Distinct profile of antiviral drugs effects in aortic and pulmonary endothelial cells revealed by high-content microscopy and cell painting assays.

Antiretroviral therapy have significantly improved the treatment of viral infections and reduced the associated mortality and morbidity rates. However, highly effective antiretroviral therapy (HAART) may lead to an increased risk of cardiovascular diseases, which could be related to endothelial toxicity. Here, seven antiviral drugs (remdesivir, PF-00835231, ritonavir, lopinavir, efavirenz, zidovudine and abacavir) were characterized against aortic (HAEC) and pulmonary (hLMVEC) endothelial cells, using high-content microscopy. The colourimetric study (MTS test) revealed similar toxicity profiles of all antiviral drugs tested in the concentration range of 1 nM-50 µM in aortic and pulmonary endothelial cells. Conversely, the drugs' effects on morphological parameters were more pronounced in HAECs as compared with hLMVECs. Based on the antiviral drugs' effects on the cytoplasmic and nuclei architecture (analyzed by multiple pre-defined parameters including SER texture and STAR morphology), the studied compounds were classified into five distinct morphological subgroups, each linked to a specific cellular response profile. In relation to morphological subgroup classification, antiviral drugs induced a loss of mitochondrial membrane potential, elevated ROS, changed lipid droplets/lysosomal content, decreased von Willebrand factor expression and micronuclei formation or dysregulated cellular autophagy. In conclusion, based on specific changes in endothelial cytoplasm, nuclei and subcellular morphology, the distinct endothelial response was identified for remdesivir, ritonavir, lopinavir, efavirenz, zidovudine and abacavir treatments. The effects detected in aortic endothelial cells were not detected in pulmonary endothelial cells. Taken together, high-content microscopy has proven to be a robust and informative method for endothelial drug profiling that may prove useful in predicting the organ-specific endothelial toxicity of various drugs.

What is the ability of inflamed endothelium to uptake exogenous saturated fatty acids? A proof-of-concept study using spontaneous Raman, SRS and CARS microscopy.

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective.

Comparison of anti-cancer effects of novel protein disulphide isomerase (PDI) inhibitors in breast cancer cells characterized by high and low PDIA17 expression.

BACKGROUND: Protein disulphide isomerases (PDIs) play an important role in cancer progression. However, the relative contribution of the various isoforms of PDI in tumorigenesis is not clear. METHODS: The content of PDI isoforms in 22 cancer cells lines was investigated using LC-MS/MS-based proteomic analysis. The effects of PDIA1, PDIA3 and PDIA17 inhibition on the proliferation, migration and adhesion of MCF-7 and MDA-MB-231 cells, identified as high and low PDIA17 expressing cells, respectively, were assessed using novel aromatic N-sulphonamides of aziridine-2-carboxylic acid derivatives as PDI inhibitors. RESULTS: PDIA1 and PDIA3 were the most abundant in cancer cell lysates and were also detected extracellularly in breast cancer cells (MDA-MB-231 and MCF-7). Some cancer cell lines (e.g., MCF-7, HT-29) showed upregulated expression of PDIA17, whereas in others (e.g., MDA-MB-231, 67NR), PDIA17 was not detected. The simultaneous inhibition of PDIA1 and PDIA3 showed similar anti-proliferative effects in MCF-7 and MDA-MB-231 breast cancer cells. However, the inhibition of PDIA1 and PDIA17 in the MCF-7 cell line resulted in more effective anti-adhesive and anti-proliferative effects. CONCLUSIONS: PDIA1 and PDIA3 represent major isoforms of multiple cancer cells, and their non-selective inhibition displays significant anti-proliferative effects irrespective of whether or not PDIA17 is present. The more pronounced anti-adhesive effects of PDI inhibition in hormone-sensitive MCF-7 cells featured by higher levels of PDIs when compared to triple-negative MDA-MB-231 cells suggests that targeting extracellular PDIA1 and PDIA3 with or without additional PDIA17 inhibition may represent a strategy for personalized anti-adhesive, anti-metastatic therapy in cancers with high PDI expression.

Astaxanthin as a new Raman probe for biosensing of specific subcellular lipidic structures: can we detect lipids in cells under resonance conditions?

Here we report a new Raman probe for cellular studies on lipids detection and distribution. It is (3S, 3'S)-astaxanthin (AXT), a natural xanthophyll of hydrophobic properties and high solubility in lipids. It contains a chromophore group, a long polyene chain of eleven conjugated C=C bonds including two in the terminal rings, absorbing light in the visible range that coincides with the excitation of lasers commonly used in Raman spectroscopy for studying of biological samples. Depending on the laser, resonance (excitation in the visible range) or pre-resonance (the near infrared range) Raman spectrum of astaxanthin is dominated by bands at ca. 1008, 1158, and 1520 cm-1 that now can be also a marker of lipids distribution in the cells. We showed that AXT accumulates in lipidic structures of endothelial cells in time-dependent manner that provides possibility to visualize e.g. endoplasmic reticulum, as well as nuclear envelope. As a non-toxic reporter, it has a potential in the future studies on e.g. nucleus membranes damage in live cells in a very short measuring time.

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD+ and ATP Depletion.

OBJECTIVES: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. CONCLUSIONS: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.

Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer.

Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection. We show their potential in the detection of lung macrometastasis, in which we already observed the ECM remodelling. The CARS image revealed a dense cell fraction typical of ECM remodeling and reduction of the TPEF signal together with an increase of fluorescence lifetime predominantly due to NAD(P)H suggesting metabolic changes in the metastatic foci. FT-IR spectroscopy allowed not only for macrometastasis detection but also their stage definition based mainly on the analysis of proteins, RNA and glycogen fractions. The multimodal approach additionally suggested partial enzymatic degradation of elastin in ECM and collagen remodelling.

Tracking Extracellular Matrix Remodeling in Lungs Induced by Breast Cancer Metastasis. Fourier Transform Infrared Spectroscopic Studies.

This work focused on a detailed assessment of lung tissue affected by metastasis of breast cancer. We used large-area chemical scanning implemented in Fourier transform infrared (FTIR) spectroscopic imaging supported with classical histological and morphological characterization. For the first time, we differentiated and defined biochemical changes due to metastasis observed in the lung parenchyma, atelectasis, fibrous, and muscle cells, as well as bronchi ciliate cells, in a qualitative and semi-quantitative manner based on spectral features. The results suggested that systematic extracellular matrix remodeling with the progress of the metastasis process evoked a decrease in the fraction of the total protein in atelectasis, fibrous, and muscle cells, as well as an increase of fibrillar proteins in the parenchyma. We also detected alterations in the secondary conformations of proteins in parenchyma and atelectasis and changes in the level of hydroxyproline residues and carbohydrate moieties in the parenchyma. The results indicate the usability of FTIR spectroscopy as a tool for the detection of extracellular matrix remodeling, thereby enabling the prediction of pre-metastatic niche formation.

Proteomic characterization of early lung response to breast cancer metastasis in mice.

INTRODUCTION: The tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma. MATERIALS AND METHODS: The studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery). RESULTS: Palpable primary tumors were noted in the 2nd week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1st week and progressed during the 2nd week, accompanied by increased concentration of 2-OH-E+, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1st week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2nd week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz). CONCLUSION: The analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics.

BACKGROUND: The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis. METHODS: Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics. RESULTS: Based on primary tumour growth and pulmonary metastases, 1-2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3-4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate ("Warburg effect"). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins. CONCLUSIONS: The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.

Nitric oxide deficiency and endothelial-mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice.

BACKGROUND: Mesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial-mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice. METHODS: NO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1-5 weeks after 4T1 cancer cell inoculation in Balb/c mice. RESULTS: Phosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation. CONCLUSIONS: Decreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis.

Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

Interactions between cancer cells and the endothelium play a crucial role during metastasis. Here we examined the effects of a carbon monoxide-releasing molecule (CORM-401) and a nitric oxide donor (PAPA NONOate) given alone or in combination on breast cancer cell adhesion and transmigration across the lung microvascular endothelium. We further explored whether the effects of CO and NO on cancer-endothelial cells interactions are linked with changes in cellular bioenergetics in breast cancer or endothelial cells. We found that CORM-401 and PAPA NONOate alone or in combination markedly decreased transmigration of breast cancer cells across human lung microvascular endothelial cells (hLMVEC), while cancer cell adhesion to the endothelium was diminished only by a combination of the two compounds. In hLMVECs, CORM-401 decreased glycolysis and stimulated mitochondrial respiration, while in breast cancer cells CORM-401 decreased both glycolysis and mitochondrial respiration. In contrast, PAPA NONOate decreased mitochondrial respiration and slightly stimulated glycolysis in both cell lines. When both donors were given together, mitochondrial respiration and glycolysis were both profoundly inhibited, and cancer-endothelial cells interactions were additively suppressed. Intercellular adhesion molecule-1 (ICAM-1), involved in breast cancer cell adhesion to hLMVECs, was downregulated by CORM-401 and PAPA NONOate, when applied alone, while a combination of both compounds did not cause any enhancement of ICAM-1 downregulation. In conclusion, our findings demonstrate that CO and NO differently affect cellular bioenergetics of cancer and endothelial cells and suggest that this phenomenon may contribute to additive anti-adhesive and anti-transmigratory effects of CO and NO. Pharmacological attenuation of metabolism represents a novel, effective way to prevent cancer cell interactions with the endothelium, that is an energy-demanding process.

Chronic heart failure induces early defenestration of liver sinusoidal endothelial cells (LSECs) in mice.

AIM: Chronic heart failure (CHF) is often linked to liver malfunction and systemic endothelial dysfunction. However, whether cardio-hepatic interactions in heart failure involve dysfunction of liver sinusoidal endothelial cells (LSECs) is not known. Here we characterize LSECs phenotype in early and end stages of chronic heart failure in a murine model. METHODS: Right ventricle (RV) function, features of congestive hepatopathy, and the phenotype of primary LSECs were characterized in Tgαq*44 mice, with cardiomyocyte-specific overexpression of the Gαq protein, at the age of 4- and 12-month representative for early and end-stage phases of CHF, respectively. RESULTS: 4- and 12-month-old Tgαq*44 mice displayed progressive impairment of RV function and alterations in hepatic blood flow velocity resulting in hepatic congestion with elevated GGT and bilirubin plasma levels and decreased albumin concentration without gross liver pathology. LSECs isolated from 4- and 12-month-old Tgαq*44 mice displayed significant loss of fenestrae with impaired functional response to cytochalasin B, significant changes in proteome related to cytoskeleton remodeling, and altered vasoprotective function. However, LSECs barrier function and bioenergetics were largely preserved. In 4- and 12-month-old Tgαq*44 mice, LSECs defenestration was associated with prolonged postprandial hypertriglyceridemia and in 12-month-old Tgαq*44 mice with proteomic changes of hepatocytes indicative of altered lipid metabolism. CONCLUSION: Tgαq*44 mice displayed right-sided HF and altered hepatic blood flow leading to LSECs dysfunction involving defenestration, shift in eicosanoid profile, and proteomic changes. LSECs dysfunction appears as an early and persistent event in CHF, preceding congestive hepatopathy and contributing to alterations in lipoprotein transport and CHF pathophysiology.

Phylloquinone improves endothelial function, inhibits cellular senescence, and vascular inflammation.

Phylloquinon (PK) and menaquinones (MK) are both naturally occurring compounds belonging to vitamin K group. Present study aimed to comprehensively analyze the influence of PK in several models of vascular dysfunction to determine whether PK has vasoprotective properties, similar to those previously described for MK. Effects of PK and MK on endothelial dysfunction were studied in ApoE/LDLR-/- mice in vivo, in the isolated aorta incubated with TNF, and in vascular cells as regard inflammation and cell senescence (including replicative and stress-induced models of senescence). Moreover, the vascular conversion of exogenous vitamins to endogenous MK-4 was analyzed. PK, as well as MK, given for 8 weeks in diet (10 mg/kg) resulted in comparable improvement in endothelial function in the ApoE/LDLR-/- mice. Similarly, PK and MK prevented TNF-induced impairment of endothelium-dependent vasorelaxation in the isolated aorta. In in vitro studies in endothelial and vascular smooth muscle cells, we identified that both PK and MK displayed anti-senescence effects via decreasing DNA damage while in endothelial cells anti-inflammatory activity was ascribed to the modulation of NFκB activation. The activity of PK and MK was comparable in terms of their effect on senescence and inflammation. Presence of endogenous synthesis of MK-4 from PK in aorta and endothelial and smooth muscle cells suggests a possible involvement of MK in vascular effects of PK. In conclusion, PK and MK display comparable vasoprotective effects, which may be ascribed, at least in part, to the inhibition of cell senescence and inflammation. The vasoprotective effect of PK in the vessel wall can be related to the direct effects of PK, as well as to the action of MK formed from PK in the vascular wall.

In vitro cytotoxicity testing of new generation oxazaphosphorines against human histiocytic lymphoma cells.

Oxazaphosphorines belong to a group of alkylating agents. Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorines. The objective of the present study was to compare the cytotoxic action of these oxazaphosphorine compounds against human histiocytic lymphoma U937 cells. The chemical structures of the oxazaphosphorines were responsible for the different responses of U937 cells. The cytotoxic effects of D-17272, D-18864, and D-19575 on U937 cells depended on the agent tested, its dose, and the time intervals after the oxazaphosphorine application. Among the oxazaphosphorine agents, D-18864 appeared to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The in vitro cytotoxic activities of the oxazaphosphorines were strongly associated with their cell death inducing potential.

In vitro effects of new generation oxazaphosphorines on human promyelocytic leukemia cells.

Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorine agents. The present investigation was undertaken to determine the activity of these three oxazaphosphorines in human promyelocytic leukemia HL-60 cells. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter and microscopy methods. Functional and morphological changes were observed after exposure of HL-60 cells to the oxazaphosphorine agents. The various patterns of temporary alterations in cell viability, size and count, and also in the frequency of leukemic cells undergoing mitotic catastrophe, apoptosis and necrosis, were shown. Different leukemic cell responses to the action of the three oxazaphosphorines were evaluated. These are the first data comparing the in vitro activity of D-17272, D-18864 and D-19575 against human promyelocytic leukemia cells.

In vitro response of human pathological hematopoietic cells to cladribine.

The influence of cladribine (2-chloro-2'-deoxyadenosine, CdA) on in vitro response of human acute lymphoblastic leukemia MOLT-4 cells, human histiocytic lymphoma U-937 cells, and human promyelocytic leukemia HL-60 cells, was determined using the MTT spectrophotometric and Beckman Coulter methods. Cell viability, cell volume and count were compared 24h and 48h after cladribine application at four concentrations--50 nM, 100 nM, 250 nM, and 500 nM. Different patterns of temporary changes in the viability, volume and count of pathological hematopoietic cells exposed to the action of CdA were found. The effects of CdA on MOLT-4, U-937, and HL-60 cells were dependent on the agent tested and its concentration, the time intervals after agent application, and the cell line used. The various in vitro cytotoxic activities of CdA against the three human pathological hematopoietic cell lines were shown.

Comparative effects of new generation oxazaphosphorines on the size and viability of human acute myeloblastic leukemia cells.

Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864), and beta-D-glucose-isophosphoramide mustard (D-19575, glufosfamide) are three new generation oxazaphosphorine agents. The aim of the present study was to compare the cell response to the action of these three oxazaphosphorines. The experiments were performed in vitro on human acute myeloblastic leukemia ML-1 cells. After exposure of ML-1 cells to the oxazaphosphorines, the size, viability and count of these cells were determined. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter method. The temporary changes in the ML-1 cell size, viability and count, were dependent on the oxazaphosphorine agent tested, its dose, and the time intervals after its application. Among the three oxazaphosphorine agents, D-18864 proved to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The results suggest the possibility of using the electronic sizing and counting method and the MTT assay as a rapid in vitro test for assessing leukemic cell sensitivity to the action of new potential chemotherapeutic agents.

High-Resolution Fourier Transform Infrared (FT-IR) Spectroscopic Imaging for Detection of Lung Structures and Cancer-Related Abnormalities in a Murine Model.

Label-free molecular imaging is a promising utility to study tissues in terms of the identification of their compartments as well as chemical features and alterations induced by disease. The aim of this work was to assess if higher magnification of optics in the Fourier transform infrared (FT-IR) microscope coupled with the focal plane detector resulted in better resolution of lung structures and if the histopathological features correlated with clustering of spectral images. FT-IR spectroscopic imaging was performed on paraffinized lung tissue sections from mice with optics providing a total magnification of 61× and 36×. Then, IR images were subjected to unsupervised cluster analysis and, subsequently, cluster maps were compared with hematoxylin and eosin staining of the same tissue section. Based on these results, we observed minute features such as cellular compartments in single alveoli and bronchiole, blood cells and megakaryocytes in a vessel as well as atelectasis of the lung. In the case of the latter, differences in composition were also noted between the tissue from the non-cancerous and cancerous specimen. This study demonstrated the ability of high-definition FT-IR imaging to evaluate the chemical features of well-resolved lung structures that could complement the histological examination widely used in animal models of disease.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg-1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Glufosfamide as a new oxazaphosphorine anticancer agent.

Glufosfamide (ß-D-glucose-isophosphoramide mustard, D-19575) belongs to the oxazaphosphorine class. Glufosfamide is a novel glucose conjugate of ifosfamide in which isophosphoramide mustard, the alkylating metabolite of ifosfamide, is glycosidically linked to the ß-D-glucose molecule. Glufosfamide represents an attractive new agent for cancer therapy. Its mode of action on normal and pathological cells is still under experimental and clinical investigations. An assessment of the anticancer potential of glufosfamide is of key importance in therapy. The researchers reviewed the current knowledge available on glufosfamide tested in the preclinical studies/clinical trials, based on a collection of the original papers and conference abstracts published and relevant articles searched in the SCOPUS and MEDLINE database and websites.