Detection Of Annexin Research Articles

The Rac1-inhibitor EHop-016 attenuates AML cell migration and enhances the efficacy of daunorubicin in MOLM-13 transplanted zebrafish larvae

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase implicated in cell migration and homing of hematopoietic cells to the hematopoietic niche, and is commonly overexpressed in acute myeloid leukemia (AML). This can lead to quiescence of leukemic blasts in the niche and reduced response to therapy. We investigated the Rac1 inhibitor EHop-016 on AML by assessing its effects on MOLM-13 cells in vitro and in zebrafish larvae, regarding cell motility and therapeutic potential in combination with daunorubicin (DNR). In vitro assessment of proliferation and viability was by measurement of 3H-thymidine incorporation and detection of Annexin V/PI positive cells. Cell motility was evaluated by measurement of migration in a transwell system. Fluorescently stained MOLM-13 cells were injected into zebrafish larvae, and individual cells followed by confocal microscopy. Cell accumulation in the caudal hematopoietic tissue (CHT) was studied using a 12-hour timelapse, while in vivo efficacy of DNR, EHop-016 or a combination was investigated over 24 h.The in vitro results showed that EHop-016 acted synergistically in combination with DNR in reducing the viability of MOLM-13 cells (Bliss synergy score above 10 %). Non-toxic concentrations of EHop-016 reduced cell migration. These findings were reproduced in zebrafish larvae: larvae receiving both DNR and EHop-016 had significantly reduced tumor burden compared to the untreated control or single treatments. The accumulation of MOLM-13 cells in the CHT was reduced in larvae receiving EHop-016 treatment.Our findings demonstrate that targeting Rac1 in AML holds promise as a complementary treatment to established chemotherapy and should be further investigated.

SAR131675 Receptor Tyrosine Kinase Inhibitor Induces Apoptosis through Bcl- 2/Bax/Cyto c Mitochondrial Pathway in Human Umbilical Vein Endothelial Cells.

Tyrosine Kinase Inhibitors (TKIs) can be used to inhibit cancer cell proliferation by targeting the vascular endothelial growth factor receptor (VEGFR) family. SAR131675 is a highly selective receptor tyrosine kinase inhibitor to VEGFR3 that reveals the inhibitory effect on proliferation in human lymphatic endothelial cells. However, the molecular mechanisms underlying this process are generally unclear. This study was performed to investigate the possible involvement of the Bcl-2/Bax/Cyto c apoptosis pathway in Human Umbilical Vein Endothelial Cells (HUVECs). In addition, the role of Reactive Oxygen Species (ROS) and mitochondrial membrane potential was evaluated. The effect of SAR131675 on HUVEC cell viability was evaluated by MTT assay. The activity of SAR131675 in inducing apoptosis was carried out through the detection of Annexin V-FITC/PI signal by flow cytometry. To determine the mechanisms underlying SAR131675 induced apoptosis, the mitochondrial membrane potential, ROS generation, the activity of caspase-3, and expression of apoptosis-related proteins such as Bcl-2, Bax, and cytochrome c were evaluated in HUVECs. SAR131675 significantly inhibited cell viability and induced apoptosis in HUVECs in a dose-dependent manner. Moreover, SAR131675 induced mitochondrial dysfunction, ROS generation, Bcl-2 down-regulation, Bax upregulation, cytochrome c release, and caspase-3 activation, which displays features of mitochondria-dependent apoptosis signaling pathway. Our present data demonstrated that SAR131675-induced cytotoxicity in HUVECs associated with the mitochondria apoptotic pathway. These results suggest that further studies are required to fully elucidate the role of TKIs in these cellular processes.

The mTOR Component, Rictor, Is Regulated By the Microenvironment to Control Dormancy and Proliferative States in Myeloma Cells

Introduction: Multiple myeloma (MM) cell dormancy and proliferative states, particularly in standard risk patients, are regulated by the BM niches and factors they secrete. Mesenchymal stem cells (MSCs) and their differentiated progeny are key microenvironmental components in MM. We established a reproducible experimental system in which normal MSCs were co-cultured with BM-dependent MM lines for 5 days, at which point MM cells were removed through a trypsinization and replating process (primed MSCs). MSCs cultured alone were similarly processed (unprimed MSCs). Conditioned media (CM) from primed MSCs, but not unprimed MSCs, consistently promoted the growth of primary MM cells from 12 newly diagnosed patients with low-risk MM and 6 patients with high-risk MM (p<0.0003). We hypothesized that primed MSCs represent a high-risk microenvironment akin to growth of MM within focal lesions. The aim of the study was to identify and study microenvironmental pathways that mediate dormancy and proliferative states in MM cells. Methods: CD138+ MM cells from 27 newly diagnosed patients were used for growth experiments and molecular characterization. BM-dependent MM lines were established through passaging MM cells from patients with advanced MM in the SCID-rab model. Unprimed and primed MSCs were molecularly characterized by global gene expression profiling (GEP) and growth factor content in CM was quantified using protein arrays. Proliferation of MSCs was determined by Ki67 immunostaining and cellular senescence by beta-galactosidase (SA-beta-Gal). MM growth was assessed after culturing primary MM cells with 50% CM from unprimed MSCs, primed MSCs or fresh media for 5 days. Cell survival and proliferation were determined by MTT assay and by detection of annexin V- and Ki67-positive MM cells by flow cytometry. Ultra low depth WGS was performed to assess copy number variation. MM cells were molecularly characterized by GEP, followed by pathway analyses using Ingenuity. Rictor activity was detected in MM cells by Western Blot and double immunostaining for Ki67 and Rictor. Adenoviral-based vectors and siRNA were used for transient RICTOR overexpression and gene expression silencing, respectively. Results: Fewer Ki67+ proliferating cells and increased numbers of SA-beta-Gal senescent cells were detected in primed MSCs compared to unprimed MSCs. Primed MSCs expressed a higher level of secreted factors such as CCL5, FGF1, IL6 and IL1B, and reduced expression of FGF7, CXCL12 (SDF1) and IGFBP2 compared to unprimed MSCs. CM of primed MSCs had a higher level of IL6, FGF1 and a lower level of IGFBP2 than CM of unprimed MSCs. There was no significant difference in the proportion of annexin V+ apoptotic MM cells cultured in CM from unprimed and primed MSCs, whereas the proportion of Ki67+ proliferating cells was 5 fold higher in MM cells treated with primed MSCs CM (p<0.03). The top genes overexpressed in MM cells treated with primed MSCs CM versus unprimed MSCs CM were related to proliferation, whereas underexpressed genes were related to dormancy including BCL2, RICTOR, and CXCR4. Pathway analyses identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways in MM cells treated with primed MSCs CM. WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes. mTOR signaling is controlled by major MM growth factors such as IL6 and IGF1, therefore we investigated the role of the mTOR2 component, Rictor, in MM growth. Blocking IL6 or IGF1 with the use of neutralizing antibodies against their receptors inhibited the stimulatory effect of primed MSCs CM on MM cell growth (p<0.01). Western blot analysis and immunostaining revealed a lower level of Rictor in MM cells treated with primed MSCs CM than unprimed MSCs CM. Furthermore, Ki67+ proliferating MM cells were negative for Rictor as assessed by double immunostaining. Transient overexpression of RICTOR inhibited the growth of 3 MM cell lines by 2-fold (p<0.001), whereas RICTOR siRNA increased the growth of primary MM cells treated with unprimed MSCs CM by 1.8-fold (n=3, p<0.03). Conclusions: Primed MSCs possess a senescence phenotype but produce MM growth factors capable of shifting MM cell status from a dormant to proliferative state through downregulation of Rictor expression in MM cells. Disclosures Walker: Celgene: Research Funding. van Rhee:Takeda: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy; Adicet Bio: Consultancy; Kite Pharma: Consultancy; Karyopharm Therapeutics: Consultancy; Sanofi Genzyme: Consultancy.

Proliferation and Molecular Risk Score of Low Risk Myeloma Cells Are Increased in High Risk Microenvironment Via Augmented Bioavailability of Growth Factors

Introduction:Multiple myeloma (MM) cells from patients with smoldering MM (SMM) and low-risk (LR) MM harbor genetic alterations typically seen in patients with high-risk (HR) disease. To test whether the bone marrow (BM) microenvironment plays a role in controlling growth of LR MM cells, we established an experimental model that mimics a HR microenvironment by co-culturing normal mesenchymal stem cells (MSCs) with HR MM cells. We previously have shown that MSC conditioned media (CM) promotes growth of MM cells more effectively than cell-cell contact, as adhesion to MSCs often promotes survival at the expense of proliferation. Therefore, we utilized CM andhypothesized that MSC CM is enriched with bioactive growth factors that facilitate proliferation of LR MM cells.The aim of the study was to test the effect of CM from unprimed and primed MSCs on the survival, growth, and molecular properties of LR MM cells, and identify molecular pathways that mediate these effects.Methods:Primed MSCs were prepared by co-culturing normal MSCs with BM-dependent MM lines for 5 days. MSCs were trypsinized, replated for 40 min followed by serial washing to remove MM cells.Molecularly classified CD138-selected LR MM cells from 8 newly diagnosed patients were treated with 50% primed CM or unprimed CM, or growth media (CONT) for 5 days. Growth and survival of primary MM cells was assessed by MTT assay and detection of annexin V/PI and KI67 by flow cytometry.Microarrays were performed on primed and unprimed MSCs (n=7) and on primary LR MM cells treated with primed and unprimed MSCs CM (n=3). Pathways were analyzed using Ingenuity. Ultra low depth WGS was performed to assess copy number variation. Protein arrays were performed to test levels of secreted factors in CM (n=7).Results:Growth of primary LR MM cells (n=8) was increased by primed CM 5.1±0.05 (p<0.0001) and 7.0±0.6 (p<0.0001) folds compared to unprimed CM or CONT, respectively. In contrast, unprimed MSC CM increased growth in these cells by 1.3 fold. Flow cytometry analyses revealed no differences in proportion of annexin V/PI+ cells. However, the proportion of KI67+ cells was increased from 0.95±0.1% in unprimed CM group to 4.6±1.5% in primed CM group (p<0.04).Primed MSC CM caused MM cell GEP70 score to increase resulting in change from LR to HR in 2 experiments and from an ultra LR score to an intermediate score in another. Pathway analyses on genes differentially expressed between primed CM- and unprimed CM-treated MM cells identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways. Top transcription regulators included FOXO3, TP53, E2F4, MYC and E2F1, whereas mir-16-5p and let-7 were the top microRNAs. Top significantly upregulated genes (>2 fold) by primed MSC CM included proliferation-related factors (MKI67, TOP2A, CCNB1, BIRC5 and RRM2), whereas underexpressed genes (< 2 fold) involved regulators of cell dormancy including BCL2 (survival), RICTOR (mTOR), HEY1 (NOTCH), JUN (AP-1) and CXCR4 (adhesion). Four genes we reported to powerfully predict progression of SMM to MM (Khan et al., Haematologica 2015) were highly upregulated in MM cells by primed MSC CM.WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes.IGF1 is a central MM growth factor and IGF binding proteins (IGFBPs) control its bioavailability. We recently reported that mesenchymal cells are the main source of IGFBPs in BM, with IGFBP2 being the most downregulated gene in MM bone (Schinke et al., CCR 2018). Expression and secretion of IGFBPs (particularly IGFBP2) by MSCs were significantly reduced by priming these cells with MM cells, whereas IGF1 levels remained unchanged. IGFBP2 markedly blocked IGF1-induced MM cell growth (p<0.0003). Addition of IGF1R inhibitor significantly blocked primed CM-induced MM cell growth (p<0.02).Conclusions:MSCs primed by HR MM cells mimic a HR microenvironment, reflected by reduced level of factors that restrain bioavailability of MM growth factors such as IGF1, resulting in shutdown of master regulators of cell dormancy, which then enable a MM cells to proliferate. Such a scenario is particularly applicable in SMM and LR disease where MM cells exhibit a low proliferative index and their expansion is accelerated in distinct HR BM microenvironmental niches such as focal lesions. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

Does the Novel Class of (2R, 4S)-N-(2, 5-Difluorophenyl)-4-Hydroxy-1-(2, 2, 2-Trifluoroacetyl) Pyrrolidine-2-Carboxamide’s Have Any Effect on Cell Viability and Apoptosis of Human Hepatocellular Carcinoma Cells?

Background: The hepatocellular carcinoma is believed to be the third cause of death due to cancer, worldwide. A new derivative of the (2R,4S)-N-(2,5-difluorophenyl)-4-hydroxy-1-(2,2,2-trifluoroacetyl) pyrrolidine-2-carboxamide compound were synthesized, employing a facile one-pot reaction of trans-4-hydroxy proline and N-(2,5-difluorophenyl)-2,2,2-trifluoroacetimidoyl chloride in the presence of TiO2 as catalysts and sodium hydrogen carbonate. Objectives: Therefore, in the present study we aimed to explore whether if the above new derivative pyrrolidine-2-carboxamide can affect cellular viability and apoptosis in cancerous HepG2 cell line. Methods: HepG2 cells were cultured in RPMI 1640 medium containing fetal bovine serum. Cultured cells were then treated with 7.8 µM to 125 µM of pyrrolidine-2-carboxamide. The toxicity of the compound was assayed by MTT method and detection of annexin V signal, using a flowcytometry system. Results: We found the pyrrolidine-2-carboxamide derived inhibition of HepG2 cells following a dose and time dependent fashion. The highest numbers of dead cells were observed in 125 µM dose, after 48 hours of treatment. Flow cytometric analysis also revealed that massive numbers of apoptotic cells were detectable in response to treatment with this pyrrolidine derivative. Conclusions: Overall, according to the data presented here, the pyrrolidine-2-carboxamide could possibly control cellular viability/apoptosis. Thus it can potentially be concerned as a useful therapeutic tool for hepatocellular carcinoma treatment.

Persian shallot, Allium hirtifolium Boiss, induced apoptosis in human hepatocellular carcinoma cells.

This study investigated the potential of Persian shallot extract as an anticancer agent in HepG2 tumor cell line, an in vitro human hepatoma cancer model system. The inhibitory effect of Persian shallot on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of Persian shallot, the activity of Persian shallot in inducing apoptosis was investigated through the detection of annexin V signal by flow cytometry and expression of some apoptosis related genes such p21, p53, puma, caspase-8 family-Bcl-2 proteins like bid, bim, bcl-2 and bax were measured by real-time PCR in HepG2 cells. Persian shallot extract inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 149μg/ml. The results of real-time PCR revealed a significant up-regulation of bid, bim, caspase-8, puma, p53, p21 and bax genes and a significant downregulation of bcl-2 gene in HepG2 cells treated with Persian shallot extract significantly. Therefore, this is the first report on an increased expression of bid, bim, caspase-8, puma, p53, p21 and bax genes and down regulation of bcl-2 gene indicating that the Persian shallot extract possibly induced the process of cell death through the intrinsic and extrinsic apoptosis pathways and triggers the programmed cell death in HepG2 tumor cell lines by modulating the expression of pro-/anti-apoptotic genes. Furthermore, we showed that Persian shallot extract increased annexin V signal and expression, resulting in apoptotic cell death of HepG2 cells after 24h treatment. Therefore, according to the results of this study, the Persian shallot extract could be considered as a potential candidate for production of drug for the prevention or treatment of human hepatoma.

Cu(o-phthalate)(phenanthroline)] Exhibits Unique Superoxide-Mediated NCI-60 Chemotherapeutic Action through Genomic DNA Damage and Mitochondrial Dysfunction.

The in cellulo catalytic production of reactive oxygen species (ROS) by copper(II) and iron(II) complexes is now recognized as a major mechanistic model in the design of effective cytotoxins of human cancer. The developmental complex, [Cu(o-phthalate)(1,10-phenanthroline)] (Cu-Ph), was recently reported as an intracellular ROS-active cytotoxic agent that induces double strand breaks in the genome of human cancer cells. In this work, we report the broad-spectrum action of Cu-Ph within the National Cancer Institute's (NCI) Developmental Therapeutics Program (DTP), 60 human cancer cell line screen. The activity profile is compared to established clinical agents-via the COMPARE algorithm-and reveals a novel mode of action to existing metal-based therapeutics. In this study, we identify the mechanistic activity of Cu-Ph through a series of molecular biological studies that are compared directly to the clinical DNA intercalator and topoisomerase II poison doxorubicin. The presence of ROS-specific scavengers was employed for in vitro and intracellular evaluation of prevailing radical species responsible for DNA oxidation with superoxide identified as playing a critical role in this mechanism. The ROS targeting properties of Cu-Ph on mitochondrial membrane potential were investigated, which showed that it had comparable activity to the uncoupling ionophore, carbonyl cyanide m-chlorophenyl hydrazine. The induction and origins of apoptotic activation were probed through detection of Annexin V and the activation of initiator (8,9) and executioner caspases (3/7) and were structurally visualized using confocal microscopy. Results here confirm a unique radical-induced mechanistic profile with intracellular hallmarks of damage to both genomic DNA and mitochondria.

Abstract 2757: Pharmaco imaging study of the effects of Debio 1143, a new orally available IAP inhibitor, in a triple negative breast cancer model

Abstract Introduction: Inhibitors of apoptosis proteins (IAPs) are key negative regulators of programmed cell death. Their frequent deregulation in most cancer types contributes to tumor cell survival and resistance to cancer therapy, making IAPs attractive therapeutic targets. Debio 1143 (formerly named AT-406), a new potent orally-available monovalent SMAC mimetic, targets multiple IAP members and is currently in clinical trials for cancer treatment. In this study, pharmaco-imaging was used to evaluate the effects of Debio 1143 on tumor cell death and metabolism in the triple negative breast cancer (TNBC) cell line MDA-MB-231. Material and Methods: The effects of Debio 1143 on caspase-3 activation (FACS), apoptosis-induced membrane changes (FACS and radioactive detection of Annexin V binding), and cell proliferation (MTS assay) were evaluated in MDA-MB-231 cells. In vivo pharmaco-imaging experiments were performed in CB17 SCID mice bearing subcutaneous MDA-MB-231 TNBC tumors. 99mTc-Annexin V SPECT/CT (Single Photon Emission Computed Tomography/Computed Tomography) imaging was performed 6 hours and 24 hours after a single treatment with vehicle, Debio 1143 (100 mg/kg per os) or paclitaxel (7.5 mg/kg intravenously), and was completed by gamma counting of organs. Tumor metabolism was evaluated by 18F-FDG (fluorodeoxyglucose) PET/CT (Positron Emission Tomography) while animals received repeated administrations of vehicle, Debio 1143 (100 mg/kg per os) or paclitaxel (7.5 mg/kg intravenously). Results: In MDA-MB-231 cells, Debio 1143 as a single agent was highly effective in inducing apoptosis. This was reflected by a dose-dependent activation of caspase-3, an increase of Annexin V cell binding, and a cytotoxic activity with a mean IC50 of 224 nM. In MDA-MB-231 tumor-bearing mice, Debio 1143 showed an effect on 99mTc-Annexin V tumor binding with an increased tumor SPECT signal and gamma counting results 6 hours after oral administration, while paclitaxel maximum effects were detected at 24 hours post-treatment. During repeated administration, oral Debio 1143 inhibited tumor growth, which was associated with a decreased tumor 18F-FDG uptake when measured during treatment (after 1 and 2 weeks of treatment). The effects on tumor growth and 18F-FDG uptake were still present 1 week after the end of the treatment period. Conclusion: This pharmaco-imaging study of the effects of Debio 1143 in TNBC showed that Debio 1143 induced apoptosis both in vitro and in vivo. Moreover, 18F-FDG PET imaging made it possible to observe the effect of Debio 1143, suggesting that this imaging technique may be useful in a clinical setting. To conclude, this study supports the use of Debio 1143 in the treatment of TNBC - a still unmet medical need. Citation Format: Alexandra Oudot, Olivier Raguin, Lucile Bauché, Francis Bichat, Anne Vaslin, Hélène Maby-El Hajjami, Claudio Zanna, Grégoire Vuagniaux, Pierre Fumoleau, François Brunotte, Bertrand Collin. Pharmaco imaging study of the effects of Debio 1143, a new orally available IAP inhibitor, in a triple negative breast cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2014-2757

THU0049 Uc-Mscs Inhibit Both T Cell Apoptosis and Autophagy in Patients with Systemic Lupus Erythematosus

BackgroundT cells from SLE patients have higher spontaneous apoptotic rate than that from healthy donors. The appearance of nucleosomes from these apoptotic cells in circulation may induce the production of...

Up-regulated lipocalin-2 in pulmonary hypertension involving in pulmonary artery SMC resistance to apoptosis.

A key feature of pulmonary hypertension (PH) is the remodeling of small pulmonary arteries due to abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. However, the cellular mechanisms underlying how PASMCs in the pathological condition of pulmonary hypertension become resistant to apoptosis remain unknown. It was recently reported that lipocalin 2 (Lcn2) is up-regulated in a wide array of malignant conditions, which facilitates tumorigenesis partly by inhibiting cell apoptosis. In this study, we observed that the expression levels of Lcn2 were significantly elevated in a rat PH model induced with monocrotaline and in patients with congenital heart disease-associated PH (CHD-PH) when compared with respective control. Therefore, we hypothesize that Lcn2 could regulate human PASMC (HPASMC) apoptosis through a mechanism. By the detection of DNA fragmentation using the TUNEL assay, the detection of Annexin V/PI-positive cells using flow cytometry, and the detection of cleaved caspase-3 and caspase-3 activity, we observed that Lcn2 significantly inhibited HPASMC apoptosis induced by serum withdrawal and H2O2 treatment. We also observed that Lcn2 down-regulated the proapoptotic protein Bax, decreased the levels of cellular ROS, and up-regulated the expression of superoxide dismutases (SOD1 and SOD2). In conclusion, Lcn2 significantly inhibits HPASMC apoptosis induced by oxidative stress via decreased intracellular ROS and elevated SODs. Up-regulation of Lcn2 in a rat PH model and CHD-PH patients may be involved in the pathological process of PH.

Comparison Of Autologous Microparticles and Fucosyltransferase-7 For Adhesion Improvement Of Hematopoietic Stem Cells To Bone Marrow Endothelial Cells In a Microfluidic Flow Chamber

The improvement of graft function and time to engraftment might help to reduce infection-related mortality in stem cell transplantation (SCT). While the concept of stem cells fucosylation for accelerated engraftment has already reached clinical study phase (for cord blood transplantation; NCT01471067), own previous work has shown an association between engraftment time and circulating microparticles bearing P-Selectin and P-Selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 contains the sialyl Lewis x (CD15s) antigen that requires fucosylation for optimal binding of P- and E-Selectin on endothelial cells. We therefore hypothesized that addition of microparticles (MP) might enhance adhesion of human stem cells (HSC) to bone marrow endothelial cells and that MP might have synergistic effects in combination with stem cell fucosylation. HSC were obtained from apheresis products of allogeneic donors, purified by Ficoll and magnetic bead separation for CD34, stained with calcein AM and perfused through an automated microfluidic flow chamber (Bioflux 200, Fluxionbio, USA) covered with a confluent layer of an immortalized human bone marrow endothelial cell line (HBMEC). Photos (and videos) were taken using a fluorescence microscope at start, 5 min and 10 min and analyzed for adherent HSC across the whole chamber (about 1.5 sqmm) using ImageJ software. Autologous MP were generated by addition of calcimycin to apheresis and isolation of MP by centrifugation. For control experiments, one part of the MP solution was passed through a 0,2µm-filter to remove MP. MP concentration (mean: 1362/µl) was assessed by detection of Annexin V binding in flow cytometry, using TrucountBeads® for quantification. Fucosylation was performed by 1h incubation of isolated CD34+ stem cells with GDP-fucose and fucosyltransferase 7 (FUT7). Successful fucosylation was controlled by CD15s staining of HSC in flow cytometry. Results of seven experiments (in duplicate) demonstrated the highest number of adherent HSC in the MPpositiv/FUT7negativ preparation (median: 32 HSC/sqmm; range: 15-78), followed by MPpositiv/FUT7positiv (30 HSC/sqmm; range: 16-38), MPnegativ/FUT7positiv (median: 25/sqmm; range: 11-27) and MPnegativ/FUT7negativ (20 HSC/sqmm; range: 0-22). Comparison of the MPpositiv/FUT7negativ and MPnegativ/FUT7negativ as well as the MPpositiv/FUT7positiv and MPpositiv/FUT7negativ preparations showed statistically significant differences in Wilcoxon rank test (p<.05) while comparison of MPpositiv/FUT7positiv vs. MPnegativ/FUT7positiv and MPnegativ/FUT7positiv vs. MPnegativ/FUT7negativ preparations did not. In summary, these results demonstrate that MP can improve HSC adhesion to bone marrow endothelial cells similar to fucosylation. The effect of fucosylation on HSC adhesion appears to be mediated by MP. However, there is not a synergistic effect between MP and fucosylation. Disclosures:No relevant conflicts of interest to declare.

Abstract 557: Identification of mitogen-activated protein kinases which are required for the growth of triple-negative breast cancers.

Abstract Background: Breast cancer is the most frequently diagnosed malignancy (besides non-melanoma skin cancer) and is the second leading cause for cancer-related death among US women. Most breast cancers overexpress the estrogen receptor (ER) or, to a lesser extent, the HER2 receptor and thus are responsive to hormonal therapies. However nearly a quarter of patients do not express ER, HER2, or progesterone receptor (“triple-negative breast cancer”) and are clinically managed as a single disease with nonspecific chemotherapies. Novel targeted therapies are urgently needed to treat these aggressive cancers. Recent large-scale studies integrating multiple dimensions of genomic data have revealed that alterations to the MAP kinase pathways are critical for the development and progression of triple-negative cancers (TNBCs). In particular, mutations in MAP3K genes have been associated with receptor-negative tumors. In this study, we hypothesized that TNBCs depend on unique MAP3K proteins distinct from those required by non-TNBCs to transduce growth signals. Methods: We performed an unbiased screen of the MAP3K family to identify genes critical for growth and tumorigenicity in six TNBC and six ER-positive cell lines. Pools of siRNA were deposited into optical microplates and cell lines were liposomally reverse-transfected. Depending on the growth rate, cells were stained with DAPI at 4 or 6 days post-transfection and nuclei were detected by high-throughput microscopy. To test for inhibition of anchorage-independent growth, cells were transfected with siRNA and the next day seeded in soft agar in 6-well dishes. Colonies were counted by machine at 1-3 weeks and compared to control siRNA-treated cells. For death assays, siRNA-treated cells were grown for 2 days and collected for western blot detection of annexin V and cleaved caspase 3. Results: We determined that MAP3K3 (MEKK3) and TAO2 (MAP3K17) are essential for anchorage-dependent growth of TNBC cells, but are not required for the proliferation of ER-positive cell lines. Among the TNBC cells, there were variable responses to knockdown of these kinases. While the growth of MDA-MB-231, HCC70, and HCC1937 cells were greatly inhibited after MAP3K3 knockdown, the growth of HCC1937 cells was unaffected by MAP3K3 knockdown but was instead suppressed by TAO2 inhibition. Knockdown of MAP3K3 and TAO2 also resulted in a dramatic reduction of anchorage-independent colony formation in these TNBC cells but not in ER-positive lines. We demonstrated that this reduction in growth is due to suppression of proliferation, and not by induction of apoptosis via annexin V apoptotic assay and detection of cleaved caspases. Conclusion: These data suggest that TNBCs evolve a dependence on specific MAP kinase pathways, and that targeting MAP3K3 and TAO2 may be an effective strategy to treat the subset of TNBCs that rely on these molecules to potentiate growth. Citation Format: Graham M. Poage, Abhijit Mazumdar, Ivan P. Uray, Powel H. Brown. Identification of mitogen-activated protein kinases which are required for the growth of triple-negative breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 557. doi:10.1158/1538-7445.AM2013-557

Limited Use of Antiseptics in Septic Surgery

ABSTRACTIn septic joint surgery, treatment with antiseptics is a standard procedure. Postinfectious degeneration of cartilage often results in early arthritis that necessitates joint replacement, even in young patients. It is still unclear whether antiseptics have only a toxic effect on chondrocytes or also induce late cell death through apoptosis. We hypothesized that commonly used antiseptics (polyhexanide, hydrogen peroxide, and chlorhexidine) induce different stages of apoptosis on human chondrocytes. Human chondrocytes were isolated and cultured. Polyhexanide, hydrogen peroxide, and chlorhexidine were added to the monolayer cultures. Early and late apoptotic cells were analyzed using flow cytometric detection of Annexin V, active caspases, and 7AAD, and fluorescence microscopy using Annexin V and propidium iodide staining. Flow cytometric analysis revealed an increase of Annexin V and active caspases expression of human chondrocytes after incubation with polyhexanide, hydrogen peroxide, and chlorhexidine. Fluorescence microscopy demonstrated a high number of apoptotic cells. Our data show that polyhexanide and chlorhexidine only promote the early stages of apoptosis without any differences in toxicity. Conversely, hydrogen peroxide induces early and later stages of apoptosis on primary human chondrocytes in vitro.

Increased susceptibility to CD8 T cell mediated killing limits the HIV-1 reservoir in naïve CD4 T cells from elite controllers (105.13)

Abstract Elite controllers are HIV-1 infected persons with undetectable levels of HIV-1 replication in the absence of antiretroviral treatment, for reasons that remain unclear. We used a flow cytometric cytotoxicity assay based on the detection of Annexin V in dying target cells to assess the susceptibility of naïve and memory CD4 T cell subsets from elite controllers, HIV progressors, HAART-treated persons and HIV-1 negative individuals to killing by identical HIV-1-specific CD8 T cells. We found that naïve CD4 T cells from elite controllers showed significantly higher susceptibilities to HIV-1-specific CD8 T cell mediated killing than naïve CD4 T cells from all other reference patient cohorts. This increased susceptibility to CD8 T cell-dependent cytotoxicity was not related to higher baseline activation or apoptosis rates. Levels of integrated HIV-1 DNA were significantly decreased in naïve CD4 T cells from elite controllers compared to the alternative patient cohorts, while integrated HIV-1 DNA in memory cell subsets were not different between the study groups. Our data suggest that a higher susceptibility of naïve CD4 T cells to the cytotoxic activities of HIV-1-specific CD8 T cells reduces the HIV-1 reservoir in naïve T cells from elite controllers, and in this way contributes to low cellular reservoirs for HIV-1 in these patients.

Abstract LB-151: Tumor VEGF-C promotes immune suppression and tolerance

Abstract The expression of the vascular endothelial growth factor, VEGF-C, is correlated with tumor invasion and hypothesized to induce metastasis through enhanced lymphangiogenesis and the creation of an “escape route”. However, new evidence suggests that VEGF-C can induce invasion in the absence of obvious tumor lymphangiogenesis, and furthermore, that lymphatic transport may play a role in modulating immunity. Still however, the role of the lymphatics as players in anti-tumor immunity and tumor invasion remains unexplored. Here we present a new model for VEGF-C induced tumor-invasion in which VEGF-C and lymphatic endothelial cells play a direct role in the suppression of anti-tumor immunity. Using a B16 F10 mouse melanoma that overexpresses VEGF-C, we demonstrated that VEGF-C induces the expression of the lymphoid homing cytokine, CCL21, within the stroma of the tumor and drives lymphocyte trafficking, fluid drainage and metastasis to the sentinel draining lymph node. Within the tumor stroma VEGF-C recruited a more regulatory immune cell infiltrate including increased regulatory T cells (CD25+FoxP3+) and M2 macrophages (CD11b+CD80-CD206+) and decreased antigen specific cytotoxic T cells (CD8+Trp2+). To test the ability of this mediated VEGF-C response to overcome host immunity we engineered a B16 F10 line to express ovalbumin (OVA) as a model tumor antigen. When we preimmunized the mice against OVA we observed that VEGF-C provided protection against vaccine (OVA/LPS) induced anti-tumor immunity. Growth of OVA bearing tumors was inhibited with vaccination and correlated with an increase in CD8+ antigen specific cytotoxic T cells. However, neither the growth nor the infiltration of cytotoxic T cells was affected by vaccination when the tumors also overexpressing VEGF-C. Additionally, to determine the fate of antigen specific CD8+ we adoptively transferred OT-I T cells by tail vein injection and observed their homing to the draining lymph nodes and tumor. In these microenvironments, when exposed to VEGF-C, adoptively transferred cells lost function over time as shown by the detection of Annexin V and decreased IFNγ expression. These results suggest that the expression of VEGF-C by a primary tumor not only promotes metastasis through the expansion of the lymphatic network but also through an immunological switch within the tumor and draining lymph node. We therefore present one of the first studies to identify an active, rather than passive, role for the lymphangiogenic growth factor, VEGF-C, and local lymphatic endothelial cells in the regulation of tumor immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2011-LB-151

Detection of Annexin A Autoantibodies in Sera From Colorectal Cancer Patients

Annexins (ANXAs) belong to a superfamily of closely related calcium and membrane-binding proteins and are overexpressed in some carcinomas. The overexpression of ANXAs presumably induces an autoantibody response, but this has not been demonstrated for sera from colorectal cancer (CRC) patients. We examined serum samples from 220 CRC patients and 216 healthy volunteers to evaluate the ANXA autoantibody response in patients by using an enzyme-linked immunosorbent assay with recombinant ANXA A4 as the antigen. The sensitivity of the anti-ANXA response from CRC patient sera was about 85% and the specificity was about 61.6%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in the same sera samples, the sensitivity and specificity values were 43.2% and 85.2%, respectively. Combined detection using ANXA autoantibodies and CEA produced better sensitivity (71.8%) and specificity (79.2%) compared with CEA sensitivity (43.2%) and anti-ANXA specificity (61.6%). The area under a receiver operating characteristic curve was 0.794 for ANXA autoantibodies, 0.666 for CEA, and 0.84 for both markers together. Importantly, in comparison to CEA (27.5% seropositivity), ANXA autoantibody showed a remarkable change (81.3% seropositivity) at the early stage of CRC. Measurement of ANXA autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.

Abstract 505: Plasma treatment induces apoptosis in U87 cells in vitro and in vivo

Abstract Background Over the past few years, nonthermal atmospheric pressure plasma has emerged as a novel promising tool in medicine. Plasma is a partially ionized gas sustained by a supply of energy in various forms (electric, thermal, radiative . . .). Plasma contains free charges (electrons, ions), free radicals, excited molecules, energetic photons (UV) and generates transient electric fields. Studies have demonstrated an antitumor effect of plasma treatment on melanoma cells lines and our group has recently demonstrated an antitumor effect of plasma treatment on U87 glioma bearing mice. The aim of this work was to better understand the mechanisms involved in the antitumor effect of plasma treatment observed on U87 glioma cells in vitro and in vivo. Methods The experiments were performed using a floating electrode DBD plasma reactor. For in vitro experiments, 24-well plate were seeded with U87 cells previously transfected by the luciferase gene (U87-Luc). Cells were treated by a single fraction of plasma. Effects of treatment on proliferation and viability were assessed by bioluminescence imaging (BLI), MTT assay and by trypan blue assay. For in vivo experiments, malignant glioma U87 model was heterotopically implanted in nude mice. When tumors reached 150 mm3, mice were randomly assigned into two groups: CTRL and Plasma. Plasma was delivered as a daily fraction of 6 min at 200Hz for five consecutive days and tumors were imaged by bioluminescence before treatment and 24h after the first and the last fraction. Treatment effects on tumor cells proliferation and apoptosis were assessed 24h after the last fraction by determination of cell cycle distribution and detection of annexin V positive cells, respectively. Results and Discussion Plasma treatment showed a significant antitumoral effect on U87 glioma in vitro and in vivo. In vitro plasma induced a decrease of BLI resulting from a massive cell death. Moreover, in vivo experiments, plasma treated tumors presented an important BLI decrease of ∼70% associated with a ∼60% reduction of tumor volume as compared to CTRL. Such results are particularly encouraging in this high chemo and radioresistant model. Cell cycle distribution and annexin V analysis showed a marked apoptosis induction in the treated group. To our knowledge, only in vitro experiments have reported an apoptosis induction of plasma treatment on melanoma cell lines. DNA double strand break and ROS formation have been reported in vitro during plasma treatment and these mechanisms may participate in the induction of apoptosis observed in vivo. Our data highlight a major antitumor effect of plasma treatment both in vitro and in vivo and this effect may be linked to an apoptosis induction. Further studies are ongoing to elucidate mechanism of action of plasma in apoptosis induction and to understand the implication of the different plasma components in the cytotoxic effect. These results will allow us to develop more efficient plasma types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 505.

Induction of Caspase-Independent Apoptosis by Sickle Cell Disease (SCD) Serum in Non-SCD Leukocytes

Increased white cell counts are associated with augmented mortality and morbidity in sickle cell disease (SCD) and leukocytes play a central role in the vaso-occlusive process of sickle cell disease by adhering to the vascular endothelium and participating in mechanisms of oxidative stress and inflammation. The relevance of neutrophil death to the pathogenesis of inflammatory disease is now recognized, since alterations in the apoptotic processes of leukocytes may affect their cellular function and inflammatory processes. As such, this study investigated the effect of serum from control individuals (CON), SCD patients in steady state (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day HU) on the apoptotic processes of neutrophils (Neu). Neu were separated from whole blood of CON individuals using a ficoll-paque gradient; serum was also separated and filtered through 0.22μm ultra-filters. Pools of serum were prepared by mixing serum from 10 individuals from each subject group. Neutrophils (4×106 cells/ml) were cultured in the presence of pooled filtered serum (10% v/v) for 16h (37°C, 5%CO2) in DMEM. Apoptosis was evaluated by detection of annexin V binding, and viability was determined by MTS assay. CON Neu cultured in the presence of pooled SCD serum demonstrated a significantly higher percentage of apoptotic cells (67.6 ± 4.3%; n=4) compared to Neu cultured with CON serum (52.3 ± 3.6%; n=4; P<0.01 comp. SCD, paired t-test) or SCDHU serum (55.4 ± 4.2%; n=4; P<0.01 comp. SCD). In contrast, Caspase-3 activity was not significantly different in CON Neu following 16h of culture in the presence of CON and SCD serum (2.46±0.13 and 2.50±0.19-fold increased caspase-3 activity at 16h compared to basal activity, respect., n>5; P>0.05); Neu cultured with SCDHU serum demonstrated a higher caspase-3 activity (4.23±0.70-fold increased activity, n=8, P>0.05 Wilcox. matched pairs), but this increase was not statistically different to that of Neu cultured with CON serum. Viability did not differ significantly following 16h-culture of CON Neu with CON, SCD, or SCDHU serum (0.70±0.05; 0.80±0.06; 0.86±0.08 OD490nm, respect., n>9, P>0.05); furthermore co-incubation of the Neu with serums in the presence of the programmed necrosis inhibitor, Necrostatin (10μM), had no significant effect on cell viability (0.80±0.05; 0.83±0.05; 0.86±0.05 OD490nm, for CON, SCD and SCDHU respect., n>9, P>0.05), indicating that SCD serum does not appear to induce a process of necroptosis in Neu. Neutrophil death in SCD, like other inflammatory diseases, appears to be regulated by a complex network of both intracellular death and survival signaling pathways that may be modulated by extracellular stimuli. We demonstrate, under the conditions described herein, that filtered SCD serum can induce an apoptotic process in non-SCD neutrophils that does not appear to be mediated by alterations in caspase-3 activity. High levels of circulating TNF-α, a cytokine that has been previously found elevated in the serum of our population of SCD patients, are known to elicit neutrophil death via a caspase-independent mechanism; likewise, intracellular reactive oxygen species (ROS) production also induces caspase-independent neutrophil apoptosis. Interestingly, activity levels of superoxide dismutase (SOD), were found in significantly lower levels in SCD serum compared to CON serum (data not shown), suggesting that SCD serum may have a greater capacity to induce ROS production in neutrophils. Notably, serum from SCDHU individuals did not stimulate increased apoptosis in control neutrophils, indicating a reduction in the serum factor(s) that elicit apoptosis in these cells. As such, Neu may be subject to both anti-and pro-apoptotic stimuli in the circulation of SCD individuals. Since alterations in Neu apoptotic processes may have significant effects on Neu function and number, in turn contributing to inflammatory processes and cellular damage at sites of inflammation, studies to understand the complex balance of these mechanisms in SCD are necessary.

Autocrine and Paracrine Chemokine Receptor 7 Activation in Head and Neck Cancer: Implications for Therapy

The chemokine receptor 7 (CCR7) mediates survival and invasiveness of metastatic squamous cell carcinoma of the head and neck (SCCHN) to regional lymph nodes. Constitutive prosurvival signaling by the phosphoinositide-3 kinase/Akt pathway has been observed in SCCHN cells independent of epidermal growth factor receptor (EGFR) signaling. Human SCCHN cell lines were treated with agents that block or activate CCR7-mediated signaling, and Akt activation, cell viability in the presence or absence of EGFR inhibition, and cisplatin-induced apoptosis (in the presence or absence of Akt inhibition) were assessed by immunoblotting, the MTT assay, and the detection of annexin V, respectively. Expression and secretion of chemokines by primary tumors, metastatic nodes, and benign nodes of patients with SCCHN were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The role of paracrine activation of CCR7 on tumor growth was analyzed by comparing the growth of orthotopic tumors derived from B7E3 murine oral carcinoma cells in wild-type BALB/c mice, in paucity of lymphoid T cell (plt, deficient in CCL19 and CCL21 expression) mice, and in plt mice in which the implanted B7E3 cells overexpressed CCR7 (n = 14 mice per group). In the absence of exogenous ligand treatment, blockade of CCR7 signaling reduced levels of phosphorylated (activated) Akt and decreased SCCHN cell viability by up to 59% (95% confidence interval [CI] = 58.2% to 59.8%), enhancing the effect of EGFR inhibition. CCR7 stimulation protected metastatic SCCHN cells from cisplatin-induced apoptosis in an Akt-dependent manner. Metastatic nodes expressed and secreted higher levels of CCL19 than benign nodes or primary tumors. CCR7-positive murine SCCHN tumors grew more slowly in plt mice than in control BALB/c mice (mean average tumor volume on day 20 = 12.2 and 26.5 mm(3), respectively; difference = 14.3 mm(3), 95% CI = 12.3 to 17.1 mm(3)). Secretion of CCL19 and CCL21 by SCCHN cells and by paracrine sources combine to promote activation of CCR7 prosurvival signaling associated with tumor progression and disease relapse. CCR7 and its cognate chemokines may be useful biomarkers of SCCHN progression, and blockade of CCR7-mediated signaling may enhance the efficacy of platinum- and EGFR-based therapies.