Bending of a lipid membrane edge by annexin A5 trimers.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

A novel coordination complex of platinum (PT) induces cell death in colorectal cancer by altering redox balance and modulating MAPK pathway.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous tumor having various genetic alterations. The current treatment options had limited impact on disease free survival due to therapeutic resistance. Novel anticancer agents are needed to treat CRC specifically metastatic colorectal cancer. A novel coordination complex of platinum, (salicylaldiminato)Pt(II) complex with dimethylpropylene linkage (PT) exhibited potential anti-cancer activity. In this study, we explored the molecular mechanism of PT-induced cell death in colorectal cancer. METHODS: Colony formation was evaluated using the clonogenic assay. Apoptosis, cell cycle analysis, reactive oxygen species, mitochondrial membrane potential and caspase-3/- 7 were assessed by flow cytometry. Glutathione level was detected by colorimetric assay. PT-induced alteration in pro-apoptotic/ anti-apoptotic proteins and other signaling pathways were investigated using western blotting. P38 downregulation was performed using siRNA. RESULTS: In the present study, we explored the molecular mechanism of PT-mediated inhibition of cell proliferation in colorectal cancer cells. PT significantly inhibited the colony formation in human colorectal cancer cell lines (HT-29, SW480 and SW620) by inducing apoptosis and necrosis. This platinum complex was shown to significantly increase the reactive oxygen species (ROS) generation, depletion of glutathione and reduced mitochondrial membrane potential in colorectal cancer cells. Exposure to PT resulted in the downregulation of anti-apoptotic proteins (Bcl2, BclxL, XIAP) and alteration in Cyclins expression. Furthermore, PT increased cytochrome c release into cytosol and enhanced PARP cleavage leading to activation of intrinsic apoptotic pathway. Moreover, pre-treatment with ROS scavenger N-acetylcysteine (NAC) attenuated apoptosis suggesting that PT-induced apoptosis was driven by oxidative stress. Additionally, we show that PT-induced apoptosis was mediated by activating p38 MAPK and inhibiting AKT pathways. This was demonstrated by using chemical inhibitor and siRNA against p38 kinase which blocked the cytochrome c release and apoptosis in colorectal cancer cells. CONCLUSION: Collectively, our data demonstrates that the platinum complex (PT) exerts its anti-proliferative effect on CRC by ROS-mediated apoptosis and activating p38 MAPK pathway. Thus, our findings reveal a novel mechanism of action for PT on colorectal cancer cells and may have therapeutic implication.

No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to "Candidatus Liberibacter solanacearum" at the Gut Interface.

"Candidatus Liberibacter solanacearum" is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between "Ca. Liberibacter solanacearum" and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two "Ca. Liberibacter solanacearum" haplotypes, LsoA and LsoB. To this end, we first quantified and localized "Ca. Liberibacter solanacearum" in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to "Ca. Liberibacter solanacearum" infection. The results showed no cellular markers of apoptosis despite the large amount of "Ca. Liberibacter solanacearum" present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to "Ca. Liberibacter solanacearum": the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either "Ca. Liberibacter solanacearum" haplotype. This study represents a first step toward understanding the interactions between "Ca. Liberibacter solanacearum" and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

Absence of Distinct Immunohistochemical Distribution of Annexin A5, C3b, C4d, and C5b-9 in Placentas From Patients With Antiphospholipid Antibodies, Preeclampsia, and Systemic Lupus Erythematosus.

INTRODUCTION: In pregnancy, the presence of preeclampsia (PEC), systemic lupus erythematosus (SLE), and/or antiphospholipid antibody syndrome (APLS) is characterized by poor obstetric outcomes, with potential adverse effects for both mother and fetus. Although the histopathologic changes observed in these entities have been well established, the pathogenic mediators associated with tissue injury are poorly understood. METHODS: Forty placentas were evaluated, including 10 patients with preeclampsia, 9 with SLE, 11 with APLS, and 10 disease-free controls. Each case was subjected to a panel of immunohistochemical markers including C3b, C4d, Annexin A5, and C5b-9. Staining was graded on intensity and distribution. RESULTS: C4d staining was distinctly different among disease groups and controls. Moreover, 6/10 PEC cases, 3/9 SLE cases, and 4/11 APLS cases showed at least focal staining for C4d. All controls were negative. Annexin A5 (AnxA5) staining showed intrinsic variability in all disease groups, while 10/10 controls showed diffuse, strong staining (2+ or 3+). C3b staining was heterogeneous among groups. DISCUSSION: Previously, antiphospholipid antibody (aPLA)-associated pregnancy complications have been thought to be a consequence of a unique aPLA-mediated pathogenic mechanism. However, the immunohistochemical similarity (increased complement and decreased AnxA5 staining) observed in placentas from patients with APLS, PEC, and SLE suggests that aPLA-associated pregnancy complications may reflect a more general autoimmune mechanism.

Annexins in Glaucoma.

Glaucoma is one of the leading causes of irreversible visual loss, which has been estimated to affect 3.5% of those over 40 years old and projected to affect a total of 112 million people by 2040. Such a dramatic increase in affected patients demonstrates the need for continual improvement in the way we diagnose and treat this condition. Annexin A5 is a 36 kDa protein that is ubiquitously expressed in humans and is studied as an indicator of apoptosis in several fields. This molecule has a high calcium-dependent affinity for phosphatidylserine, a cell membrane phospholipid externalized to the outer cell membrane in early apoptosis. The DARC (Detection of Apoptosing Retinal Cells) project uses fluorescently-labelled annexin A5 to assess glaucomatous degeneration, the inherent process of which is the apoptosis of retinal ganglion cells. Furthermore, this project has conducted investigation of the retinal apoptosis in the neurodegenerative conditions of the eye and brain. In this present study, we summarized the use of annexin A5 as a marker of apoptosis in the eye. We also relayed the progress of the DARC project, developing real-time imaging of retinal ganglion cell apoptosis in vivo from the experimental models of disease and identifying mechanisms underlying neurodegeneration and its treatments, which has been applied to the first human clinical trials. DARC has potential as a biomarker in neurodegeneration, especially in the research of novel treatments, and could be a useful tool for the diagnosis and monitoring of glaucoma.

Synthesis and evaluation of a radiolabeled bis-zinc(II)-cyclen complex as a potential probe for in vivo imaging of cell death.

The exposition of phosphatidylserine (PS) from the cell membrane is associated with most cell death programs (apoptosis, necrosis, autophagy, mitotic catastrophe, etc.), which makes PS an attractive target for overall cell death imaging. To this end, zinc(II) macrocycle coordination complexes with cyclic polyamine units as low-molecular-weight annexin mimics have a selective affinity for biomembrane surfaces enriched with PS, and are therefore useful for detection of cell death. In the present study, a 11C-labeled zinc(II)-bis(cyclen) complex (11C-CyclenZn2) was prepared and evaluated as a new positron emission tomography (PET) probe for cell death imaging. 11C-CyclenZn2 was synthesized by methylation of its precursor, 4-methoxy-2,5-di-[10-methyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester] phenol (Boc-Cyclen2) with 11C-methyl triflate as a prosthetic group in acetone, deprotection by hydrolysis in aqueous HCl solution, and chelation with zinc nitrate. The cell death imaging capability of 11C-CyclenZn2 was evaluated using in vitro cell uptake assays with camptothecin-treated PC-3 cells, biodistribution studies, and in vivo PET imaging in Kunming mice bearing S-180 fibrosarcoma. Starting from 11C-methyl triflate, the total preparation time for 11C-CyclenZn2 was ~40 min, with an uncorrected radiochemical yield of 12 ± 3% (based on 11C-CH3OTf, n = 10), a radiochemical purity of greater than 95%, and the specific activity of 0.75-1.01 GBq/µmol. The cell death binding specificity of 11C-CyclenZn2 was demonstrated by significantly different uptake rates in camptothecin-treated and control PC-3 cells in vitro. Inhibition experiments for 18F-radiofluorinated Annexin V binding to apoptotic/necrotic cells illustrated the necessity of zinc ions for zinc(II)-bis(cyclen) complexation in binding cell death, and zinc(II)-bis(cyclen) complexe and Annexin V had not identical binding pattern with apoptosis/necrosis cells. Biodistribution studies of 11C-CyclenZn2 revealed a fast clearance from blood, low uptake rates in brain and muscle tissue, and high uptake rates in liver and kidney, which provide the main metabolic route. PET imaging using 11C-CyclenZn2 revealed that cyclophosphamide-treated mice (CP-treated group) exhibited a significant increase of uptake rate in the tumor at 60 min postinjection, compared with control mice (Control group). The results indicate that the ability of 11C-CyclenZn2 to detect cell death is comparable to Annexin V, and it has potential as a PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy.

Characterization of surface antigens of reticulated immature platelets.

Reticulated platelets (RPs) are immature platelets with high dense granules content and a residual amount of megakaryocyte-derived of mRNA. Increased level of RPs has been found to be an independent predictor of cardiovascular ischemic events, and has been associated with impaired response to various anti-platelet drugs. The study aimed to characterize and compare the surface antigenic properties of reticulated versus mature platelets. Platelets from healthy individuals and diabetic patients were tested at rest and after activation with adenosine diphosphate (ADP). For each patient, we calculated the proportion of RPs and mature platelets using flow cytometry analysis with thiazole orange staining (for RPs) and CD42b platelet-specific antibody. We also tested the surface expression of P-selectin and Annexin V, by double staining flow cytometry in RPs versus mature platelets. A total of 20 subjects were recruited (10 healthy individuals, 10 diabetics). Activation with ADP did not cause a significant change in the proportion of RPs. Following activation, RPs demonstrated a significant increase in the expression of both P-selectin and Annexin V, while mature platelets exhibited a non-significant increase in both markers. These findings were consistent in both healthy subjects and patients with diabetes. In conclusion, RPs have a significantly higher capacity to increase the expression of platelet activation markers compared with mature platelets.

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70.

Effects of 12C6+ heavy ion beam irradiation on the p53 signaling pathway in HepG2 liver cancer cells.

The heavy ion beam is considered to be the ideal source for radiotherapy. The p53 tumor suppressor gene senses DNA damage and transducts intracellular apoptosis signals. Previous reports showed that the heavy ion beam can trigger complex forms of damage to cellular DNA, leading to cell cycle arrest and apoptosis of HepG2 human liver cancer cells; however, the mechanisms remains unclear fully. In order to explore whether the intrinsic or extrinsic pathway participates this process, HepG2 cells were treated with 12C6+ HIB irradiation at doses of 0 (control), 1, 2, 4, and 6 Gy with various methods employed to understand relevant mechanisms, such as detection of apoptosis, cell cycle, and Fas expression by flow cytometry, analysis of apoptotic morphology by electron microscopy and laser scanning confocal microscopy, and screening differentially expressed genes relating to p53 signaling pathway by PCR-array assay following with any genes confirmed by western blot analysis. This study showed that 12C6+ heavy ion beam irradiation at a dose of 6 Gy leads to endogenous DNA double-strand damage, G2/M cell cycle arrest, and apoptosis of human HepG2 cells via synergistic effect of the extrinsic and intrinsic pathways. Differentially expressed genes in the p53 signaling pathway related to DNA damage repair, apoptosis, cycle regulation, metastasis, deterioration and radioresistance were also discovered. Consequently, the expressions of Fas, TP53BP2, TP53AIP1, and CASP9 were confirmed upregulated after 12C6+ HIB irradiation treatment. In conclusion, this study demonstrated the mechanisms of inhibition and apoptosis induced by 12C6+ heavy ion beam irradiation on HepG2 cancer cells is mediated by initiation of the biological function of p53 signaling pathway including extrinsic and intrinsic apoptosis pathway.

Apoptosis in Acanthamoeba castellanii belonging to the T4 genotype.

Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade.

Matrigel and Activin A promote cell-cell contact and anti-apoptotic activity in cultured human retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is a leading cause of blindness among the aging population. Currently, replacement of diseased retinal pigment epithelium (RPE) cells with transplanted healthy RPE cells could be a feasible approach for AMD therapy. However, maintaining cell-cell contact and good viability of RPE cells cultured in vitro is difficult and fundamentally determines the success of RPE cell transplantation. This study was conducted to examine the role of Matrigel and Activin A (MA) in regulating cell-cell contact and anti-apoptotic activity in human RPE (hRPE) cells, as assessed by atomic force microscopy (AFM), scanning electron microscope (SEM), immunofluorescence staining, quantitative polymerase chain reaction (qPCR) analysis, Annexin V/propidium iodide (PI) analysis, mitochondrial membrane potential (â–³Ψ m) assays, intracellular reactive oxygen species (ROS) assays and Western blotting. hRPE cells cultured in vitro could maintain their epithelioid morphology after MA treatment over at least 4 passages. The contact of N-cadherin to the lateral cell border was promoted in hRPE cells at P2 by MA. MA treatment also enhanced the expression of tight junction-associated genes and proteins, such as Claudin-1, Claudin-3, Occludin and ZO-1, as well as polarized ZO-1 protein distribution and barrier function, in cultured hRPE cells. Moreover, MA treatment decreased apoptotic cells, ROS and Bax and increased â–³Ψ m and Bcl2 in hRPE cells under serum withdrawal-induced apoptosis. In addition, MA treatment elevated the protein expression levels of ß-catenin and its target proteins, including Cyclin D1, c-Myc and Survivin, as well as the gene expression levels of ZO-1, ß-catenin, Survivin and TCF-4, all of which could be down-regulated by the Wnt/ß-catenin pathway inhibitor XAV-939. Taken together, MA treatment could effectively promote cell-cell contact and anti-apoptotic activity in hRPE cells, partly involving the Wnt/ß-catenin pathway. This study will benefit the understanding of hRPE cells and future cell therapy.

Inula Viscosa Extracts Induces Telomere Shortening and Apoptosis in Cancer Cells and Overcome Drug Resistance.

Telomerase is activated in human papillomavirus (HPV) positive cervical cancer and targeting telomeres offers a novel anticancer therapeutic strategy. In this study, the telomere targeting properties, the cytotoxic as well as the pro-apoptotic effects of hexane (IV-HE) and dichloromethane (IV-DF) fractions from Inula viscosa L. extracts were investigated on human cervical HeLa and SiHa cancer cells. Our data demonstrate that IV-HE and IV-DF extracts were able to inhibit cell growth in HeLa and SiHa cells in a dose-dependent manner and studied resistant cell lines exhibited a resistance factor less than 2 when treated with the extracts. IV-HE and IV-DF extracts were able to inhibit telomerase activity and to induce telomere shortening as shown by telomeric repeat amplification protocol and TTAGGG telomere length assay, respectively. The sensitivity of fibroblasts to the extracts was increased when telomerase was expressed. Finally, IV-HE and IV-DF were able to induce apoptosis as evidenced by an increase in annexin-V labeling and caspase-3 activity. This study provides the first evidence that the IV-HE and IV-DF extracts from Inula viscosa L. target telomeres induce apoptosis and overcome drug resistance in tumor cells. Future studies will focus on the identification of the molecules involved in the anticancer activity.

Cu2+-labeled dansyl compounds as fluorescent and PET probes for imaging apoptosis.

Compound DNSTT-Cu2+, a novel chelate of Cu2+ with DOTA conjugated to a fluorescent dansyl fragment, is developed for imaging cell apoptosis. Apoptotic U-87MG cells could be selectively visualized by the fluorescence of DNSTT-Cu2+ from cytoplasm of cells, confirmed by the fluorescence of apoptosis cells co-labeled with Alexa Fluor 568-labeled annexin V, a conventional probe for selectively labeling membranes of apoptosis cells. A radioactive 64Cu2+ analog, DNSTT-64Cu2+, was easily synthesized, providing a potential PET probe for imaging apoptosis in vivo.

Increased alveolar soluble annexin V promotes lung inflammation and fibrosis.

The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesised that alveolar soluble annexin V contributes to lung fibrosis, based on the observation that human IPF bronchoalveolar lavage fluid (BALF) containing high annexin V levels promoted fibroblast involvement in alveolar epithelial wound healing that was reduced when annexin V was depleted from the BALF. Conditioned medium from annexin V-treated alveolar epithelial type 2 cells (AEC2), but not annexin V per se, induced proliferation of human fibroblasts and contained pro-fibrotic, IPF-associated proteins, as well as pro-inflammatory cytokines that were found to correlate tightly (r>0.95) with annexin V levels in human BALF. ErbB2 receptor tyrosine kinase in AECs was activated by annexin V, and blockade reduced the fibrotic potential of annexin V-treated AEC-conditioned medium. In vivo, aerosol delivery of annexin V to mouse lung induced inflammation, fibrosis and increased hydroxyproline, with activation of Wnt, transforming growth factor-ß, mitogen-activated protein kinase and nuclear factor-κB signalling pathways, as seen in IPF. Chronically increased alveolar annexin V levels, as reflected in increased IPF BALF levels, may contribute to the progression of IPF by inducing the release of pro-fibrotic mediators.

Quantitative detection of eryptosis in human erythrocytes using tunable resistive pulse sensing and annexin-V-beads.

Toxicological assessments of human red blood cells (RBCs) are important in human health because RBCs are the most abundant cell type in our body. Erythrotoxicology testing guidelines using hemolysis have been established as a standard (e.g. by the ASTM International). However, many xenobiotics promote eryptosis (apoptosis in human RBCs) without causing hemolysis. Based on the major features of eryptosis, i.e. cell shrinkage and translocation of phosphatidylserine (PS) to the outer lipid bilayer of the plasma membrane, we report here a novel approach utilizing the quantitative tunable resistive pulse sensing (TRPS) technology, a widely adopted technique for characterizing nanoparticles in the field of nanotechnology, to measure the degree of eryptosis in a non-optical manner. With the TRPS system, we were able to determine PS externalization with microbeads functionalized with annexin-V for PS binding, cell swelling and shrinkage in physiological buffers (cell volume: 86 ± 12 fL) and solutions of different osmolarities with or without apoptotic trigger. After setting these standards, we then evaluated the toxicity of Polyphyllin D (PD), a potential anti-cancer drug that kills more liver cancer cells with multi-drug resistance, in erythrocytes to prove our concept. Data revealed that PD induced PS externalization and shrinkage in RBCs in a dose-dependent manner. Moreover, another feature of eryptosis, as small as 5 fL, was detected thus showing the PD-induced erythrotoxicity in human cells. Taken together, our results indicate that our approach using annexin-V-beads and TRPS is simple, safe and convenient, using only a small volume (35 µL) to evaluate the erythrotoxicity of xenobiotics.

Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

Site-specifically 11C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death.

BACKGROUND: Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging. METHODS: AnxA5 and the control protein were recombinantly expressed with a C-terminal "Sel-tag", the tetrapeptide -Gly-Cys-Sec-Gly-COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with 11C for dynamic in vivo PET studies. RESULTS: AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The 11C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease. CONCLUSIONS: These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy. GENERAL SIGNIFICANCE: The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of 11C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.

Storage time affects umbilical cord blood viability.

BACKGROUND: Cryopreserved umbilical cord blood (CB) is increasingly used as a cell source to reconstitute marrow in hematopoietic stem cell transplant patients. Delays in cryopreservation may adversely affect cell viability, thereby reducing their potential for engraftment after transplantation. STUDY DESIGN AND METHODS: The impact of delayed cryopreservation for up to 3 days on the viability of both CD45+ and CD34+ cell populations in 28 CB donations with volumes of 58.40 ± 15.4 mL (range, 39.4-107.4 mL) was investigated to establish whether precryopreservation storage time could be extended from our current time of 24 to 48 hours in line with other CB banks. Viability was assessed on 3 consecutive days, both before and after cryopreservation, by flow cytometry using 7-aminoactinomycin D (7-AAD) and annexin V methods. RESULTS: The results using 7-AAD and annexin V indicated the viability of CD34+ cells before cryopreservation remained high (>92.33 ± 4.11%) over 3 days, whereas the viability of CD45+ cells decreased from 86.36 ± 4.97% to 66.24 ± 7.78% (p < 0.0001) by Day 3. Storage time significantly affected the viability of CD34+ cells after cryopreservation. Using 7-AAD, the mean CD34+ cell viability decreased by approximately 5% per extra day in storage from 84.30 ± 6.27% on Day 1 to 79.01 ± 7.44% (p < 0.0057) on Day 2 and to 73.95 ± 7.54% (p < 0.0001) on Day 3. With annexin V staining CD34+ cell viability fell by approximately 7% per extra day in storage from 77.17 ± 8.47% on Day 1 to 69.56 ± 13.30% (p < 0.0194) on Day 2 and to 62.89 ± 15.22% (p < 0.0002) on Day 3. CONCLUSION: This study demonstrates that extended precryopreservation storage adversely affects viability and should be avoided.

Pulsatilla saponin A, an active molecule from Pulsatilla chinensis, induces cancer cell death and inhibits tumor growth in mouse xenograft models.

BACKGROUND: Many natural compounds possess antitumor growth activities. Pulsatilla chinensis is an herb used in traditional Chinese medicine to treat infectious diseases. More recently, extracts from P chinensis have been shown to contain antitumor activities. MATERIALS AND METHODS: In this study, we isolated Pulsatilla saponin A as an active compound from P chinensis extracts and tested its anticancer activity in vitro and in vivo. RESULTS: In cell culture, Pulsatilla saponin A significantly inhibited the growth of human hepatocellular carcinoma SMCC-7721 cells and pancreatic BXPC3 and SW1990 cancer cells. Similar inhibitory activities were observed when the compound was tested in mouse xenograft tumor models using human hepatocellular carcinoma Bel-7402 and pancreatic cancer SW1990 cells. In Comet assay and flow cytometric analysis of cell cycle distribution and annexin V expression, DNA damage, G2 arrest, and apoptosis were identified in Pulsatilla saponin A-treated cancer cells. Based on the results of Western blotting, p53 and cyclin B protein levels were higher, whereas Bcl-2 protein levels were lower in Pulsatilla saponin A-treated cancer cells than in vehicle-treated cells. CONCLUSIONS: Pulsatilla saponin A may exert its antitumor effect by inducing DNA damage and causing G2 arrest and apoptosis in cancer cells. Pulsatilla saponin A and its derivatives may be developed as a new class of anticancer agents.

Francisella tularensis inhibits the intrinsic and extrinsic pathways to delay constitutive apoptosis and prolong human neutrophil lifespan.

Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ∼80% of infected neutrophils remained viable at 48 h compared with ∼50% of control cells, and ∼40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.