Detection Of Phosphatidylserine Research Articles

Detection of phosphatidylserine by using liquid crystal supported on the gold-deposited waveform surfaces with the annexin V-based signal enhancement

Tumor cells expose phosphatidylserine (PS) on their plasma membrane; thus, secreted PS-positive exosomes in the bloodstream could be used as biomarkers for cancer. In this study, we report a label-free liquid–crystal-mediated PS sensor employing PS antibodies, which were immobilized on waveform surfaces, with annexin V-based signal enhancement. When PS-annexin V clusters bind to the antibody, there is an orientational transition of the liquid crystal, which is supported on PS antibody-immobilized surfaces. The binding of PS-annexin V clusters to PS antibodies was confirmed by analyzing the surface topography using atomic force microscopy and an ellipsometer; the binding kinetics were determined using the surface plasmon resonance technique. This sensor could sensitively detect PS at the threshold of 0.9 ng/mL in HEPES buffer and 0.92 ng/mL in human serum and showed high selectivity, due to its specific interaction with the antibody. Thus, the proposed method is a promising platform for PS detection, not only for clinical diagnostics but also for the identification of low levels of PS in a biological environment.

Carbon quantum dots-Annexin V probe: photoinduced electron transfer mechanism, phosphatidylserine detection, and apoptotic cell imaging.

An annexin V-based probe is designed and fabricated using carbon quantum dot as highly stable and biocompatible fluorescent crystals for real-time fluorescence imaging of apoptotic cells. Carbon quantum dots were synthesized, characterized, and conjugated to annexin V. The fluorescence of CQDs at 450nm (excitation at 350nm) is quenched due to the photoinduced electron transfer between "carbon quantum dots" and two amino acids (tyrosine and tryptophan) in the annexin structure as quencher. The probe shows very strong and bright fluorescence emission in the presence of phosphatidylserine on the outer layer of the apoptotic cell membrane. It was shown that using fluorescence spectroscopy, the probe can be applied to sensitivephosphatidylserine determination and using fluorescence microscopy, it is possible to monitor cell apoptosis in real time.

Detection of Phosphatidylserine by Using Liquid Crystal Supported on the Gold-Deposited Waveform Surfaces with the Annexin V-Based Signal Enhancement

Detection of Phosphatidylserine by Using Liquid Crystal Supported on the Gold-Deposited Waveform Surfaces with the Annexin V-Based Signal Enhancement

Tyro3, Axl, Mertk receptor-mediated efferocytosis and immune regulation in the tumor environment.

Three structurally related tyrosine receptor cell surface kinases, Tyro3, Axl, and Mertk (TAM) have been recognized to modulate immune function, tissue homeostasis, cardiovasculature, and cancer. The TAM receptor family appears to operate in adult mammals across multiple cell types, suggesting both widespread and specific regulation of cell functions and immune niches. TAM family members regulate tissue homeostasis by monitoring the presence of phosphatidylserine expressed on stressed or apoptotic cells. The detection of phosphatidylserine on apoptotic cells requires intermediary molecules that opsonize the dying cells and tether them to TAM receptors on phagocytes. This complex promotes the engulfment of apoptotic cells, also known as efferocytosis, that leads to the resolution of inflammation and tissue healing. The immune mechanisms dictating these processes appear to fall upon specific family members or may involve a complex of different receptors acting cooperatively to resolve and repair damaged tissues. Here, we focus on the role of TAM receptors in triggering efferocytosis and its consequences in the regulation of immune responses in the context of inflammation and cancer.

Апоптоз и фенотип тромбоцитов у больных с острым ишемическим инсультом

Aim. To develop of knowledge of platelet apoptosis and phenotype in patients with acute ischemic stroke and to study the prognostic value of some membrane-bound proteins. Method. The study included 18 patients aged 59-78 with acute ischemic stroke. Platelet were isolated by immonomagnetic separation within the first 24 hours and at 7 days of ischemic stroke. The expression of surface antigens was measured using monoclonal antibodies. For detection of phosphatidylserine (PS) externalization platelets were incubated with Annexin V. The mitochondrial membrane potential (MMP) was measured using JC-1 during platelets apoptosis. Immediately after the incubation, platelets were analyzed by flow cytometry. Results. On day 1 of stroke, the expression of CD62p (P-selectin) was higher than in healthy subjects (18.3% vs . 4.1%, respectively, p<0.05). The PS externalization on platelets from patients with stroke was significantly higher than in healthy subjects (20.6% vs. 3.2%, respectively, p<0.01). A decrease in MMP to 70% was observed in platelets from patients with stroke compared to 94% in the control group (p<0.05). Conclusion. Therefore, acute ischemic stroke was associated with the following platelet abnormalities: increased PS expression and declined MMP in parallel with increased P-selectin expression, which reflects platelet activation. Further studies of platelet phenotype and apoptosis biomarkers will provide a deeper insight into the pathogenetic role of platelets in ischemic stroke and contribute to the development of methods for monitoring effects of antiplatelet therapy.

Inhaled TRIM72 Protein Protects Ventilation Injury to the Lung through Injury-guided Cell Repair.

Studies showed that TRIM72 is essential for repair of alveolar cell membrane disruptions, and exogenous recombinant human TRIM72 protein (rhT72) demonstrated tissue-mending properties in animal models of tissue injury. Here we examine the mechanisms of rhT72-mediated lung cell protection in vitro and test the efficacy of inhaled rhT72 in reducing tissue pathology in a mouse model of ventilator-induced lung injury. In vitro lung cell injury was induced by glass beads and stretching. Ventilator-induced lung injury was modeled by injurious ventilation at 30 ml/kg tidal volume. Affinity-purified rhT72 or control proteins were added into culture medium or applied through nebulization. Cellular uptake and in vivo distribution of rhT72 were detected by imaging and immunostaining. Exogenous rhT72 maintains membrane integrity of alveolar epithelial cells subjected to glass bead injury in a dose-dependent manner. Inhaled rhT72 decreases the number of fatally injured alveolar cells, and ameliorates tissue-damaging indicators and cell injury markers after injurious ventilation. Using in vitro stretching assays, we reveal that rhT72 improves both cellular resilience to membrane wounding and membrane repair after injury. Image analysis detected rhT72 uptake by rat alveolar epithelial cells, which can be inhibited by a cholesterol-disrupting agent. In addition, inhaled rhT72 distributes to the distal lungs, where it colocalizes with phosphatidylserine detection on nonpermeabilized lung slices to label wounded cells. In conclusion, our study showed that inhaled rhT72 accumulates in injured lungs and protects lung tissue from ventilator injury, the mechanisms of which include improving cell resilience to membrane wounding, localizing to injured membrane, and augmenting membrane repair.

Assay of Single-Cell Apoptosis by Ensemble and Single-Molecule Fluorescence Methods: Annexin-V/Polyethylene Glycol-Functionalized Quantum Dots as Probes

Apoptosis plays a critical role in many biological processes and the etiology of various diseases of the immune system. The study of apoptosis would allow both improving the diagnosis of certain diseases and serving as a target of drug screening. In this paper, we developed a sensitive assay of single-cell apoptosis using semiconductor quantum dots (QDs) as fluorescent-labeling probes. The principle of this assay is based on the detection of phosphatidylserine (PS) exposed on the plasma membrane during the drug-induced apoptosis. The QD-labeled annexin V (AV) was prepared to specifically target PS on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer, and single-molecule fluorescence correlation spectroscopy (FCS). We developed the procedures for conjugation of QDs to AV and for purification of their conjugates by gel chromatography. The obtained conjugates were characterized by FCS, capillary electrophoresis, and zeta potential analyzer. We studied the nonspecific adsorption of cells to different surface-modified QDs and found that the nonspecific adsorption effects were significantly reduced by modification of QDs with polyethylene glycol in the detection of apoptosis. In this assay, the results obtained by flow cytometry were consistent with the commercial test kit. Furthermore, a home-built single-molecule FCS system was developed for in situ study the drug-induced apoptosis. We observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. Compared with conventional methods, the fluorescent methods represented here possess high sensitivity because of the use of high photo stability and brightness QDs as labeling probes and provide the temp-spatial information on a single apoptotic cell.

Апоптоз тромбоцитов при глобальной ишемии головного мозга у крыс

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

Preparation, characterization and anti-proliferative effects of sclareol-loaded solid lipid nanoparticles on A549 human lung epithelial cancer cells

Here, sclareol-loaded solid lipid nanoparticles (sclareol-SLNs) was formulated and tested for potential geno-cytotoxicity upon A549 lung cancer cells. Sclareol-SLNs was formulated using hot homogenization. Different physicochemical features including size, zeta potential, dispersity, drug loading (DL), and encapsulation efficiency (EE) as well as in vitro drug release behavior were determined. Genotoxicity and cytotoxicity of the developed sclareol-SLNs were evaluated using DNA ladder and MTT assay, respectively. The cell death types were determined by detection of phosphatidylserine, dsDNA integrity, and proportion of different amounts in the associated cell cycle. Electron microscopy and particle size analysis showed dispersed and homogenous NPs with an average diameter of 88 nm and a high percent of EE (89%) and drug loading (42.47 mg/g). Sustained drug release was achieved at a physiologic pH, and long-term stability in terms of the size, zeta, dispersity, and EE of NPs was achieved. Cytotoxicity assay results showed that the plain sclareol inhibited A549 growth with an IC50 value of 19 μg/mL after 24 h, yet sclareol-SLNs inhibition was perceived after 48 h. DNA fragmentation assay using gel electrophoresis showed clear fragmentation in the chromatin within the cell's treated by sclareol and NPs. Flow cytometry analyses determined early and late apoptosis in sclareol and sclareol-SLNs treated cells.

MTDH and MAP3K1 are direct targets of apoptosis-regulating miRNAs in colorectal carcinoma

Artificially designed miRNAs mimics and inhibitors that specifically target known oncogenes have attracted significant research attention. Herein, we aimed to explore whether MIR-375, MIR-145, and MIR-224 are involved in induction of apoptosis of CRC cells by regulating apoptosis-mediating genes MTDH, MAP3K1, PDK1, BAX, and BCL-XL. MTT assay was used to assess cell growth. Apoptosis was determined in terms of caspase activity measurement and phosphatidylserine detection using annexin V staining by flow cytometry. Quantitative real time PCR, Western blotting, and luciferase reporter assay were carried out to validate genes regulation and targeting by miRNAs. We found that ectopic expression of MIR-375 and MIR-145, and inhibition of MIR-224 can decrease cell growth and induce cell ability to undergo early apoptosis. At mRNA level, transfected cells displayed down-regulation of MTDH, PDK1 and BCL-XL, while BAX and MAP3K1 were up-regulated. Protein expression of MTDH was decreased in cells transfected with MIR-145 mimic and MIR-224 inhibitor but remained unchanged in MIR-375 mimic-transfected cells. Furthermore, MAP3K1 protein expression exibited a decreased level after MIR-375 transient expression with no significant change after MIR-145 mimic or MIR-224 inhibitor transfection. Luciferase reporter assay revealed that MIR-375 and MIR-145 can bind to 3′UTR of MTDH, supporting that MTDH is directly targeted by both miRNAs. Similarly, MAP3K1 was found to be directly regulated by MIR-375. The study concluded that the expression modulation of tumor suppressors MIR-375 and MIR-145, and oncomiR MIR-224 have the ability to induce apoptosis of CRC cells through regulation of apoptosis mediating genes MTDH, MAP3K1, PDK1, BCL-XL and BAX.

Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts

ABSTRACTAnnona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D).

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy

BackgroundPatients with germ cell tumors (GCTs) receiving cisplatin-based chemotherapy are at increased risk of thrombosis, but the underlying cellular and molecular mechanisms remain obscure. ObjectiveTo study baseline tissue factor (TF) expression by GCT cell lines and its modulation by cisplatin treatment. MethodsTF expression was assessed by single-stage clotting and thrombin generation assay, flow cytometry, ELISA, and Western blot analysis. Cell cycle analysis and detection of phosphatidylserine (PS) membrane exposure were carried out by flow cytometry. TF mRNA was analyzed by quantitative RT-PCR. ResultsSignificant expression of TF-specific procoagulant activity (PCA) was detected on three non-seminoma (NT2, 2102Ep, NCCIT) and one seminoma cell line (TCam-2). Treatment with 0.4μM cisplatin (corresponding to the IC50) for 48hrs increased TF PCA on NT2 cells 3-fold, an effect that was largely independent of PS exposure and that could not be explained by translocation of active TF from intracellular storage pools. Cisplatin-induced TF PCA expression in NT2 cells did not occur before 12hrs, but was steady thereafter and accompanied by a 2-fold increase in total and surface-located TF antigen. Importantly, increased TF gene transcription or production and release of an intermediate factor were not involved in this process. Cell cycle analysis suggested that cisplatin-induced G2/M arrest resulted in an accumulation of procoagulant TF on the membrane surface of NT2 cells. ConclusionsIn addition to induction of apoptosis/necrosis with PS-mediated activation of preformed TF, cisplatin may alter the procoagulant phenotype of GCT cells through an increase in total cellular TF antigen.

Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH

Phosphatidylserine (PS) is preferentially located in the inner leaflet of the cell membrane, and translocation of PS oxidized in fatty acyl chains to the outside of membrane has been reported as signaling to macrophage receptors to clear apoptotic cells. It was recently shown that PS can be oxidized in serine moiety of polar head-group. In the present work, a targeted lipidomic approach was applied to detecting OxPS modified at the polar head-group in keratinocytes that were exposed to the radical generator AAPH. Glycerophosphoacetic acid derivatives (GPAA) were found to be the major oxidation products of OxPS modified at the polar head-group during oxidation induced by AAPH-generated radicals, similarly to previous observations for the oxidation induced by OH radical. The neutral loss scan of 58Da and a novel precursor ion scan of m/z 137.1 (HOPO3CH2COOH) allowed the recognition of GPAA derivatives in the total lipid extracts obtained from HaCaT cells treated with AAPH. The positive identification of serine head group oxidation products in cells under controlled oxidative conditions opens new perspectives and justifies further studies in other cellular environments in order to understand fully the role of PS polar head-group oxidation in cell homeostasis and disease.

Scutellaria litwinowii Induces Apoptosis Through Both Extrinsic and Intrinsic Apoptotic Pathways in Human Promyelocytic Leukemia Cells

Scutellaria is a genus of Lamiaceae with known antiproliferative potentials. Scutellaria litwinowii Bornm. & Sint. ex Bornm. is one of the Iranian species of Scutellaria. Although there are widespread reports about the cytotoxic and antitumor effects of some species of this genus, research on the molecular mechanism responsible for the anticancer effects of S. litwinowii has not yet been conducted. In the current study, the apoptotic effects of S. litwinowii on 2 myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspases-3, -8, and -9, poly (ADP ribose) polymerase (PARP) cleavage, detection of phosphatidylserine on the outer layer of cell membrane and sub-G1 peak in the flow cytometry histogram of treated cells, suggested the induction of apoptosis. S. litwinowii also increased the Bax/Bcl-2 ratio. It could be concluded that S. litwinowii induced apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells and it might be considered as a novel candidate in the treatment of hematological malignancies.

Reduction of Phosphatidylserine Exposure through Copper Chelation Leads to Reduced Fibrin Deposition After Laser Induced Vascular Injury In Vivo.

Abstract 3049Poster Board II-1025Colocalization and assembly of blood coagulation factors in the presence of negatively charged phospholipids leads to a 1,000-fold increase in the rate of thrombin generation compared to the solution reaction. We have established prothrombin fragment 1, the region of prothrombin containing the γ-carboxy-glutamic acid domain, as a probe for anionic phospholipids including phosphatidylserine. Prothrombin fragment 1 binds with high affinity to phosphatidylserine-containing membranes in vitro and identifies phosphatidylserine exposure relevant for the site of assembly of coagulation complexes in vivo. In order to determine the effect of phosphatidylserine exposure on thrombus formation during the laser injury model in vivo, we treated mice orally with the Cu2+ chelator tetrathiomolybdate for one week prior to study. This treatment has been shown to suppress phosphatidylserine exposure in rats (PNAS, 100: 6700-05, 2003). After copper chelator treatment, normal partial thromboplastin times (39 sec vs 42 sec, p=0.5) and whole blood counts in treated versus untreated mice were similar, suggesting that copper chelation did not affect the function of coagulation factors or total blood cell counts. Annexin V and Prothrombin fragment 1 were also used to measure phosphatidylserine exposure after thrombin (1 U/ml) stimulation of washed platelets using flow cytometric analysis. Platelets from untreated mice exhibited 2-fold increase in binding of both Annexin V and Prothrombin fragment 1 after thrombin stimulation; these values are similar to those previously reported. In contrast, the platelets of treated mice did not expose phosphatidylserine upon thrombin stimulation. Treatment with copper chelator did not affect platelet degranulation, as determined by surface exposure of P-selectin in flow cytometry. In addition, total phospholipid content and the ratio of outer to inner membrane phospholipids was not affected by treatment with copper chelator, suggesting that any reduction in detection of phosphatidylserine was due to reduction in exposure on the cell surface in response to an appropriate stimulus rather than reduced biosynthesis. Fluorescently conjugated Prothrombin fragment 1 or fluorescently conjugated antibody directed against phosphatidylserine were used as probes to follow the kinetics of phosphatidylserine exposure after the laser injury of cremaster muscle arterioles of a living mouse using high speed fluorescence intravital microscopy. Endogenous platelets were labeled with a fluorescently conjugated Fab fragment of an anti-CD41 antibody and fibrin deposition was measured using a fluorescently conjugated antibody that recognizes fibrin but not fibrinogen. We observed a 42% reduction (median of 18 thrombi, p=0.02) in Prothrombin fragment 1 binding and a 60% reduction (median of 27 thrombi, p=0.0002) in anti-phosphatidylserine binding after laser injury compared to untreated animals (n=58 thrombi). The accumulation of platelets during thrombus formation was not affected by the treatment when compared to untreated mice (p=0.4). On the other hand, fibrin deposition was reduced by 64% in treated mice (median of 38 thrombi, p=0.001) when compared to untreated animals (39 of thrombi). These data suggest that suppression of phosphatidylserine exposure reduces assembly of coagulation complexes resulting in a suboptimal concentration of thrombin for full fibrin generation but sufficient thrombin to activate platelets to yield a normal platelet thrombus. This emphasizes the importance of the exposure of anionic phospholipids as the surface for the colocalization of the coagulation complexes in vivo. DisclosuresNo relevant conflicts of interest to declare.

Propolis Induced Apoptosis in MC Coy-Plovdiv Cells

ABSTRACTPropolis is a natural product with different biological activities. Current research is focused on its ability to suppress tumor growth by induction of apoptosis. However, its selective influence on normal and transformed cells is not fully studied yet.Our previous investigations determined the McCoy-Plovdiv cell line as a model system for in vitro examination of propolis. In the present study we analysed ethanol extracts of propolis from the region of East Rodopi mountains in final concentrations 0,001; 0,1; 1,0; 10 μg/ml.The proliferative activity was examined with MTT and NR cell vitality tests. The type of cell dead was proven via TUNEL test and by fluorescent detection of phosphatidylserine (PS) externalization. Cytotoxicity tests showed decreased cell vitality from 100 to 0% increasing with propolis concentration. The TUNEL test proved the presence of apoptotic cells. Propolis in concentrations 0,001-1,0 μg/ml induced apoptosis in a large number of cells. The results from PS externalization confirmed the same observation. Increasing propolis concentrations augment the intensity of the fluorescence signal and the number of apoptotic cells.Our results evidence that Bulgarian propolis induces apoptosis in McCoy-Plovdiv cells.

Contribution à l’étude de l’apoptose par la cytométrie en flux des cellules souches hématopoïétiques CD34+ avant et après le processus de congélation

Apoptosis represents a particular form of programmed cell death which appears in all the damaged cells and potentially hazardous. It plays a crucial role in the development of multicellular organisms by assuring and maintaining the cellular homeostasis. Thus, apoptosis intervenes not only in the normal process of organisms’ development but also in immune defence and in cancerous cells detection. Indeed, any blockage in the program of the apoptotic machinery would be responsible of some neurodegenerative and auto-immune diseases and could play a crucial role in different steps of carcinogenesis. Some researchers were very interested in studying apoptosis in hematopoietic stem cells CD34+ which could be intended to be reinfused to patients suffering from malignant diseases. They have noted that kinetic study of apoptosis of the hematopoietic stem cells CD34+ after the process of cryoconservation is also necessary. Such study permits to quantify the real and exact number of the viable hematopoietic stem cells CD34+ and therefore to eliminate such risk which would be associated with the reinfusion of apoptotic cells to patients. In this paper, we describe our contribution to hematopoietic stem cells CD34+ study by flow cytometry before and after cryopreservation by using annexin V as a specific probe allowing detection of phosphatidyl serine, one of the major features of apoptosis. But, we have noted a pronounced induction of apoptosis in peripheral mobilized blood compared to cytapheresis (after cryopreservation: 29.79% of apoptotic HSC CD34+ in peripheral mobilized blood but only 11.67% apoptotic HSC CD34+ in cytapheresis). Besides, we have noticed that hematopoietic stem cells CD34+ have had a statute of viability better than other mononuclear cells. These results put in value the reliability, the simplicity and the efficiency of flow cytometry for the analysis of apoptosis in hematopoietic stem cells CD34+ by following the intensity of fluorescence of annexin V.

In vitro evaluation of dichloro-bis(pyrazole)palladium(II) and dichloro-bis(pyrazole)platinum(II) complexes as anticancer agents

Cisplatin (cis-diamminedichloroplatinum) was first identified for its anti-bacterial activity, and was later also shown to be an efficient anticancer agent. However, the therapeutic use of this anticancer drug is somewhat limited by its toxic side effects, which include nephrotoxicity, nausea, and vomiting. Furthermore the development of drug-resistant tumours is commonly observed following therapy with cisplatin. Hence there is a need for improved platinum derived drugs to overcome these limitations. Apoptosis contributes significantly to the cytotoxic effects of anticancer agents such as cisplatin; therefore in this study the potential anticancer properties of a series of pyrazole palladium(II) and platinum(II) complexes, [(3,5-R(2)pz)(2)PdCl(2)] [R = H (1), R = Me (2)] and [(3,5-R(2)pz)(2)PtCl(2)] [R = H (3), R = Me (4)], were evaluated by assessment of their pro-apoptotic activity. The induction of apoptosis was measured in CHO cells by the detection of phosphatidylserine (PS) exposure using the annexin V and APOPercentage assays; DNA fragmentation using the Terminal deoxynucleotide transferase dUTP Nick End Labelling (TUNEL) assay; and the detection of activated caspase-3. The platinum complexes were shown to be considerably more active than the palladium complexes, with complex 3 demonstrating the highest level of cytotoxic and pro-apoptotic activity. The LD(50) values for complex 3 and cisplatin were 20 and 70 microM, respectively, demonstrating that the cytotoxic activity for complex 3 was three times higher than for cisplatin. Various human cancer cell lines, including CaSki, HeLa, as well as the p53 mutant Jurkat T cell line were also shown to be susceptible to complex 3. Collectively, this in vitro study provides insights into action of palladium and platinum complexes and demonstrates the potential use of these compounds, and in particular complex 3, in the development of new anticancer agents.

Apoptotic pathway induced by noscapine in human myelogenous leukemic cells

It has been shown that noscapine, an opium-derived phthalideisoquinoline alkaloid that is currently being used as an oral antitussive drug, induces apoptosis in myeloid leukemia cells. The molecular mechanism responsible for the anticancer effects of noscapine is poorly understood. In the current study, the apoptotic effects of noscapine on two myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspase-2, -3, -6, -8 and -9, poly(ADP ribose) polymerase cleavage, detection of phosphatidylserine on the outer layer of the cell membrane, nucleation of chromatin, and DNA fragmentation suggested the induction of apoptosis. Noscapine increased the Bax/Bcl-2 ratio with a significant decrease of Bcl-2 expression accompanied with Bcl-2 phosphorylation. Using an inhibitory approach, the activation of the caspase cascade involved in the noscapine-induced apoptosis was analyzed. We observed no inhibitory effect of the caspase-8 inhibitor on caspase-9 activity. In view of these results and taking into consideration that K562 cells are Fas-null, we suggested that caspase-8 is activated in a Fas-independent manner downstream of caspase-9. In conclusion, noscapine can induce apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells, and it can be a novel candidate in the treatment of hematological malignancies.

Transactivator of Transcription Fusion Protein Transduction Causes Membrane Inversion

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT.GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.