Apoptosis In Target Cells Research Articles

Effect of modulation of epithelial-mesenchymal transition regulators Snail1 and Snail2 on cancer cell radiosensitivity by targeting of the cell cycle, cell apoptosis and cell migration/invasion.

Cancer is one of the leading causes of cancer-associated mortality worldwide. Several strategies of treatment, including radiotherapy, have been developed and used to treat this disease. However, post-treatment metastasis and resistance to treatment are two major causes for the limited effectiveness of radiotherapy in cancer patients. Epithelial-mesenchymal transition (EMT) is regulated by SNAIL family transcription factors, including Snail1 and Snail2 (Slug), and serves important roles in progression and cancer resistance to treatment. Snail1 and Slug also have been shown to be implicated in cancer treatment resistance. For resolving the resistance to treatment problems, combining the modulation of gene expression with radiotherapy is a novel strategy to treat patients with cancer. The present review focuses on the effect of Snail1 and Slug on cancer radiosensitivity by targeting cell apoptosis, the cell cycle and cell migration/invasion.

Newcastle disease virus V protein inhibits apoptosis in DF-1 cells by downregulating TXNL1

Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.

Actin Cytoskeleton Remodeling Drives Breast Cancer Cell Escape from Natural Killer-Mediated Cytotoxicity.

Elucidation of the underlying molecular mechanisms of immune evasion in cancer is critical for the development of immunotherapies aimed to restore and stimulate effective antitumor immunity. Here, we evaluate the role of the actin cytoskeleton in breast cancer cell resistance to cytotoxic natural killer (NK) cells. A significant fraction of breast cancer cells responded to NK-cell attack via a surprisingly rapid and massive accumulation of F-actin near the immunologic synapse, a process we termed "actin response." Live-cell imaging provided direct evidence that the actin response is associated with tumor cell resistance to NK-cell-mediated cell death. High-throughput imaging flow cytometry analyses showed that breast cancer cell lines highly resistant to NK cells were significantly enriched in actin response-competent cells as compared with susceptible cell lines. The actin response was not associated with a defect in NK-cell activation but correlated with reduced intracellular levels of the cytotoxic protease granzyme B and a lower rate of apoptosis in target cells. Inhibition of the actin response by knocking down CDC42 or N-WASP led to a significant increase in granzyme B levels in target cells and was sufficient to convert resistant breast cancer cell lines into a highly susceptible phenotype. The actin response and its protective effects were fully recapitulated using donor-derived primary NK cells as effector cells. Together, these findings establish the pivotal role of actin remodeling in breast cancer cell resistance to NK-cell-mediated killing.Significance: These findings establish the pivotal role of the actin cytoskeleton in driving breast cancer cell resistance to natural killer cells, a subset of cytotoxic lymphocytes with important roles in innate antitumor immunity. Cancer Res; 78(19); 5631-43. ©2018 AACR.

Evaluation of Serum Interferon-gamma Level in Vitiligo Patients

Background: Vitiligo is an acquired depigmentation disorder of the skin, resulting from a loss of functioning epidermal melanocytes. Multiple pathogenetic factors have been proposed, including the neural theory, genetic predisposition and impaired anti-oxidative defense. Cytokines are small immuneregulatory molecules that can generate an inappropriate immune response when imbalanced. IFN-γ is a pleiotropic cytokine that is a key regulator of the immune system. In addition to host defense, IFN-γ also contributes to autoimmune pathology by inducing autoantibodies, activating autologous cytotoxic T cells and inducing target cell apoptosis. It plays an important role to induce depigmentation in vitiligo. Aim: In this study, we aimed to determine whether vitiligo is associated with alterations in serum level of IFN-γ or not and to find out its association with disease course, severity, and duration. Methods: This case control study included 40 patients presented with stable (N=22) and active (N=18) vitiligo (non-segmental and segmental), diagnosed on the basis of typical clinical features, were selected as patient group. Forty age and sex matched apparently healthy individuals were also included representing the control group. Also patients with previous skin cancer or premalignant skin lesions, or taking immunosuppressive drugs as methotrexate and patients with hepatitis viral infection and those on INF therapy were excluded. They were 25 females (62.5%) and 15 males (37.5 %), their ages ranged from 18 to 45 years old (mean of 33.53). The controls were 20 females (50%) and 20 males (50%), their ages ranged from 18 to 46 years old (mean of 32.85). Results: The distribution of vitiligo in patients, 60% was vitiligo vulgaris, 25% were segmental vitiligo and 15% were acro-fascial vitiligo. The results showed no statistical significant difference (p-value > 0.05) between patients and control as regard age, sex and family history and showed statistical significant difference (p-value < 0.001) between patients and control as regard serum concentration of IFN-γ. Results also showed statistical significant (p-value < 0.001) positive (r = 0.63) correlation between IFN-γ levels and VASI score in patients group and statistical significant difference (p-value < 0.001) between IFN-γ levels and duration, clinical types and activity of the disease in patients group. Conclusion: This study proved high serum level of IFN-γ may be risk factor for vitiligo progression suggesting that it could be used as a marker for assessing vitiligo activity and may open the way for further therapeutic approaches for vitiligo. Serum IFN-γ is positively correlated with disease duration and severity, although it does not seem to be influenced by age, sex and family history of the patient.

Evaluation of Serum Interferon-gamma Level in Vitiligo Patients

Background: Vitiligo is an acquired depigmentation disorder of the skin, resulting from a loss of functioning epidermal melanocytes. Multiple pathogenetic factors have been proposed, including the neural theory, genetic predisposition and impaired anti-oxidative defense. Cytokines are small immuneregulatory molecules that can generate an inappropriate immune response when imbalanced. IFN-γ is a pleiotropic cytokine that is a key regulator of the immune system. In addition to host defense, IFN-γ also contributes to autoimmune pathology by inducing autoantibodies, activating autologous cytotoxic T cells and inducing target cell apoptosis. It plays an important role to induce depigmentation in vitiligo. Aim: In this study, we aimed to determine whether vitiligo is associated with alterations in serum level of IFN-γ or not and to find out its association with disease course, severity, and duration. Methods: This case control study included 40 patients presented with stable (N=22) and active (N=18) vitiligo (non-segmental and segmental), diagnosed on the basis of typical clinical features, were selected as patient group. Forty age and sex matched apparently healthy individuals were also included representing the control group. Also patients with previous skin cancer or premalignant skin lesions, or taking immunosuppressive drugs as methotrexate and patients with hepatitis viral infection and those on INF therapy were excluded. They were 25 females (62.5%) and 15 males (37.5 %), their ages ranged from 18 to 45 years old (mean of 33.53). The controls were 20 females (50%) and 20 males (50%), their ages ranged from 18 to 46 years old (mean of 32.85). Results: The distribution of vitiligo in patients, 60% was vitiligo vulgaris, 25% were segmental vitiligo and 15% were acro-fascial vitiligo. The results showed no statistical significant difference (p-value > 0.05) between patients and control as regard age, sex and family history and showed statistical significant difference (p-value < 0.001) between patients and control as regard serum concentration of IFN-γ. Results also showed statistical significant (p-value < 0.001) positive (r = 0.63) correlation between IFN-γ levels and VASI score in patients group and statistical significant difference (p-value < 0.001) between IFN-γ levels and duration, clinical types and activity of the disease in patients group. Conclusion: This study proved high serum level of IFN-γ may be risk factor for vitiligo progression suggesting that it could be used as a marker for assessing vitiligo activity and may open the way for further therapeutic approaches for vitiligo. Serum IFN-γ is positively correlated with disease duration and severity, although it does not seem to be influenced by age, sex and family history of the patient.

Rice Defensin OsAFP1 is a New Drug Candidate against Human Pathogenic Fungi

Fungal infections, such as candidiasis and aspergillosis, are some of the most frequent infections in humans. Although antifungal drugs are available for the treatment of these infections, antifungal agents with new mechanisms of action should be developed because of the increasing incidence of drug-resistant pathogens in recent years. In this study, a basic functional analysis of rice defensin OsAFP1, a novel antifungal drug candidate, was conducted. OsAFP1 exerted fungicidal activity against Candida albicans, the most common pathogenic fungus in humans, at 4 μM concentration, but it did not inhibit the growth of human pathogenic bacteria. In addition, OsAFP1 retained structural stability after heat treatment at 100 °C for 10 min and after serum treatment at 37 °C for 24 h. A propidium iodide (PI) uptake assay and mutational analysis revealed that amino acid residues within the C-terminal γ-core motif of OsAFP1, particularly Leu-39 and Lys-41, play an important role in its antifungal activity. Further, PI uptake and apoptosis assays suggested that OsAFP1 exerts its antifungal activity by inducing apoptosis of target cells. Immunohistochemistry showed that the OsAFP1 target molecule was located in the cell wall. These findings indicate that OsAFP1 may be developed into a potent antifungal drug.

Cardiovascular Effects of Incretin-Based Therapies and Their Therapeutic Potential

Antidiabetic drugs with incretin activity in addition to pronounced hypoglycemic activity cause moderate reduction in blood pressure and fat mass as well as improve the lipid profile in patients with type 2 diabetes mellitus (T2DM). In clinical trials the addition of glucagon-like peptide-1 (GLP-1) analogues to standart T2DM therapy leads to significantly reduce the risk of fatal and nonfatal cardiovascular complications. According to the results of many experimental and clinical studies it was shown that GLP-1 analogs protect endothelium in diabetic patients and protect cardiomyocytes after ischemia-reperfusion lesion. Pleiotropic effects of GLP-1-based therapies are realized due to the presence of GLP-1-receptor in endothelial cells, cardiomyocytes, neurons, monocytes and macrophages, as well as due to the connection of the receptor with the most important intracellular signaling cascades (through activation of protein kinase A and B). Whereby GLP-1-based therapies affect the functional condition as well as processes of regeneration and apoptosis of target cells. This review presents the results of studies the cardiovascular effects of GLP-1-based therapies of diabetes. Described proposed nowadays mechanisms of endothelium protective and cardioprotective action of GLP-1 analogs that associated with the action on endothelial function, vascular wall inflammation (the expression of adhesion molecules and inflammatory cytokines), and apoptosis of endothelial cells and cardiomyocytes.

Antioxidant and Antiapoptotic effect of aqueous extract of Pueraria tuberosa (Roxb. Ex Willd.) DC. On streptozotocin-induced diabetic nephropathy in rats

BackgroundOxidative stress and renal apoptosis play a significant role in the progression of diabetic nephropathy. The tubers of Pueraria tuberosa (Roxb. ex Willd.) DC. has been traditionally used as anti-ageing and health promotive tonic. The purpose of this study was to investigate its nephroprotective effect and mechanism via antioxidant and antiapoptotic potential in Streptozotocin-induced diabetic nephropathy (DN) in rats.MethodsThe chemical composition of aqueous extract of Pueraria tuberosa (PTY-2r) was analyzed by gas chromatography-mass spectrometry (GC-MS). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (55 mg/kg body weight) in rats. After 60 days, the rats were randomly divided into 3 groups (n = 6/each group), namely DN control (DN) group-2, DN rats treated with PTY-2r at the dose of 50 mg/100 g, group-3 and 100 mg/100 g, group-4 p.o. for 20 days. The normal rats were chosen as a normal control (NC) group-1. PTY-2r was orally given to the rats for 20 days. Reactive oxygen species (ROS), lipid peroxidation (LPO) and the activity of ROS-scavenging enzymes – superoxide dismutase (SOD), catalase (CAT) & glutathione peroxidase (GPx) were determined in the kidney tissue of DN rats. The expression of apoptosis-related proteins was measured by immunofluorescence.ResultsGC-MS analysis of PTY-2r indicated the presence of 37 compounds among them 5-Hydroxymethylfurfural (17.80%), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (17.03%), n-Hexadecanoic acid (5.18%) and 9-Octadecenoic acid (Z) - (6.69%) were found in the higher amount. A significant increase in ROS and LPO was observed along with the decreased activity of antioxidant enzymes, responsible for oxidative stress in the kidney of DN rats. Since, high oxidative stress induces apoptosis in target cells, as shown by significantly decreased expression of Bcl-2 along with increased expression of Bax, active Caspase-3 & cleaved PARP-1 in DN control rats, suggesting apoptosis. The PTY-2r treatment significantly raised the activity of antioxidant enzymes, suppressed oxidative stress and apoptosis thus, prevented urinary albumin excretion in a dose-dependent manner.ConclusionsThe findings suggest that PTY-2r exerted the nephroprotective potential against STZ induced DN rats via suppressing oxidative stress and apoptosis due to the presence of different bioactive compounds.Graphical ᅟ

Inhibition of granzyme B activity blocks inflammation induced by lipopolysaccharide through regulation of endoplasmic reticulum stress signaling in NK92 cells.

Granzyme B (GrB) is a serine protease that is expressed in the lytic granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), and which has been widely reported to serve a crucial role for target cell apoptosis. GrB may serve a non‑cytotoxic role in inflammation, but the evidence remains unclear. The present study aimed to establish an inflammatory cell model by using NK92 cells stimulated with lipopolysaccharide (LPS) to investigate whether GrB was involved in the development of inflammation. The extracellular levels of tumor necrosis factor‑α (TNF‑α), interleukin‑1β (IL‑1β) and GrB were examined by ELISA, and it was demonstrated that LPS treatment increased the extracellular levels of TNF‑α, IL‑1β and GrB, and these increased expression levels were inhibited by pretreatment with the GrB inhibitor serpin A3N (SA3N). The protein expression levels of glucose‑regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), nuclear factor‑κB (NF‑κB), inhibitor of NF‑κB (IκBα) and GrB were examined by western blot analysis. The results demonstrated that LPS stimulation increased the expression levels of GRP78, CHOP, NF‑κB and GrB, and decreased the expression of IκBα, and these changes were inhibited by SA3N, which indicated that inhibition of GrB activity may suppress endoplasmic reticulum (ER) stress signaling. Therefore, it was suggested that GrB may be a potential pro‑inflammatory factor, and inhibition of GrB activity may aid the prevention of the development of inflammation by suppressing ER stress signaling.

Targeting Cell Necroptosis and Apoptosis Induced by Shikonin via Receptor Interacting Protein Kinases in Estrogen Receptor Positive Breast Cancer Cell Line, MCF-7.

Recognition of a new therapeutic agent may activate an alternative programmed cell death for the treatment of breast cancer. Here, it has been tried to evaluate the effects of Shikonin, a naphthoquinone derivative of Lithospermum erythrorhizon, on the induction of necroptosis and apoptosis mediated by RIPK1-RIPK3 in the ER+ breast cancer cell line, MCF-7. In the current study, cell death modalities, cell cycle patterns, RIPK1 and RIPK3 expressions, caspase-3 and caspase-8 activities, reactive oxygen species and mitochondrial membrane potential have been evaluated in the Shikonin-treated MCF-7 cells. Necroptosis and apoptosis have been occurred by Shikonin, with a significant increase in RIPK1 and RIPK3 expressions, although necroptosis was the major rout in MCF-7 cells. Shikonin significantly increased the percentage of the cells in sub-G1 and also those in the later stages of cell cycle, which represents an increase in necroptosis and apoptosis. Under caspase inhibition by Z-VAD-FMK, Shikonin has stimulated necroptosis, which could be arrested by Nec-1. An increase in ROS levels and a decrease in the mitochondrial membrane potential have also been observed. On the basis of present findings, Shikonin has been suggested as a good candidate for the induction of cell death in ER+ breast cancer, although further investigations, experimental and clinical, are required.

Glioblastoma Cell Lines Display Different Sensitivities to Plasma-Activated Medium

Plasma-activated medium (PAM) is a novel chemotherapy that induces reactive oxygen species (ROS) and cell death in a wide range of cancer cell types, suggesting that PAM may be a promising therapeutic option for cancer treatment. However, dose response experiments suggest that PAM sensitivity is cell line specific. We examined the sensitivities of three glioblastoma cell lines to PAM, and found a wide variation in cell killing that was linked to differences in PAM induced ROS and apoptosis. These results indicate that the PAM sensitivity of glioblastoma cells, and potentially cancer cells more generally, is heterogeneous and likely to be dependent on the regulation of apoptosis and antioxidant pathways in target cells.

Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.

Human regulatory T cells undergo self-inflicted damage via granzyme pathways upon activation.

Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show - by using cytometry by time of flight (CYTOF) - an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non-GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.

Innate immunity protein Tag7 (PGRP-S) activates lymphocytes capable of Fasl-Fas-dependent contact killing of virus-infected cells.

The innate immunity protein Tag7 (PGRP-S, PGLYRP1) is involved in antimicrobial and antitumor defense. As shown in our previous studies, Tag7 specifically interacts with the major heat shock protein Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against tumor cells. A stable complex of Tag7 with the calcium-binding protein Mts1 (S100A4) stimulates migration of lymphocytes. Moreover, Tag7 can activate cytotoxic lymphocytes that recognize and kill HLA-negative tumor cells. Here, we have shown that Tag 7 treatment of human peripheral blood mononuclear cells (PBMCs) results in activation of different cytotoxic lymphocyte populations-natural killer (NK) cells and CD8+ NKG2D+ T lymphocytes-that kill Moloney murine leukemia virus (MMLV) infected SC-1 cells using different mechanisms of cell death induction. This mechanism in NK cells is based on the release of granzymes, which activate apoptosis in target cells, while CD8+ NKG2D+ T lymphocytes recognize the noncanonical MicA antigen on the surface of virus-containing cells and kill them via the FasL-Fas interaction, triggering the apoptotic or necroptotic cell death pathway. Preliminary incubation of PBMCs with virus-infected cells and following incubation with Tag7 results in activation of lymphocytes with a different phenotype. These lymphocytes change the spectrum of target cells and the mechanism of cell death induction, and their interaction with target cells is not species-specific. © 2017 IUBMB Life, 69(12):971-977, 2017.

Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection

CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with retroviruses. Similar to textbook knowledge the most important function of CD4+ T cells during an acute retrovirus infection seems to be their helper function for other immune cells. Whereas there was no direct anti-viral activity of CD4+ T cells during acute Friend Virus (FV) infection, they were absolutely required for the control of chronic infection. During chronic FV infection a population of activated FV-specific CD4+ T cells did not express cytotoxic molecules, but Fas Ligand that can induce Fas-induced apoptosis in target cells. Using an MHC II-restricted in vivo CTL assay we demonstrated that FV-specific CD4+ T cells indeed mediated cytotoxic effects against FV epitope peptide loaded targets. CD4 + CTL killing was also detected in FV-infected granzyme B knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies against FasL, which identified the Fas/FasL pathway as critical cytotoxic mechanism during chronic FV infection. Interestingly, targeting the co-stimulatory receptor CD137 with an agonistic antibody enhanced CD4+ T cell cytotoxicity. This immunotherapy may be an interesting new approach for the treatment of chronic viral infections.

Elevated levels of granzyme B correlated with miR-874-3p downregulation in patients with acute myocardial infarction.

Granzyme B could induce apoptosis of target cell in acute myocardial infarction (AMI) and was identified as the target of miR-874-3p by searching the miRNA database. We aim to determine the levels of granzyme B and miR-874-3p as well as assay their correlations or predict powers in AMI. We measured levels of plasma granzyme B and miR-874-3p in 80 AMI patients or 40 healthy controls and assayed their correlations or predicted powers for AMI. Elevated levels of granzyme B (16.71 ± 7.23 ng/l vs 9.27 ± 3.90 ng/l) correlated with miR-874-3p downregulation (0.20- ± 0.17-fold vs 1.00- ± 0.79-fold) in AMI patients. Plasma miR-874-3p might target granzyme B and it might be an additional biomarker for AMI.

Abstract 2648: CD47 antibody-induced engulfment of human T-cell leukemia cells by bone marrow-derived macrophages

Abstract CD47 is a trans-membrane “don’t-eat-me” signaling protein that enables tumor cells to evade clearance by neighboring phagocytes. Blocking CD47 allows phagocytes to identify and clear tumor cells and is a promising new approach for cancer immunotherapy. In this study, we characterized anti-CD47 antibody-mediated engulfment of living tumor cells (CCRF-CEM) by mouse bone-marrow derived macrophages (BMDMs) or immortalized mouse macrophages (J774A.1). Phagocytosis was quantified using a pH-sensitive cell-labeling fluorescent probe, pHrodo, and automated kinetic live-cell analysis (IncuCyte). CCRF-CEM cells were first labeled using pHrodo (250ng ml-1 for 1h), washed and then treated with antibody for 1 h. Target cells were then added to BMDMs or J774A.1 that had been seeded overnight on 96-well plates. Phase- and fluorescence images were captured and quantified every 15min. Anti-CD47 antibody (B6H12.2, 0.04-5μg ml-1), but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30’-4h), as evidenced by an increase in intracellular fluorescence as the label accumulates in the acidic phagosome. After 4h the red fluorescence area was increased by 25-fold (1975 ± 391 μm2 vs 80 ± 41 μm2). From close inspection of the time-lapse images cellular engulfment could be clearly observed, coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 as the effector cell. Interestingly, the rate and degree of engulfment appeared effector cell-dependent. The mechanism of engulfment was not via induction of target cell apoptosis since anti-CD47 did not induce PS externalization (Annexin V) or activate caspase 3/7. Anti-CD47 had no direct effect on CCRF-CEM proliferation for the first 4 h but upon longer exposures (&amp;gt;8 h) cell growth was attenuated. Our experimental findings substantiate the known pro-phagocytic effects of anti-CD47 antibodies, and provide a model system and method for quantitative functional analysis and mechanistic insight of CD47 modulators as cancer therapeutics. Citation Format: Gillian Lovell, Clare Szybut, Kalpana Patel, Hinnah Campwala, Nicola Bevan, Dan Appledorn, Tim James Dale, Derek John Trezise. CD47 antibody-induced engulfment of human T-cell leukemia cells by bone marrow-derived macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2648. doi:10.1158/1538-7445.AM2017-2648

Peptide-functionalized nanoparticles for the selective induction of apoptosis in target cells.

The study developed a prohibitin (PHB) targeted nanotherapy for selective induction of apoptosis in target cells. Gold nanoparticles (AuNPs) were bifunctionalized with adipose homing and proapoptotic peptides. The efficacy and mode of cell death induced by the AuNPs were investigated in vitro on three cancer cell lines. The antiproliferative activity of PHB-targeted bifunctionalized AuNPs was more pronounced on cells that express the PHB receptor, and demonstrated receptor-mediated targeting and selectivity. The bifunctionalized AuNPs induced cell death by apoptosis. The PHB-targeted nanotherapy under study could potentially be used for treatment of diseases that are characterized by overexpression of PHB. As such, further investigations will be conducted in vivo.

A Combination of Three Repurposed Drugs Administered at Reperfusion as a Promising Therapy for Postischemic Brain Injury.

Cerebral ischemia leads to multifaceted injury to the brain. A polytherapeutic drug that can be administered immediately after reperfusion may increase protection to the brain by simultaneously targeting multiple deleterious cascades. This study evaluated efficacy of the combination of three clinically approved drugs: lamotrigine, minocycline, and lovastatin, using two mouse models: global and focal cerebral ischemia induced by transient occlusion of the common carotid arteries or the middle cerebral artery, respectively. In vitro, the combination drug, but not single drug, protected neurons against oxygen-glucose deprivation (OGD)-induced cell death. The combination drug simultaneously targeted cell apoptosis and DNA damage induced by ischemia. Besides acting on neurons, the combination drug suppressed inflammatory processes in microglia and brain endothelial cells induced by ischemia. In a transient global ischemia model, the combination drug, but not single drug, suppressed microglial activation and inflammatory cytokine production, and reduced neuronal damage. In a transient focal ischemia model, the combination drug, but not single drug, attenuated brain infarction, suppressed infiltration of peripheral neutrophils, and reduced neurological deficits following ischemic stroke. In summary, the combination drug confers a broad-spectrum protection against ischemia/reperfusion (I/R) injury and could be a promising approach for early neuroprotection after out-of-hospital cardiac arrest or ischemic stroke.

CD47 antibody-induced engulfment of human T cell leukemia cells by bone marrowderived macrophages

Abstract CD47 is a trans-membrane “don’t-eat-me” signaling protein that enables tumor cells to evade clearance by neighboring phagocytes. Blocking CD47 allows phagocytes to clear tumor cells and is a promising new approach for cancer immunotherapy. In this study, we characterized anti-CD47 antibody-mediated engulfment of living tumor cells (CCRF-CEM) by mouse bone-marrow derived macrophages (BMDMs) or immortalized mouse macrophages (J774A.1). Phagocytosis was quantified using a pH-sensitive cell-labeling fluorescent probe, pHrodo, and automated kinetic live-cell analysis (IncuCyte). CCRF-CEM cells were labeled, washed and then treated with antibody. Target cells were then added to BMDMs or J774A.1 cells in 96-well plates. Phase- and fluorescence images were captured and quantified over time. Anti-CD47 antibody, but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30’-4h), as evidenced by a significant increase in intracellular fluorescence. Close inspection of the time-lapse images revealed clear cellular engulfment coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 macrophages. Interestingly, the rate and degree of engulfment was effector cell-dependent. The mechanism of engulfment was not via induction of target cell apoptosis since anti-CD47 did not induce PS externalization (Annexin V) or activate caspase 3/7. Our experimental findings substantiate the known pro-phagocytic effects of anti-CD47 antibodies, and provide a model system and method for quantitative functional analysis and mechanistic insight of CD47 modulators as cancer therapeutics.