Detection of A5 single-positive cells during necroptosis.

<p><b>(A–C)</b> L929 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s) and RIPK3 (gsk872) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. <b>(A)</b> 10⁶ cells were harvested 3 hours after stimulation of cell death, and cell death key factors pMLKL and CC3 were detected using western blot. <b>(B)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(C)</b> Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. <b>(D–F)</b> U937 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ) or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s), RIPK3 (gsk872) and pMLKL (NSA) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. <b>(D)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(E)</b> Example smoothed flow cytometry density plots. <b>(F)</b> Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. <b>(G–H)</b> 3 x 10⁴ HaCaT cells per well in 96-well plate were stimulated, and PI and A5-FITC were added. The plate was placed on IncuCyteZOOM apparatus and 2 images per well were recorded every 30–45 minutes. <b>(G)</b> Normalized PI- or A5-positive objects per image. <b>(H)</b> Example single A5-positive necroptotic cells, imaged using IncuCyteZOOM apparatus. <b>(I)</b> HaCaT cells were harvested, and the cell death key factors pMLKL and CC3 were detected using western blot. Representative data are shown from 1 of at least 2 independent experiments. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s008" target="_blank">S1 Data</a>. A5, annexin V, CC3, cleaved caspase 3; Con, control; FITC, fluorescein isothiocyanate; MLKL, mixed lineage kinase domain-like; Nec-1s, necroptotic inhibitor of RIPK1; NSA, necrosulfonamide; PI, propidium iodide; pMLKL, phosphorylated mixed lineage kinase-like; RIPK1, receptor-interacting protein kinase-1; RIPK3, receptor-interacting protein kinase-3; Sd, protein ladder; SD, standard deviation; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD.</p

Necroptotic A5 single-positive cell death requires pMLKL membrane translocation.

<p>U937 cells were stimulated for necroptosis using TNFα, birinapant (SMAC mimetic) and zVAD (TBZ). After 2 hours, cells were treated with necroptotic inhibitors RIPK1 (Nec-1s), RIPK3 (gsk872), and pMLKL (NSA), or left untreated (Con). <b>(A)</b> Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). <b>(B)</b> Cell counts were measured using the Attune NxT flow cytometer (mean ± SD). Statistic comparisons between total cell numbers in each group to total cell numbers at the 2 hour time point in the control group was carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p</i> < <i>0</i>.<i>05</i>. Data are representative of 1 experiment from at least 3 independent ones. <b>(C–F)</b> U937 cells were stimulated as in (A) for 2 hours, prior to A5/PI staining. Cells were sorted into live cells (double-negative) or PS-exposed-necroptotic (A5-positive PI-negative). <b>(C)</b> Necroptotic cell death key factor pMLKL was detected in the different sorted populations using western blot. Cell viability was measured at different time points after sorting in the A5⁻PI⁻ <b>(D)</b> and A5⁺PI⁻ <b>(E)</b> populations, using A5/PI staining, and then analyzed by flow cytometry (mean ± SD). Where indicated, RIPK1 (Nec-1s), RIPK3 (gsk872), and pMLKL (NSA) inhibitors were added to the collection tubes. Dashed lines represent the state of cells prior to the addition of inhibitors. Data are taken from 3 independent experiments. <b>(E)</b> U937 cells were stimulated and sorted into PS-exposed-necroptotic (A5-positive PI-negative) as above. Viable cells (A5⁻PI⁻) were measured and counted at 6 days after sorting. Data are taken from 2 independent experiments. (mean ± SD). Statistic comparisons between total cell numbers was carried using parametric paired Student <i>t</i> test. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s012" target="_blank">S5 Data</a>. A5, annexin V; Con, control; Nec-1s; necroptotic inhibitor of RIPK1; MLKL, mixed lineage kinase domain-like; NSA; necrosulfonamide; PI; propidium iodide; pMLKL; phosphorylated mixed lineage kinase-like protein; PS, phosphatidylserine; RIPK1, receptor-interacting protein kinase-1; RIPK3, receptor-interacting protein kinase-3; SD, standard deviation; SMAC, second mitochondrial-derived activator of caspase; Std, protein ladder; TBZ, TNFα + birinapant + zVAD; zVAD, Z-VAD-FMK.</p

Phagocytosis of PS-exposed-necroptotic cells.

<p><b>(A)</b> U937 cells were first stained with CFSE prior to stimulation for apoptosis and necroptosis using a combination of TNFα, birinapant (SMAC mimetic), and zVAD. PS exposure was tested every 30 minutes until exposure reached 40% in both the apoptotic and necroptotic samples (determined by A5/PI staining). Cells were washed twice and resuspended in DMEM before adding on IFN-γ treated BMDMs at a 2.5:1 ratio. Phagocytosis was analyzed by flow cytometry (mean ± SD). BMDMs with or without addition of live cells served as negative controls. Data are taken from 3 independent experiments. <b>(B)</b> U937 cells treated as above were added on TG peritoneal macrophages at a 2.5:1 ratio, and phagocytosis was analyzed by flow cytometry (mean ± SD). <b>(C)</b> Viability of the U937 cells, which were used as target to (B), is shown. TG peritoneal macrophages with no addition of cells or with addition of live cells served as negative controls. <b>(D–I)</b> Supernatants from the phagocytic TG peritoneal macrophages from (B) were collected and then analyzed for cytokines and chemokines using ELISAs. Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(J–K)</b> U937 cells were left untreated or stimulated for apoptosis or necroptosis. PS exposure was tested every 30 minutes until reaching 40% in apoptosis and necroptotic samples (determined by A5/PI staining). 2 x 10⁶ cells in 100 μl per mouse were IP injected. One hour after injection, CD11b peritoneal cells were analyzed for CFSE fluorescence <b>(J)</b> and phagocytosis of CFSE target cells <b>(K)</b> by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. <b>(L)</b> Illustration of competitive in vivo phagocytosis assay. CFSE- or Hoescht-stained L929 cells were left untreated or stimulated for necroptosis (TSZ). PS exposure was tested every 30 minutes until reaching 40% in necroptotic samples (determined by A5/PI staining). A 1:1 ratio mix of live and necroptotic cells was IP injected (total of 2 x 10⁶ cells in 100 μl per mouse). (M) One hour after injection, F4/80 peritoneal cells were analyzed for phagocytosis of the live and/or necroptotic cells by flow cytometry (<i>N</i> = 4, mean ± sem). Statistic comparisons between each injected target cells populations were carried using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p < 0</i>.<i>05</i>. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s013" target="_blank">S6 Data</a>. A5, annexin V; BMDM, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; DMEM, dulbecco’s modified eagle’s medium; IP, intraperitoneally; PI, propidium iodide; MFI, mean fluorescence intensity; PS, phosphatidylserine; SD, standard deviation; SMAC, second mitochondrial-derived activator of caspase; TG, thioglycolate; zVAD, Z-VAD-FMK.</p

Necroptotic cells expose PS to the outer plasma membrane prior to its permeabilization.

<p>U937 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). <b>(A–D)</b> Cell viability was measured at different time points after stimulation of cell death by flow cytometry using <b>(A)</b> A5 and LD, <b>(B)</b> A5- and PI-staining, <b>(C)</b> A5, LD, and PI or <b>(D)</b> A5, Z, and PI triple staining. <b>(E)</b> Four hours after stimulation of apoptosis (TB) or necroptosis (TBZ), U937 cells were triple stained with A5, Z, and PI, and analyzed by imagestream flow cytometry. Example imagestream flow plots are shown, brightness contrast for each channel between each population was identical. Representative data are shown from 1 of at least 2 independent experiments that were carried out. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s009" target="_blank">S2 Data</a>. A5, annexin V; BF, bright field; Con, control; LD, LiveDead; PI, propidium iodide; PS, phosphatidylserine; SSC, side scatter; TB, TNFα + birinapant; TBZ, TNFα + birinapant + zVAD; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD; Z, Zombie.</p

PS-exposed necroptotic cells do not show membrane leakage.

<p><b>(A–B)</b> CFSE labeled U937 cells were stimulated for either (i) apoptosis (TB), necroptosis (TBZ) or (ii) left untreated (Con). <b>(A)</b> Three hours after stimulation, cell viability was measured using A5, Z, and PI triple staining, and then analyzed by flow cytometry. <b>(B)</b> Geometric mean of CFSE fluorescent in the different populations from (A). Representative data are shown from 1 of at least 3 independent experiments that were carried out. <b>(C-D)</b> GFP expressing HaCaT cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). <b>(C)</b> Nine hours after stimulation, cell viability was measured using A5, Z, and PI triple staining, and then analyzed by flow cytometry. <b>(D)</b> Geometric mean of GFP fluorescent in the different populations from (A) were calculated using FloJo software. Representative data are shown from 1 of at least 2 independent experiments that were carried out. <b>(E)</b> Necroptosis (TBZ) U937 cells were isolated into 3 different populations according to their A5, Z, and PI triple staining by FACSAria using FACSDiva software (BD Biosciences). Sorted cells and untreated cells (live cells) were fixed and prepared for SEM analysis. Statistic comparisons between each population to the A5⁻ population were carried out for each treatment using ANOVA, followed by a Tukey’s multiple comparison test, * <i>p</i> < <i>0</i>.<i>05</i>. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s010" target="_blank">S3 Data</a>. A5, annexin V; Con, control; CFSE, carboxyfluorescein succinimidyl ester; GFP, green fluorescent protein; MFI, geometric mean fluorescence intensity; PI, propidium iodide; PS, phosphatidylserine; SEM, scanning electron microscopy; TB, TNFα + birinapant; TBZ, TNFα + birinapant + zVAD; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD; Z, Zombie.</p

Necroptotic cells release PS-exposed ECVs and pMLKL.

<p><b>(A-C)</b> CFSE labeled U937 cells were stimulated for either (i) apoptosis (TB), necroptosis (TBZ), or (ii) left untreated (Con). Three hours after stimulation, cells <b>(A)</b> and microparticles <b>(B)</b> were divided by their A5, Z, and PI staining, and the geometric mean of CFSE fluorescence in the different populations was then calculated using FloJo software. <b>(C)</b> Calculated total number of microparticles in the different treatments. Data are taken from 3 independent experiments. <b>(D)</b> CFSE and Hoescht prelabeled U937 cells were stimulated as above. Three hours after stimulation, apoptotic/necroptotic bodies were isolated and then analyzed for CFSE and Hoescht staining using flow cytometry. <b>(E–F)</b> ECVs from CFSE labeled U937 necroptotic cells were isolated using a size exclusion column (qEV, ZION), (fractions 7–9). <b>(E)</b> ECV (right panel) size was compered to submicron beads of known size (left panel) and <b>(F)</b> further stained for A5 and PI, and then analyzed using flow cytometry. <b>(G)</b> The different isolated fractions (qEV, ZION) from U937 necroptotic cells were concentrated, and the cell death key factors pMLKL and CC3 were detected using western blot. <b>(H)</b> U937 cells were stimulated for apoptosis (TS) or necroptosis (TSZ). Treated cells and supernatants were fractionated as illustrated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s005" target="_blank">S5C Fig</a>. Cell death key factors pMLKL and CC3 were detected in the different fractions using western blot. <b>(I–K)</b> 5 x 10⁶ U937 cells were stimulated for either (i) apoptosis (TB), necroptosis (TBZ), or (ii) left untreated (Con). ECVs from treated supernatants were isolated using ultracentrifuge and their concentration <b>(I)</b> and size <b>(J)</b> were analyzed using NanoSight. <b>(K)</b> Example NanoSight histogram and detection of pMLKL in ECVs. All raw data for the data summarized under this Fig can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002711#pbio.2002711.s011" target="_blank">S4 Data</a>. APC, allophycocyanin; A5, annexin V; CC3, cleaved caspase 3; Con, control; CFSE, carboxyfluorescein succinimidyl ester; ECV, extracellular vesicle; MFI, geometric mean fluorescence intensity; MLKL; mixed lineage kinase domain-like; NSA, necrosulfonamide; PI, propidium iodide; pMLKL, phosphorylated mixed lineage kinase-like; PS, phosphatidylserine; Std, protein ladder; SN, supernatant; SSC, side scatter; TB, TNFα + birinapant; TBZ, TNFα + birinapant + zVAD; TS, TNFα + SMAC mimetic; TSN, total supernatant; TSZ, TNFα + SMAC mimetic + zVAD; Z, Zombie; zVAD, Z-VAD-FMK.</p

Effect of AKBA treatment on NF-ĸB-related protein expression in an ectopic GBM model.

<p>Formalin-fixed paraffin-embedded sections were stained immuno-histochemically for IĸB-α. Representative microphotographs from each treatment group are shown (original magnification x200).</p

Effect of AKBA treatment on proliferation and intra-tumoral microvessel density in an ectopic GBM model.

<p>Formalin-fixed paraffin-embedded sections were stained immunohistochemically for Ki-67 (proliferation marker) and CD31 (endothelial marker). Representative microphotographs from each treatment group are shown (original magnification x400 –Ki-67, x200- CD31).</p

Combined acetyl-11-keto-β-boswellic acid and radiation treatment inhibited glioblastoma tumor cells

<div><p>Glioblastoma multiforme (GBM) is the most common and most aggressive subtype of malignant gliomas. The current standard of care for newly diagnosed GBM patients involves maximal surgical debulking, followed by radiation therapy and temozolomide chemotherapy. Despite the advances in GBM therapy, its outcome remains poor with a median survival of less than two years. This poor outcome is partly due to the ability of GBM tumors to acquire adaptive resistance to therapy and in particular to radiation. One of the mechanisms contributing to GBM tumor progression and resistance is an aberrant activation of NF-ĸB, a family of inducible transcription factors that play a pivotal role in regulation of many immune, inflammatory and carcinogenic responses. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic terpenoid extracted from the gum Ayurvedic therapeutic plant Boswellia serrata. AKBA is anti-inflammatory agent that exhibits potent cytotoxic activities against various types of tumors including GBM. One of the mechanisms underlying AKBA anti-tumor activity is its ability to modulate the NF-ĸB signaling pathway. The present study investigated <i>in vitro</i> and <i>in vivo</i> the effect of combining AKBA with ionizing radiation in the treatment of GBM and assessed AKBA anti-tumor activity and radio-enhancing potential. The effect of AKBA and/or radiation on the survival of cultured glioblastoma cancer cells was evaluated by XTT assay. The mode of interaction of treatments tested was calculated using CalcuSyn software. Inducing of apoptosis following AKBA treatment was evaluated using flow cytometry. The effect of combined treatment on the expression of PARP protein was analysed by Western blot assay. Ectopic (subcutaneous) GBM model in nude mice was used for the evaluation of the effect of combined treatment on tumor growth. Immunohistochemical analysis of formalin-fixed paraffin-embedded tumor sections was used to assess treatment-related changes in Ki-67, CD31, p53, Bcl-2 and NF-ĸB-inhibitor IĸB-α. AKBA treatment was found to inhibit the survival of all four tested cell lines in a dose dependent manner. The combined treatment resulted in a more significant inhibitory effect compared to the effect of treatment with radiation alone. A synergistic effect was detected in some of the tested cell lines. Flow cytometric analysis with Annexin V-FITC/PI double staining of AKBA treated cells indicated induction of apoptosis. AKBA apoptotic activity was also confirmed by PARP cleavage detected by Western blot analysis. The combined treatment suppressed tumor growth <i>in vivo</i> compared to no treatment and each treatment alone. Immunohistochemical analysis showed anti-angiogenic and anti-proliferative activity of AKBA <i>in vivo</i>. It also demonstrated a decrease in p53 nuclear staining and in Bcl-2 staining and an increase in IĸB-α staining following AKBA treatment both alone and in combination with radiotherapy. In this study, we demonstrated that AKBA exerts potent anti-proliferative and apoptotic activity, and significantly inhibits both the survival of glioblastoma cells <i>in vitro</i> and the growth of tumors generated by these cells. Combination of AKBA with radiotherapy was found to inhibit factors which involved in cell death regulation, tumor progression and radioresistence, therefore it may serve as a novel approach for GBM patients.</p></div

Effect of AKBA treatment on p53 and Bcl-2 expression in an ectopic GBM model.

<p>Formalin-fixed paraffin-embedded sections were stained immunohistochemically for p53 and Bcl-2. Representative microphotographs from each treatment group are shown (original magnification x200).</p