Cells exposed to TTFields often result in mitotic disruption and subsequent cell death.

<p>Upon their removal from the TTFields (right panel), cells exhibited significantly signs of cell stress including altered morphology compared to sham-treated controls (left panel) including increased size and vacularity (<b>A</b>). Insets show enlargement of cells in the field for detail. This increase in vacularity was also evident by increased side scatter by FACS (<b>B</b>). HeLa (upper panels) or MCF-7 cells (lower panels) were synchronized using aphidicolin, plated on gridded glass bottom dishes and either Sham-treated or exposed to TTFields during either mitosis or the G₁ phase. After removal from the TTFields, cells were counted in individual grids at 4 and 24 hours after the termination of treatment and the resulting ratios were measured as a metric of proliferation. Most cells present at 4 hours remained at 24 hours. The proliferation of cells was significantly lower following exposure to TTFields during the M phase compared to sham-treated cultures. Both the TTFields-treated and Sham-treated cells exhibited similar proliferation when treated in G₁ (<b>C</b>). HCT116 p53^+/+ (upper panels) or HCT116 p53^-/- cells (lower panels) were incubated for 24 hours either without (left panels) or with TTFields-exposure (right panels) and then incubated for an additional 24 hours. Cells were allowed to incorporate BrdU into their DNA as a measure of cells in S phase (<b>D</b>). To test if cells exposed to TTFields exhibited a higher incidence of apoptosis. HCT-116 p53^+/+ cells were treated with TTFields for 24 hours and then further incubated at 37°C and then stained with FITC-labeled Annexin V at 18, 36, and 60 hours after the midpoint of their treatment. Annexin V binding to cells was visualized by fluorescence microscopy and scored for the presence of Annexin V positive cells. Cells were observed to undergo apoptosis after 18 hours of removal from TTFields with a peak at 36 hours (<b>E</b>). In order to test the effect of p53 depletion on TTFields-induced apoptosis, the responses of HCT-116 p53^+/+ were compared with HCT116 p53^-/- cells at 36 hours following TTFields treatment. p53^+/+ with exposure to TTFields exhibited higher levels of apoptotic cells than their p53^-/- counterparts (<b>F</b>).</p

Cells exposed to TTFields during M-phase exhibit chromosomal disordering during the metaphase to anaphase progression.

<p>HeLa cells were partially synchronized by treating with aphidicolin, then stained with DRAQ5 to visualize their chromosomes and subjected to fluorescence and phase contrast time lapse microscopy. Cells were imaged as they transited through mitosis either with or without TTFields exposure by both phase contrast and fluorescence during exposure to TTFields and time lapse series were captured. Single frames extracted from the time-lapse series of either Sham-treated (<b>A</b>) or TTFields-treated (<b>B</b>) cells visualized by phase contrast (left panels) or DRAQ5 (middle panels) at intervals of 240 seconds showed cells undergoing violent mitotic contractions that appeared coincident with the separation of daughter chromosomes at the onset of anaphase. Measurement of the time intervals between chromatid condensation and formation of the metaphase plate (<b>C</b>) and from the formation of the metaphase plate to either evidence of anaphase or TTFields-induced membrane contractions (<b>D</b>) were similar, 49.39 ± 1.988 min vs. 43.26 ± 2.088 min for plate formation and 47.04 ± 1.196 min vs. 44.36 ± 2.037 min for metaphase exit in Sham-treated vs TTFields-treated cultures, respectively. However, the outcome of mitosis was markedly different in Sham-treated (n = 130) vs. TTFields-treated (n = 85) cultures (<b>E</b>). Mitotic spindles and metaphase plates formed normally in TTFields treated cells (<b>F</b>), however, cells exiting mitosis in TTFields-treated cultures exhibited increased in abnormal nuclei with many cell possessing multiple micronuclear structures (<b>G</b>). Scale bar = 20 μm.</p

Septin 7 localization is perturbed by TTFields.

<p>Crystal structure of Septin complex composed of Septins 2, 6, and 7 showing the direction of the dipole vector (2711Debyes) relative to the longitudinal axis of the heterotrimer (<b>A</b>). Synchronized cells were cultured in the absence of TTFields, TTFields delivered at 150 kHz (n = 30) or 500 kHz (n = 12), fixed and stained with antibodies against α-tubulin and Septin 7 and counter stained with DAPI. Confocal microscopy of cells captured in mid anaphase under these conditions revealed that Septin 7 localization was reduced and the midlines of anaphase spindles were perturbed by TTFields of 150 kHz but to a lesser extent at TTFields at 500 kHz compared to control cells (n = 27) (<b>B</b>). The average intensity of Septin 7 staining was separately determined within the region of the midline and for the entire cell. The ratio of the average intensity of Septin 7 staining at midline that of the total staining within the cell in Sham (n = 10) and presence of TTFields at 150kHz (n = 9) or 500kHz (n = 8) showed a significant decrease in Septin accumulation at the midlines in cells treated with 150 kHz TTFields (<b>C</b>). In contrast to Septin 7, PLK1 localization to the anaphase spindle midline appeared unperturbed by TTFields at 150 kHz (n = 11) (<b>D</b>). Scale bar = 10 μm. Septin organizes filamentous actin within the CCF during anaphase, in 150kHz TTFields treated cells F-actin accumulation were significantly decrease at the midline (E and F), n = 16 sham and n = 17 TTFields. To test whether TTFields interfered with the ability of Septins to re-localize to interphase microtubules during attachment and spreading on fibronectin, cells were allowed to spread for 9 hours under Sham conditions, or treated with TTFields at either 150 kHz, or 500 kHz (<b>G</b>). The Septin localization to the cytoskeleton was scored based on the striation of Septin 7 at the base of the cell with 1 equating to no diffuse staining with no striations, 2 equating to moderate localization and striations, and 3 equating to strong striations. While cells were allowed to re-attach and spread, cells in the absence of TTFields exposure exhibited strong Septin 7 association with microtubules (n = 70) while cells that were exposed to TTFields at 150 kHz (n = 152) exhibited significantly less Septin 7 localization to microtubules. Cell spreading while exposed to TTFields of 500 kHz (n = 104) exhibited an intermediate amount of microtubule association. Data was accumulated from three separate experiments (<b>H</b>). Scale bar = 50 μm.</p

Cells exposed to TTFields exhibit an inability to progress through anaphase successfully resulting in cellular derangement.

<p>MDA-MB-231 cells were synchronized using double aphidicolin block and then allowed to re-enter the cell cycle in the absence of TTFields (top panels), exposed to TTFields (middle panels) or treated with 3 μM paclitaxel (bottom panels). Cells were harvested and analyzed by FACS for transit through mitosis by staining with antibodies against Cyclin B antibodies and pH3 at 4, 7, 9, 11 and 13 hours after aphidicolin removal. Cells treated with TTFields did not exhibit a marked accumulation of mitotic cells showing a slight increased accumulation of cells at the 9 and 11 hour time points compared to control cultures suggesting a reduction in the rate of mitotic exit (<b>A</b>). Results are representative of 4 separate experiments. MDA-MB-231 cells were synchronized using aphidicolin followed by RO3306 treatment and were then allowed to progress through mitosis n the presence or absence of TTFields following drug removal. Cells were then collected at 60, 120 and 150 min and stained with antibodies against pH3 and 7-AAD for DNA content for analysis by FACS (<b>B</b>).</p

Tempol inhibits dendritic cell (DC) functions.

<p>Bone marrow-derived DCs (10⁶ cells/mL) were preincubated overnight with medium or Tempol at indicated concentrations and stimulated with lipopolysaccharide (LPS) (200 ng/mL) for 24 hours. Detection of <b>A,</b> TNF-α was evaluated in cell-free culture supernatants after six hours, and the levels of <b>B,</b> IL-6 and <b>C,</b> IL-12p70 were evaluated after 24 hours. <b>D,</b> Cells were stained with fluorochrome-labeled monoclonal antibodies to CD11c, CD40, CD80, CD86, and MHC class II and analyzed by flow cytometry. <b>E,</b> DCs incubated with medium or Tempol were pulsed with OVA (100 µg/mL) plus LPS (200 ng/mL) for four hours. After repeated washings, DCs were co-incubated with purified CD4⁺ cells from DO11.10 mice (DC:CD4⁺ ratio  = 1:4) for 72 hours, and proliferation was measured as described in Materials and Methods. <b>F,</b> IL-2 and <b>G</b>, IFN-γ were evaluated in the culture supernatants from the proliferation assay. *, P≤0.05 versus control group (^–/–); ^#, P≤0.05 versus LPS group (^+/−) (analysis of variance).</p

Agaphelin inhibits inflammation and neutrophil accumulation <i>in vivo</i>.

<p>A) Paw edema in mice. Carrageenan (2%) was administered to mice, in the presence of saline or Agaphelin (1 or 10 mg/Kg). Agaphelin diluted in saline was injected as a control (without carrageenan). Edema formation was evaluated at 0, 4, and 24 h after as increase in paw thickness. B) Neutrophil recruitment in inflamed footpads was evaluated by measuring tissue myeloperoxidase activity, expressed as units of activity/g of tissue, after injection of carrageenan as above. Animals were euthanized 4 hours after. Statistical significance: *, <i>p</i><0.05 or **, <i>p</i><0.01 (one-way ANOVA followed by Tukey's post hoc test; <i>n</i> = 5 in each group), compared with carrageenan only.</p

Pathologic events in malaria lead to radical oxygen species (ROS) generation and inflammation.

<p><i>Plasmodium falciparum</i> infection is associated with coagulation and complement activation, cytokine release, host response to infection, and activation of different cells types, including endothelial cells, platelets, dendritic cells and monocytes, and neutrophils. It is also associated with release of <i>Pf</i>-GPI, iron overload, heme release and Fenton reaction, and diminished levels of antioxidants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Miller1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Ruf1" target="_blank">[9]</a>. Superoxide contributes to platelet aggregation, tissue factor expression, cytokine release, NF-κB activation, DNA damage, NET formation, recruitment of inflammatory cells, conversion of nitric oxide (NO) to peroxynitrate (ONOO⁻) which in turn leads to DNA damage, PARP activation, lipid peroxidation, and protein nitration. These events result in sustained inflammation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Finkel1" target="_blank">[52]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Smith1" target="_blank">[56]</a>. NO, nitric oxide; O₂⁻, superoxide; ONOO⁻, peroxynitrate.</p

Agaphelin inhibits thrombosis <i>in vivo</i>, without impairing hemostasis.

<p>A) Arterial thrombosis. A paper filter infused with 7.5% FeCl₃ was applied to the carotid artery, and blood flow was monitored with a perivascular flow probe for 60 min or until stable occlusion took place. Fifteen minutes before injury, Agaphelin was injected into the caudal veins of the mice. Each symbol represents one animal. *, <i>p</i><0.05 (ANOVA with Dunnett post-test). B) Bleeding time. Bleeding was caused by a tail transection after intravenous injection of Agaphelin at the indicated concentrations. Absorbance at 540 nm (hemoglobin concentration) was used to estimate blood loss. *, <i>p</i><0.05 (ANOVA with Dunnett post-test).</p

Agaphelin inhibits elastase-mediated platelet aggregation, TFPI-cleavage by elastase, and neutrophil-induced coagulation.

<p>A) Platelet aggregation. Washed human platelets were stimulated with elastase only (500 nM, left panel), or cathepsin G only (110 nM, middle tracing), or elastase followed by cathepsin G (right tracing). In some experiments, Agaphelin (1 or 10 µM) was added to platelets followed by addition of elastase for 1 min, and cathepsin G. B) Agaphelin does not inhibit cathepsin G (200 nM) or C) collagen (0.6 µg/ml)-induced platelet aggregation. Aggregation response was monitored by turbidimetry using a Lumi-Aggregometer. D) TFPI cleavage. Agaphelin (0.1 µM and 1 µM) was incubated with 1 µg of TFPI in the presence of PBS or human neutrophil elastase (0.8 µg/ml). After 2 h, reactions were stopped by addition of LDS loading buffer (under reducing conditions, 10 mM DTT), boiled for 5 min, and loaded in 4–12% Nu-PAGE gel. Gels were Coomassie Blue-stained. E) Neutrophil-induced coagulation. Neutrophils (5×10⁵ cells/well) were incubated with Agaphelin (1 µM), PBS (control) or DNAse (Dornase alfa, 4 µg/ml) for 1 hour, followed by addition of PMA (50 nM) for 3 hrs. Fifty µl of this suspension was added to 50 µl of plasma, and reactions were started by addition of CaCl₂ (12.5 mM, final concentration)(*, p<0.05).</p

Tempol inhibits increase of permeability of the blood brain barrier (BBB).

<p><i>Plasmodium berghei</i> Anka parasitized red blood cells (10⁶) were injected into mice intraperitoneally (i.p.) Tempol (100 µL; 20 mg/kg), was started at day 1 or day 4 post infection (p.i.). At day 6 p.i., all animals were euthanized and the brain immediately dissected and fixed. Permeability of the BBB was detected as extravasation of IgG as described in Materials and Methods. <b>A,</b> A typical experiment is shown: <b><i>i,</i></b> uninfected mice; <b><i>ii,</i></b> infected and treated with Tempol (day 4); <b><i>iii,</i></b> infected and treated with Tempol (day 1); and <b><i>iv,</i></b> infected, non-treated mice. <b>B,</b> Quantification of inflammation of the whole brain. <b>C,</b> Quantification of inflammation of the olfactory bulbs. Quantification was performed as described in Materials and Methods. Ten mice were analyzed per group. *, P≤0.05 (ANOVA, Tukey post-test).</p