Microparticle formation by platelets exposed to high gas pressures - An oxidative stress response.

This investigation explored the mechanism for microparticles (MPs) production by human and murine platelets exposed to high pressures of inert gases. Results demonstrate that MPs production occurs via an oxidative stress response in a dose-dependent manner and follows the potency series N2>Ar>He. Gases with higher van der Waals volumes or polarizability such as SF6 and N2O, or hydrostatic pressure, do not cause MPs production. Singlet O2 is generated by N2, Ar and He, which is linked to NADPH oxidase (NOX) activity. Progression of oxidative stress involves activation of nitric oxide synthase (NOS) leading to S-nitrosylation of cytosolic actin. Exposure to gases enhances actin filament turnover and associations between short actin filaments, NOS, vasodilator-stimulated phosphoprotein (VASP), focal adhesion kinase (FAK) and Rac1. Inhibition of NOS or NOX by chemical inhibitors or using platelets from mice lacking NOS2 or the gp91phox component of NOX diminish generation of reactive species, enhanced actin polymerization and MP generation by high pressure gases. We conclude that by initiating a sequence of progressive oxidative stress responses high pressure gases cause platelets to generate MPs.

Ascorbic acid supplementation diminishes microparticle elevations and neutrophil activation following SCUBA diving.

Predicated on evidence that diving-related microparticle generation is an oxidative stress response, this study investigated the role that oxygen plays in augmenting production of annexin V-positive microparticles associated with open-water SCUBA diving and whether elevations can be abrogated by ascorbic acid. Following a cross-over study design, 14 male subjects ingested placebo and 2-3 wk later ascorbic acid (2 g) daily for 6 days prior to performing either a 47-min dive to 18 m of sea water while breathing air (∼222 kPa N2/59 kPa O2) or breathing a mixture of 60% O2/balance N2 from a tight-fitting face mask at atmospheric pressure for 47 min (∼40 kPa N2/59 kPa O2). Within 30 min after the 18-m dive in the placebo group, neutrophil activation, and platelet-neutrophil interactions occurred, and the total number of microparticles, as well as subgroups bearing CD66b, CD41, CD31, CD142 proteins or nitrotyrosine, increased approximately twofold. No significant elevations occurred among divers after ingesting ascorbic acid, nor were elevations identified in either group after breathing 60% O2. Ascorbic acid had no significant effect on post-dive intravascular bubble production quantified by transthoracic echocardiography. We conclude that high-pressure nitrogen plays a key role in neutrophil and microparticle-associated changes with diving and that responses can be abrogated by dietary ascorbic acid supplementation.

The FLT3 inhibitor quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions.

The oral second-generation bis-aryl urea fms-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) has favorable kinase selectivity and pharmacokinetics. It inhibits mutant and wild-type FLT3 in vivo at 0.1 and 0.5 µM, respectively, and has shown favorable activity and tolerability in phase I and II trials in acute myeloid leukemia, with QT prolongation as the dose-limiting toxicity. Co-administration with chemotherapy is planned. We characterized interactions of quizartinib with the ATP-binding cassette (ABC) proteins ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein). Its effects on uptake of fluorescent substrates and apoptosis were measured by flow cytometry, binding to ABCB1 and ABCG2 drug-binding sites by effects on [¹²5I]iodoarylazidoprazosin ([¹²5I]-IAAP) photolabeling and ATPase activity, and cell viability by the WST-1 colorimetric assay. Quizartinib inhibited transport of fluorescent ABCG2 and ABCB1 substrates in ABCG2- and ABCB1-overexpressing cells in a concentration-dependent manner, from 0.1 to 5 µM and from 0.5 to 10 µM, respectively, and inhibited [¹²5I]-IAAP photolabeling of ABCG2 and ABCB1 with IC50 values of 0.07 and 3.3 µM, respectively. Quizartinib at higher concentrations decreased ABCG2, but not ABCB1, ATPase activity. Co-incubation with quizartinib at 0.1 to 1 µM sensitized ABCG2-overexpressing K562/ABCG2 and 8226/MR20 cells to ABCG2 substrate chemotherapy drugs in a concentration-dependent manner in cell viability and apoptosis assays. Additionally, quizartinib increased cellular uptake of the ABCG2 substrate fluoroquinolone antibiotic ciprofloxacin, which also prolongs the QT interval, in a concentration-dependent manner, predicting altered ciprofloxacin pharmacokinetics and pharmacodynamics when co-administered with quizartinib. Thus quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. These interactions should be considered in the design of treatment regimens combining quizartinib and chemotherapy drugs and in choice of concomitant medications to be administered with quizartinib.

The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms.

Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 µM decreased the IC(50)s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC(50) of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization.

The novel BCR-ABL and FLT3 inhibitor ponatinib is a potent inhibitor of the MDR-associated ATP-binding cassette transporter ABCG2.

Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations, including T315I, and also against fms-like tyrosine kinase 3. We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nmol/L and the MDR-associated ATP-binding cassette (ABC) proteins ABCB1, ABCC1, and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1. Ponatinib potently inhibited [(125)I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC(50) values of 0.04 and 0.63 µmol/L, respectively. Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low concentrations, consistent with it being a substrate of both proteins at pharmacologically relevant concentrations. The ponatinib IC(50) values of BCR-ABL-expressing K562 cells transfected with ABCB1 and ABCG2 were approximately the same as and 2-fold higher than that of K562, respectively, consistent with ponatinib being a substrate of both proteins, but inhibiting its own transport, and resistance was also attenuated to a small degree by ponatinib-induced downregulation of ABCB1 and ABCG2 cell-surface expression on resistant K562 cells. Ponatinib at pharmacologically relevant concentrations produced synergistic cytotoxicity with ABCB1 and ABCG2 substrate chemotherapy drugs and enhanced apoptosis induced by these drugs, including daunorubicin, mitoxantrone, topotecan, and flavopiridol, in cells overexpressing these transport proteins. Combinations of ponatinib and chemotherapy drugs warrant further testing.

YBX1 expression and function in early hematopoiesis and leukemic cells.

Hematopoietic transcription factors play a critical role in directing the commitment and differentiation of hematopoietic stem cells along a particular lineage. Y-box protein (YBX1) is a transcription factor which is widely expressed throughout development and is involved in erythroid cell development; however, its role in early hematopoietic differentiation is not known. This study aims to investigate the role of YBX1 expression in early hematopoietic differentiation and leukemia. Here, we show that YBX1 is highly expressed in mouse erythroid myeloid lymphoid-clone 1 (EML), a hematopoietic precursor cell line, but is down-regulated in myeloid progenitors and GM-CSF-treated EML cells during the course of myeloid differentiation. Moreover, we found that lineage(-)/IL-7R(-)/c-kit(+)/Sca1(+) (LKS; enriched fraction of hematopoietic stem cells) and lineage(-)/IL-7R(-)/c-kit(+)/Sca1(-) myeloid progenitor cells showed high level of YBX1 expression as compared to the differentiated cells like granulocytes in mouse bone marrow. Also, YBX1 protein was expressed at high levels in myeloid leukemic cell lines blocked at different stages of myeloid development. We further investigated the role of YBX1 in leukemic cells by knockdown studies and observed that down-regulation of YBX1 expression in K562 leukemic cells inhibited their proliferation ability, induced apoptosis, and differentiation towards megakaryocytic lineage upon arsenic trioxide treatments relative to untreated. Overall, our data indicates that YBX1 is down-regulated during myeloid differentiation and the aberrant YBX1 expression in leukemic cells could be a contributing factor in the development of leukemia by blocking their differentiation. Thus, YBX1 protein could be an excellent molecular target for therapy in myeloproliferative disorders and leukemia.