green-synthesized by Alhagi maurorum aqueous extract in the treatment of several types of leukemia, i.e. acute T cell leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia. Different techniques such as transmission electron microscopy (TEM), fourier-transform infrared spectroscopy (FTIR), and ultraviolet–visible spectroscopy analysis were used to characterize AuNPs. The TEM images show a spherical morphology for AuNPs with the range size of 21 to 59 for the synthetic nanoparticles. In the antioxidant test, the IC50 of AuNPs and butylated hydroxytoluene (BHT) against DPPH free radicals were 117 and 87 µg/mL, respectively. In the oncological part of the recent study, the treated cells with AuNPs and Cytarabine were assessed by MTT assay for 48 h about the cytotoxicity and anti-leukemia properties on normal (HUVEC) and leukemia cell lines i.e., acute myeloid leukemia (Human HL-60/vcr and 32D-FLT3-ITD), acute lymphoblastic leukemia (MOLT-3 and TALL-104), and acute T cell leukemia (J.RT3-T3.5 and Jurkat, Clone E6-1). The IC50 of AuNPs were 242, 297, 383, 207, 234, and 218 µg/mL against acute myeloid leukemia (Human HL-60/vcr and 32D-FLT3-ITD), acute lymphoblastic leukemia (MOLT-3 and TALL-104), and acute T cell leukemia (J.RT3-T3.5 and Jurkat, Clone E6-1) cell lines, respectively. In addition, the IC50 of Cytarabine were 117, 113, 145, 119, 131, and 135 µg/mL against acute myeloid leukemia (Human HL-60/vcr and 32D-FLT3-ITD), acute lymphoblastic leukemia (MOLT-3 and TALL-104), and acute T cell leukemia (J.RT3-T3.5 and Jurkat, Clone E6-1) cell lines, respectively. The viability of malignant leukemia cell line reduced dose-dependently in the presence of AuNPs and Cytarabine.

Molecular mechanisms of aberrant neutrophil differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: (i) abnormal lipid metabolism causing a delayed metabolic reprogramming and (ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

Two new α-pyrone-containing polyketides isolated from the fungus Aspergillus aureoterreus

Two undescribed α-pyrone-containing polyketide derivatives designated aurovertins V (1) and W (2), and a known analogue (3), were isolated from the fungus Aspergillus aureoterreus. Their structures including the absolute configuration were elucidated on the basis of extensive spectroscopic methods and theoretical ECD calculation. Compound 1 is the first example of aurovertins with a 7R configuration, whereas 2 comprises a S configuration for C-6 and a Z geometry of the double bond Δ8. Both 1 and 2 showed no cytotoxicity against human cancer cell lines HL-60, SU-DHL-2 and U266) at the concentration of 20.0 μM.

Isolation and Structure Elucidation of New Cytotoxic Macrolides Halosmysins B and C from the Fungus Halosphaeriaceae sp. Associated with a Marine Alga.

Two new cytotoxic metabolites, halosmysins B and C, have been isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4 separated from the marine alga Sargassum thunbergii. These chemical structures have been elucidated by 1D and 2D NMR, and HRFABMS spectral analyses. The new compounds had the same 14-membered macrodiolide skeleton as halosmysin A, which was isolated from this fungal strain previously. As the unique structural feature, a diketopiperazine derivative and a sugar are conjugated to the 14-membered ring of halosmysins B and C, respectively. The absolute stereostructures of them were elucidated by the chemical derivatization such as a hydrolysis, the comparison with the known compounds (6R,11R,12R,14R)-colletodiol and halosmysin A, and a HPLC analysis of sugar. In addition, their cytotoxicities were assessed using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. Halosmysin B was shown to be potent against all of them, with IC50 values ranging from 8.2 ± 1.8 to 20.5 ± 3.6 μM, though these values were slightly higher than those of halosmysin A.

Guaiane-type sesquiterpenes from Curcumawenyujin

Eight undescribed sesquiterpenes including seven guaianes and one pseudoguaiane which were named as wenyujinols A-H, along with ten known guaianes, were isolated from rhizomes of Curcuma wenyujin Y. H. Chen et C. Ling. The structures of wenyujinols A-H were elucidated by 1D and 2D nuclear magnetic resonance (NMR) data, high resolution mass spectrum (HRMS), electronic circular dichroism (ECD) spectra, and X-ray single crystallographic analysis. All of the isolated compounds were evaluated for antioxidant activity via activation of the Nrf2-ARE pathway in human embryonic kidney (HEK) 293 cells, for inhibitory effects on NO production in RAW 264.7 cells, and for cytotoxicity against three human cancer cell lines A549, HL60, and MCF7 in vitro. The results indicated that procurcumenol (50–200 μM) and 9-oxo-neoprocurcumenol (25–200 μM) exhibited antioxidant activity via activation of the Nrf2-ARE pathway in a dose-dependent manner.

Phorneroids A–M, diverse types of diterpenoids from Euphorbia neriifolia

A chemical investigation on the aerial parts of Euphorbia neriifolia led to the identification of thirteen undescribed diterpenoids, phorneroids A–M, including ent-abietane (A–D), ent-kaurane (E–G), ent-atisane (H–K), and ent-isopimarane (L and M) types, together with three known compounds. Phorneroid A represents the first example of 8-spiro-fused 9,10-seco-ent-abietane diterpenoid lactone featuring a unique 6/5/6/5 spirocyclic framework. Biological assays showed that some of the compounds displayed moderate cytotoxicity against two human tumor cell lines, A549 and HL-60.

Nine new sesquiterpenes from Curcuma wenyujin rhizomes

Nine new sesquiterpenes wenyujinones A-I (1–9), along with ten known ones (10–19), were isolated from the rhizomes of Curcuma wenyujin Y. H. Chen et C. Ling (C. wenyujin). The chemical structures of compounds 1–9 were elucidated by NMR spectroscopic and mass spectrometric data, electronic circular dichroism (ECD) spectra analysis. Furthermore, all compounds were evaluated for the protective effects against hydrogen peroxide (H2O2)-induced injury in human hepatic L02 cells, for the inhibitory effects on nitric oxide (NO) production in RAW 264.7 cells, and for their cytotoxicity against three human cancer cell lines A549, HL60, and MCF7 in vitro. As a result, compounds 2, 4, 14 markedly weakened the oxidative damage induced by H2O2 in L02 cells via strengthening the cell viability.

Prenylated cyclohexene-type meroterpenoids and sulfur-containing xanthones produced by Pseudopestalotiopsis theae

Chemical investigation of the fungal endophyte Pseudopestalotiopsis theae isolated from leaves of Caloncoba welwitschii, collected in Cameroon, resulted in two previously undescribed sulfur-containing xanthone derivatives sydoxanthones D and E, in addition to three previously undescribed monomeric diisoprenyl-cyclohexene-type meroterpenoids biscognienynes D-F and five known natural products. The structures of the undescribed compounds were unambiguously identified by their mass spectra and by extensive 1D and 2D NMR spectroscopic analysis. Mosher's reaction was performed to determine the absolute configuration of sydoxanthones D and E while TDDFT-ECD calculations were used to assign the configuration of biscognienyne D. Biscognienynes B and D showed significant cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values of 7.7 and 6.7 μM and against the human leukemic cell lines HL60, and Hal-01 with IC50 values ranging from 4.3 to 12.1 μM.

Acetylshikonin induces autophagy-dependent apoptosis through the key LKB1-AMPK and PI3K/Akt-regulated mTOR signalling pathways in HL-60 cells.

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.

Anti-plasmodial, Cytotoxic and Antioxidant Activities of Selected Ghanaian Medicinal Plants.

Malaria affects about half of the world's population. The sub-Saharan African region is the most affected. Plant natural products have been a major source of antimalarial drugs; the first (quinine) and present (artemisinin) antimalarials are of natural product origin. Some secondary metabolites demonstrate adjuvant antioxidant effects and selective activity. The focus of this study was to investigate the anti-plasmodial activity, cytotoxicities and antioxidant properties of eight (8) Ghanaian medicinal plants. The anti-plasmodial activity was determined using the SYBR green assay and the tetrazolium-based colorimetric assay (MTT) was employed to assess cytotoxicity of extracts to human RBCs and HL-60 cells. Antioxidant potential of plant extracts was evaluated using Folin-Ciocalteu and superoxide dismutase assays. Phytochemical contstituents of the plant extracts were also assessed. All the extracts demonstrated anti-plasmodial activities at concentrations <50 μg/ml. Parkia clappertoniana and Terminalia ivorensis elicited the strongest anti-plasmodial activities with 50% inhibitory concentrations (IC50) of 1.13 μg/ml and 0.95 μg/ml, respectively. This is the first report on anti-plasmodial activities of Baphia nitida, Tabernaemontana crassa and Treculia Africana. T. Africana showed moderate anti-plasmodial activity with IC50 value of 6.62 µg/mL. Extracts of P. clappertoniana, T. Africana and T. ivorensis (0.4 mg/mL) showed >50% antioxidant effect (SOD). The extracts were not cytotoxicity towards RBCs at the concentration tested (200 μg/ml) but were weakly cytotoxic to HL-60 cell. Selectivity indices of most of the extracts were greater than 10. Our results suggest that most of the plant extracts have strong anti-plasmodial activity and antioxidant activity which warrants further investigations.

Tanshinone IIA Induces Apoptosis of Leukemia Cancer Cells and Inhibits Tumor Growth In Vivo Through Mitochondrial Pathway

To investigate the effect of Tanshinone IIA on apoptosis of leukemic cancer cells and tumor growth inhibition in vivo. Human leukemia cell line HL-60 cells were divided into model group, low-dose group, medium dose group and high-dose group and transfected according to the groups. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide method was used to calculate the inhibition rate of cell growth, flow cytometry was used to detect the apoptosis rate and mitochondrial membrane potential and Western blot was used to detect mitochondrial membrane potential related protein. The treatment group was treated with Tanshinone IIA and the control group was given phosphate-buffered saline+dimethyl sulfoxide intraperitoneal injection. 2 w later, the tumor size and weight were measured and weighed. The 24 h and 48 h proliferation inhibition rates of the experimental group were significantly higher than those of the model group and increased with the increase of the dose (p<0.05). The early apoptosis rate and late apoptosis rate of the experimental group were significantly higher than those of the model group and increased with the increase of the dose (p<0.05). The level of integral optical density in the experimental group was significantly lower than that in the model group and decreased with the increase of the dose (p<0.05). The levels of caspase-9, caspase-3 and B-cell lymphoma 2 related X protein in the experimental group were significantly higher than those in the model group, while the level of B-cell lymphoma 2 in the experimental group was significantly lower than that in the model group and there was drug dependence (p<0.05). The tumor size and weight of the treatment group were significantly lower than those of the control group (p<0.05). Tanshinone IIA can induce apoptosis and tumor growth of leukemia cancer cells in vivo and slow down the proliferation of cancer cells, which may be related to the regulation of mitochondrial pathway and the inhibition of mitochondrial membrane potential related protein.

Decoding the Mechanism of Shen Qi Sha Bai Decoction in Treating Acute Myeloid Leukemia Based on Network Pharmacology and Molecular Docking

Traditional Chinese Medicine (TCM) has been shown to be efficacious in treating leukemia for thousands of years. It has been shown that Shen Qi Sha Bai Decoction (SQSBD) has been extensively used in the treatment of acute myeloid leukemia (AML). However, the mechanism of SQSBD in treating AML remains unclear. In this study, we employed network pharmacology to analyze the potential active components and elucidate molecular mechanism of SQSBD in treating AML. A total of 268 active components were identified from SQSBD, among which 9 key components (Quercetin, luteolin, kaempferol, licochalcone A, formononetin, wogonin, β-sitosterol, oroxylin A, naringenin, and baicalein) were hit by the 6 hub targets (CDK1, MAPK1, JUN, PCNA, HSB1, STAT3) associated with leukemia. Molecular docking showed that two core active components, quercetin and licochalcone A, exhibited the highest component-like properties (DL), and could bind well to CDK1 and MAPK1 protein. The experimental validation of these two components showed that quercetin inhibited cell growth through CDK1 dephosphorylation-mediated cell cycle arrest at G2/M phase in human AML U937 and HL60 cells, and licochalcone A induced cell differentiation in these leukemia cells via activation of MAPK1 and upregulation of CD11b. All these results indicate that SQSBD is effective in the treatment of AML, and quercetin and licochalcone A are the major candidate compounds for AML treatment.

RBM38 Mediates the Proliferation of Acute Myeloid Leukemia Cells HL-60 by Regulating FZD1 mRNA Stability

To explore the regulatory function of RNA binding motif protein 38 (RBM38) in human acute myeloid leukemia cells HL-60 and its mechanism. The lentivirus carriers of overexpressed and knockdown RBM38 were constructed. After HL-60 cells were transfected, Western blot was used to analyze the expression level of RBM38 in HL-60 cells. The cell proliferation and cycle of HL-60 were detected by CCK-8 assay and flow cytometry assay, respectively. RNA immunoprecipitation coupled real-time PCR (RIP-qPCR) was used to detect the combination of RBM38 with mRNAs. Actinomycin D treatment followed by real-time PCR (AcD-qPCR) was used to detect the effect of RBM38 on the stability of target mRNAs. RBM38 in HL-60 cells was overexpressed or inhibited by lentivirus transduction. Overexpressed RBM38 promoted the cell cycle and proliferation of HL-60, while RBM38 knockdown repressed the two processes. RBM38 showed an interaction with FZD1 mRNA and enhancement of its stability. RBM38 can regulate cell proliferation of HL-60 by improving the stability of FZD1 mRNA.

Antitumor and apoptotic effects of new-generation platinum compounds on human leukemia cell lines HL-60 and K562

Antitumor and apoptotic effects of new-generation platinum compounds on human leukemia cell lines HL-60 and K562

Acorenone C: A New Spiro-Sesquiterpene from a Mangrove-Associated Fungus, Pseudofusicoccum sp. J003.

Mangrove-derived endophytes are rich in bioactive secondary metabolites with a variety of biological activities. Recently, a fungus Pseudofusicoccum sp. J003 was first isolated by our research group from mangrove species Sonneratia apetala Buch.-Ham. The subsequent chemical investigation of the methanol extract of the culture broth of this strain has led to the isolation of a new sesquiterpenoid named acorenone C (1), two alkaloids (2–3), four phenolic compounds (4–7), and four steroid derivatives (8–11). The new structure of 1 was established by extensive spectroscopic analysis, including 1D, 2D NMR spectroscopy, and HRESIMS. Its absolute configuration was elucidated by experimental ECD and ECD calculation. The in vitro AChE inhibitory, anti-inflammatory, and cytotoxic activities of the selected compounds were evaluated. The results showed that compound 1 showed mild AChE inhibitory activity, with an inhibition rate of 23.34% at the concentration of 50 μM. Compound 9 exerted a significant inhibitory effect against nitric oxide (NO) production in LPS-stimulated RAW 264.7 mouse macrophages, with an inhibition rate of 72.89% at the concentration of 25 μM, better than that of positive control L-NMMA. Compound 9 also displayed obvious inhibition effects on the growth of two human tumor cell lines, HL-60 and SW480 (inhibition rates 98.68 ± 0.97% and 60.40 ± 4.51%, respectively). The antimicrobial activities of the compounds (1–11) against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were also tested; however, none of them showed antimicrobial activities.

N-Succinyl-S-Farnesyl-L-Cysteine (SFC): A Novel Isoprenylcysteine Analog with In Vitro Anti-Inflammatory Activity and Clinical Skin Protecting Properties

Over the past 15 years, small molecule isoprenylcysteine (IPC) analogs have been identified as a potential new class of topical anti-inflammatories. Clinical studies have demonstrated that IPCs are both safe and effective in promoting healthy skin when applied topically. This work aims to demonstrate N-Succinyl-S-farnesyl-L-cysteine (SFC) as a novel IPC molecule that provides a broad spectrum of benefits for skin. Human promyelocytic cell line HL-60, human dermal microvascular endothelial cells (HDMECs), human dermal fibroblasts (HDFs), and normal human epidermal keratinocytes (NHEKs) were exposed in culture to various inducers to trigger reactive oxygen species, cytokines, or collagenase production. A 49-subject randomized double-blind, vehicle-controlled, split face trial was performed with 1% SFC gel, or 5% niacinamide and vehicle applied for 12 weeks to evaluate anti-wrinkle and anti-aging endpoints. We demonstrated that SFC inhibited GPCR and TLR-induced pro-inflammatory cytokine release in NHEKs and HDMECs from several inflammatory inducers such as UVB, chemicals, cathelicidin, and bacteria. SFC successfully reduced GPCR-induced oxidation in differentiated neutrophils. Moreover, photoaging studies showed that SFC reduced UVA-induced collagenase (pro-MMP-1) production in HDFs. Clinical assessment of 1% SFC gel demonstrated improvement above the vehicle for wrinkle reduction, hydration, texture, and overall appearance of skin. N-Succinyl-S-farnesyl-L-cysteine (SFC) is a novel anti-inflammatory small molecule and is the first farnesyl-cysteine IPC shown to clinically improve appearance and signs of aging, while also having the potential to ameliorate inflammatory skin disorders.

Multioxidized aromatic polyketides produced by a soil-derived fungus Penicillium canescens

Under OSMAC strategy, seven unreported multioxidized aromatic polyketides, penicanesins A‒G, were discovered from a soil-derived fungus Penicillium canescens along with seven known compounds. Their structures were assigned by extensive 1D and 2D NMR spectra in combination with HRESIMS and single crystal X-ray diffraction. Absolute stereochemistry of penicanesins A and D were elucidated by theoretical ECD calculation. (±)-Penicanesins A and B are two pairs of racemic aromatic polyketides with an unusual 6/6/6/6 heterotetracyclic ring core. In bioassay, (−)-penicanesin A shows potential cytotoxicity against human cancer cell lines HL-60 and SW480 with IC50 values at 13.8 ± 0.6 and 12.5 ± 0.9 μM, respectively, whereas the enantiomer (+)-penicanesin A is less active.

In Vitro Cytotoxicity and Clinical Correlates of MRSA Bacteremia.

Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections are associated with significant morbidity and mortality. MRSA secretes a number of virulence factors and pore-forming toxins that enable tissue invasion. Prior studies have found associations between decreased toxin production and poor outcomes in invasive MRSA infection, particularly in pneumonia. In this retrospective observational cohort study of MRSA bacteremia in adult patients from 2007 to 2015, we examined whether cytotoxicity was associated with 30-day mortality. Isolates were obtained from 776 patients and screened for cytotoxicity in a human HL-60 cell model, antimicrobial susceptibility, and spa type, and clinical data were abstracted from charts. We did not find an association between low cytotoxic activity and 30-day mortality in univariate logistic regression analyses. There was a difference in distribution of the genotypes across cytotoxicity phenotypes, with spa-CC008 accounting for a larger proportion of isolates in the high cytotoxicity group. Isolates with a skin and soft tissue primary infective site had a higher median cytotoxicity. There was no association between cytotoxicity and host factors such as age or comorbidity burden. The isolates in our study came from heterogeneous primary sites of infection and were predominantly from spa-CC002 and spa-CC008 lineages, so it is possible that findings in prior studies reflect a different distribution in genotypes and clinical syndromes. Overall, in this large study of cytotoxicity of MRSA bloodstream isolates, we did not find the low cytotoxicity phenotype to be predictive of poor outcomes in MRSA bacteremia.

Using NK-Derived Exosomes to Treat Leukemia

Natural killer cells (NK) are components of the innate immune system which play an important role in surveillance of cancer cells. Despite promising results in clinical trials, the use of NK-based therapies is limited due to safety issues. Exosomes are nanosized extracellular vesicles secreted by all types of cells. In recent years, exosomes have emerged as a powerful, natural therapeutic tool due to their low immunogenicity, low cytotoxicity, prolonged bioavailability and superior targeting ability. Since exosomes are known to carry cargo that reflects their cell of origin, we were prompted to test whether NK-derived exosomes (NK exo) maintain the anti-leukemia capacity of NK cells. To test the feasibility of this approach we undertook a set of preliminary experiments and evaluated the anti-leukemic potential of NK exo in-vitro on a wide variety of leukemic cell lines and patient-derived samples while also striving to assess their killing potential in-vivo using a murine xenograft model . This study aims to provide the pre-clinical evidence needed to test the NKexo approach in future clinical studies with the intention of ultimately developing an acellular “off-the-shelf” product to treat leukemia.Initially, we isolated NK exo from the engineered NK92MI cell line by ultracentrifugation and demonstrated the presence of large amounts of 100-200nm cap-shaped particles by means of nano-tracking-analysis and transmission-electron-microscopy. We then showed that these particles express the exosomal-characteristic CD63- and CD81- and NK-characteristic CD56-biomarkers. Next, we evaluated the effect of NK exo on CML (K562), T-ALL (Jurkat), B-ALL (UoC) and AML (HL-60 and KG1a) cell lines. NK exo treatment initiated a time- and dose-dependent reduction in viability and induction of apoptosis in all cell lines tested. The effect of NK exo exposure on cell viability and apoptosis was reduced in the presence of the pan-caspase inhibitor Z-VAD-FMK suggesting that NK exo exert their cytotoxic effect in a caspase-dependent manner. In addition, exposure to NK exo led to a significant increase in apoptosis in treatment naive AML, CLL and ALL patient-derived mononuclear cells. Moreover, we witnessed a significant reduction in colony formation of mononuclear cells harvested from treatment naive CML and AML patients in the presence of NK exo. While leukemia cells were targeted and severely affected by NK exo, healthy B-cells remained unaffected, indicating a selective effect. The selective trait of NK exo was also confirmed via an exosome uptake assay which demonstrated that NK exo were taken up specifically by leukemic cells but not by healthy B-cells.In an in-vivo xenograft model for AML; NOD-SCID IL2Rgama null (NSG) mice were engrafted with the well characterized human AML cell line HL60, and were then intravenously injected with NK exo. Our preliminary results indicate that NK exo treatment improves overall mice survival indicating an anti-leukemic effect of NK exo in-vivo.In this “proof-of-concept” study, we show that NK exo, similar to their cell-of-origin, have a strong and selective anti-leukemia effect. To provide the pre-clinical evidence needed to test the NK exo approach in future clinical studies, we are currently continuing to test the anti-leukemic potential of NK exo in-vivo on a xenograft murine model. Our preliminary results propose the use of NK exo as a novel therapeutic platform to combat leukemia. More broadly, since NK cells target a wide range of cancer cells, not just leukemia cells, and since we have shown that NK exo maintain the anti-leukemic capacity of their donor cells, this approach may become applicable in other hematological and solid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.