annexin-V Expression Research Articles

Prunella vulgaris and Tussilago farfara demonstrate anti-inflammatory activity in rabbits and protect human adipose stem cells against thermal stress in vitro

Ethnopharmacological relevancePrunella vulgaris L.(PV) and Tussilago farfara (TF) are perennial herbs rich in flavonoids and phenolic compounds with immense medicinal value. PV extract (PV-E) possesses potent antipyretic, anti-inflammtory, antioxidant, antiseptic, anti-cancer and immune stimulatory properties and have been traditionally known for the treatment of wounds, ulcers and sores. TF extract (TF-E) has been known for antibacterial, antioxidant, anti-inflammatory, anti-viral, anti-diabetic, anti-cancer, anti-obesity and wound healing effects. Additionally, TF-E infusions have been used for asthma, cough, and bronchopneumonia treatments. Aim of the studyThe therapeutic efficacy of transplanted human adipose stem cells (hASCs) is abrogated under the deteriorating effects of heat stress offered by burn wounds. Earlier researches has documented antioxidant priming as an effective strategy to enhance stem cell performance. As both PV-E and TF-E are known for their potent antioxidant effects. The present study aims to examine the cryoprotective effects of PV-E and TF-E priming on hASCs against in-vitro heat-induced thermal stress. Moreover, we determined the anti-inflammatory potential of both PV-E and TF-E on rabbits. MethodsAntioxidant capacity of both PV-E and TF-E is examined via DPPH assay and anti-inflammatory activity is assessed in rabbits using carrageen-induced paw edema model of inflammation. Next, we investigate the efficacy of different doses (1.25–100 μg/ml) of PV-E and TF-E on hASCs; MTT, LDH, calcein AM staining, and wound scratch assay were used to assess cell viability, cytotoxicity, proliferation ability and cell migration potential in the cells. Then, hASCs were pretreated for 24 h with optimum doses of PV-E and TF-E determined from MTT assay results and were subsequently exposed to in-vitro thermal injury (51 °C,10 min). The cytoprotective effects of both PV-E and TF-E priming under thermal stress were investigated via MTT, LDH, annexin-V staining and gene expression analysis. ResultsBoth PV-E and TF-E extracts demonstrated potent antioxidant and effective anti-inflammatory activities, with a clear reduction in inflammation. Study on hASCs exhibited improved cell viabilities, enhanced cell proliferation and migration abilities of both extracts. While heat stress data revealed that PV-E (2.5 μg/ml) and TF-E (5 μg/ml) pretreatment significantly ameliorated effects of thermal-injuries in hASCs as depicted by significantly enhanced cell viabilities, low LDH release profile, and lower annexin-V expression and regulated gene expression of the pretreated cells. ConclusionPV-E and TF-E priming effectively enabled hASCs to combat thermal injury by significantly promoting cell survival than untreated cells. Hence, these findings suggest that PV-E and TF-E priming could be used to attain improved cellular responses and enhanced therapeutic efficacy in burnt tissue.

The effects of the combination of temozolomide and Eribulin on T98G human glioblastoma cell line: an ultrastructural study

ABSTRACT Glioblastoma tumors are the most aggressive primary brain tumors that develop resistance to temozolomide (TMZ). Eribulin (ERB) exhibits a unique mechanism of action by inhibiting microtubule dynamics during the G2/M cell cycle phase. We utilized the T98G human glioma cell line to investigate the effects of ERB and TMZ, both individually and in combination. The experimental groups were established as follows: control, E5 (5 nM ERB), T0.75 (0.75 mM TMZ), T1 (1.0 mM TMZ), and combination groups (E5+T0.75 and E5+T1). All groups showed a significant decrease in cell proliferation. Apoptotic markers revealed a time-dependent increase in annexin-V expression, across all treatment groups at the 48-hour time point. Caspase-3, exhibited an increase in the combination treatment groups at the 48-hour mark. Transmission electron microscopy (TEM) revealed normal ultrastructural features in the glioma cells of the control group. However, treatments induced ultrastructural changes within the spheroid glioblastoma model, particularly in the combination groups. These changes included a dose-dependent increase in autophagic vacuoles and apoptotic morphology of the cells. In conclusion, the similarity in the mechanism of action between ERB and TMZ suggests the potential for synergistic effects when combined. Our results highlight that this combination induced severe damage and autophagy in glioma spheroids after 48 hours.

Significance of Oct‐4 transcription factor as a pivotal therapeutic target for CD44+/24− mammary tumor initiating cells: Aiming at the root of the recurrence

Breast cancer (BC) remains one of the deadliest and frequently diagnosed metastatic cancers worldwide. Cancer stem cells (CSCs) are the cell population within the tumor niche, having an epithelial to mesenchymal (EMT) transition phenotype, high self-renewal, vigorous metastatic capacity, drug resistance, and tumor relapse. Identification of targets for induction of apoptosis is essential to provide novel therapeutic approaches in BC. Our earlier studies showed that Vitamin C induces apoptotic cell death by losing redox balance in TNBC CSCs. In this study, we have attempted to identify previously unrecognized CSC survival factors that can be used as druggable targets for bCSCs apoptosis regulators isolated from the TNBC line, MDA MB 468. After a thorough literature review, Oct-4 was identified as the most promising marker for its unique abundance in cancer and absence in normal cells and the contribution of Oct-4 to the sustenance of cancer cells. We then validated a very high expression of Oct-4 in the MDA MB 468 bCSCs population using flow-cytometry. The loss of Oct-4 was carried out using small interfering RNA(siRNA)-mediated knockdown in the bCSCs, followed by assessing for cellular apoptosis. Our results indicated that Oct-4 knockdown induced cell death, changes in cellular morphology, inhibited mammosphere formation, and positive for Annexin-V expression, thereby indicating the role of Oct-4 in bCSC survival. Moreover, our findings also suggest the direct interaction between Oct-4 and Vitamin C using in silico docking. This data, hence, contributes towards novel information about Oct-4 highlighting this molecule as a novel survival factor in bCSCs.

Differential Erythrocyte Phosphatidylserine Exposure Is Associated with Variations in Clinical and Haemostatic Parameters: A Study Involving E-β and β Thalassemia Compared to Normal Population

Introduction: Thalassemia is characterized by an ineffective erythropoiesis (IE). Increased exposure of phosphatidylserine (PS) on red blood cell (RBC) surface following membrane injury by free radical generation is an important pathway of IE triggering apoptotic signal, which is also associated with hypercoagulable state of thalassemia. The current study aimed to assess PS exposure on RBC surface using annexin-V as a surrogate marker comparing thalassemic patients with healthy controls based on different clinical & laboratory parameters. Methods: The assay was based on flowcytometric detection using FITC-conjugated annexin-V antibody for PS exposure and PC-7 conjugated glycophorin A for RBC identification. The study cohort consisted of 60 Thalassemia patients including Beta (10, 17%) and E-Beta (50, 83%), attending Department of Haematology at NRS Medical College & Hospital, Kolkata, India between March 2020 to December 2021 and matched healthy controls (n= 30). Patients were broadly segregated based on age, sex, Thalassaemia type (Beta/E-Beta), transfusion dependent (TDT) / Non-transfusion dependent (NTDT) and splenectomy status. Enrolled patient and control population were assessed for PS exposure using flow cytometer from Navios EX, Beckman Coulter and its correlation with clinical parameters like transfusion dependence, thrombotic episodes, splenectomy status; as well as laboratory parameters including hemoglobin (Hb) level, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, serum ferritin level. These were assessed using a battery of statistical tools. Independent t test and Mann-Whitney U test were done for two groups and ANOVA for more than two groups. Qualitative variables were correlated using Chi-Square test. Multivariate linear regression was used to analyze the correlation between multiple variables. P-value <0.05 was considered as statistically significant. Result: The median annexin expression was significantly high (P<0.001) in thalassemia patients with Beta (18.15%) and E-Beta (9.2%), compared to control (0.4%) although the difference was not significant between Beta and E-Beta. Similarly, a significant difference (P<0.001) in annexin-V expression was observed in TDT (18%) compared to NTDT (5.4%) as well as in splenectomised (23%; IQR 19.3-28) and non-splenectomised (8.1%; IQR 4.2-18.3) groups (P=0.002). The TDT patients showed a significant risk of iron overload characterized as serum ferritin >1000 ng/ml (37/38, 97.4%) as compared to NTDT (3/22, 13.7%), and also associated with significantly increased annexin expression (18% vs 5.4%, P<0.001). Comparable observations were obtained for pooled thalassemic population with significantly low Hb, prolonged PT and aPTT (Hb: 7.2 ± 1.3 gm/dl, PT: 17.4 ± 4.4 sec, aPTT: 45.6 ± 24.9 sec) compared to healthy control (Hb: 12.6 ± 0.7 gm/dl, PT: 12.6 ± 0.8 sec, aPTT: 26.6 ± 1.9 sec). Similar variations were also significant between TDT & NTDT. The former observation was in concordance to annexin expression as well showing significantly high (P<0.001) annexin-V expression in individuals with low hemoglobin ≤7 gm/dl, prolonged PT and aPTT. Significant annexin expression was not observed with fibrinogen level and no obvious clinical manifestations of hypercoagulability in any patients were detected. Conclusion: Therefore, it can be said that RBC PS exposure was significantly high in Thalassaemia patients and it is associated with degree of anemia, transfusion dependence, iron overload, prolonged PT / aPTT and splenectomy status. There is no significant difference in PS expression between E-Beta and Beta population. But contrary to the available literature annexin expression was more in TDT compared to NTDT patients in our study. This discrepancy might be due to suboptimal transfusions in TDT patients leading to inadequate suppression of ineffective erythropoiesis and incomplete replacement of damaged RBCs by healthy erythrocytes. Studies involving larger population are needed to validate this observation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.

Vitreous microparticles contain apoptotic signals suggesting a diabetic vitreopathy

To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.

Canine dental pulp and umbilical cord-derived mesenchymal stem cells as alternative sources for cell therapy in dogs

The use of regenerative medicine for pets has been growing in recent years, and an increasing number of studies have contributed to the widespread use of cell therapies in clinical veterinary medicine. Mesenchymal stem cells (MSCs) can be isolated from different sources such as dental pulp and umbilical cord. Aiming safety and reproducibility of cell therapy in clinical practice by using sources easily obtained that are usually discarded, this study isolated, characterized, and evaluated the proliferation and colony formation potential of canine dental pulp-derived mesenchymal stem cells (cDPSCs) and canine umbilical cord tissue (cUCSCs). Three samples from each source were collected, isolated, and cultured. MSCs were differentiated into three lineages and quantified by spectrophotometry. For immunophenotypic characterization, antibodies were used to analyze the expression of cell surface markers, and 7-AAD and Annexin-V were used to analyze cell viability and apoptosis, respectively. For the clonogenic assay, cells were cultured, the colonies were stained, and counted. For the proliferation assay, the cells were plated in flasks for three days and added EdU nucleoside. cDPSCs and cUCSCs showed plastic adherence and fibroblastic morphology after cultivation. Both sources showed differentiation potential and showed CD29 and CD44 positivity and CD14, CD45, CD34 and HLA-DR negativity, and low mortality and apoptosis rates. There was no difference in proliferation rates between sources. Overall, although cUCSCs had a higher number of colony-forming units than cDPSCs, both sources presented MSCs characteristics and can be used safely as alternative sources in cell therapy.

Chemotherapy-induced Endothelial Cell Apoptosis And Wound Repair Disruption Is Attenuated By Exercise Serum Preconditioning

PURPOSE: To determine if there are protective effects of habitual physical activity on chemotherapy-induced vascular toxicity. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with 5% serum from healthy trained (n = 3) and untrained (n = 3) females. After 24 hours of serum preconditioning, HUVECs were exposed to physiological concentrations of 5-fluorouracil (5-FU), epirubicin, cyclophosphamide, and docetaxel (FEC-T). At 12 hours, the effects of exercise and chemotherapy on HUVEC viability and apoptosis were measured using intracellular flow cytometry. To assess endothelial repair, the preconditioned endothelial monolayer was scraped and exposed to FEC-T drugs. Wounds were imaged at 0, 3, 4, 6, 12, 24, and 48 hours to determine rate of gap closure. RESULTS: FEC-T chemotherapy drugs decreased viability of HUVECs by 67 ± 6%, increased caspase-3 activity by 78 ± 4% and annexin-V expression by 88 ± 3% (all p < 0.05), compared to no drug control. Preconditioning HUVECs with serum from exercise trained females prior to chemotherapy exposure resulted in improved viability of HUVECs, and a reduction in caspase-3 and annexin-V expression compared to preconditioning with serum from untrained females (5-FU: viable HUVECs for trained: 73 ± 7% vs untrained: 54 ± 3%, p = 0.03; caspase-3 expression for trained: 27 ± 6% vs untrained: 60 ± 9%, p = 0.04; epirubicin: caspase-3 expression for trained: 34 ± 5% vs untrained 51 ± 1%, p = 0.04; annexin V expression for trained: 13 ± 1% vs untrained: 18 ± 1%, p = 0.03). FEC-T also resulted in impaired HUVEC wound healing by 100 ± 1% (p = 0.01). Preconditioning HUVECs with serum from trained individuals attenuated this impairment favourably over serum from untrained individuals (untrained Vmigration impairment: 10 ± 4% vs trained Vmigration impairment: 4 ± 1%, p = 0.02). CONCLUSION: Our results confirm that FEC-T drugs commonly used in breast cancer treatment cause apoptosis the disruption of endothelial cell repair. Chemotherapy-induced endothelial cell apoptosis and impaired wound healing may be attenuated in exercise trained individuals.

Intrinsic and extrinsic apoptosis responses in leukaemia cells following daunorubicin treatment

BackgroundDaunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course.MethodsThis study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins.ResultsDaunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells.ConclusionsThis study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.

Xao tam phan (Paramignya trimera) methanol extract induced apoptosis in hepatocellular carcinoma HepG2 cell line in vitro

Introduction: Xao Tam Phan (Paramignya trimera) has long been used in Viet Nam as an herbal medicine for the treatment of Hepatitis, hepatocellular carcinoma, and diabetes. This study aimed to determine the anti-proliferation effect of Paramignya trimera extract (P. trimera extract) on HepG2 hepatocellular carcinoma cells.&#x0D; Methods: AlamarBlue assay was used to determine the IC50 values of P. trimera extract on HepG2 cells. Adipose-derived stem cells (ADSCs) was used as normal cell control. For apoptosis examination, P. trimera extract-treated HepG2 cells were incubated with Annexin V/Propidium iodide (PI). Then they have been analyzed their expression of Annexin-V and PI by flow cytometry. The cell nuclear degradation also was evaluated by PI/Hoechst 33342 staining assay.&#x0D; Results: Doxorubicin and P. trimera extract IC50 values on HepG2 cells were 55.13 +/- 2.028 ng/ml and 582.533 +/- 16.521 mg/ml, respectively. Those on ADSCs were 5.96 +/- 0.56 ng/ml and 268.976 +/- 19.325 mg/ml, respectively. Side effect index value (SEI) of P. trimera extract was 2.175 +/- 0.12, and the SEI of doxorubicin was 8.71 +/- 0.36. Flow cytometry analysis indicated significant apoptosis on P. trimera extract-treated HepG2 cells at a dose of 500 mg/ml (32.39 +/- 2.28% apoptotic cells, and 14.63 +/- 1.59% necrotic cells). Nuclear aggregation and degradation was seen on 500 mg/ml P. trimera treated HepG2 cells.&#x0D; Conclusion: P. trimera extract could inhibit HepG2 hepatocellular carcinoma cell proliferation by inducing apoptosis.&#x0D;

Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed ‘isocarbostyril alkaloid’. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.

Lactobacillus casei CRL 431 improves endothelial and platelet functionality in a pneumococcal infection model.

Streptococcus pneumoniae is able to activate coagulation and induce platelet aggregation, both of which are typical responses to systemic inflammation. The interactions between inflammation and coagulation and between soluble adhesion molecules and endothelial cells are important in the pathogenesis of an unbalanced haemostatic system. Therefore, an exaggerated and/or insufficiently controlled haemostatic activity may appreciably contribute to the severity of the disease. The aim of the present study was to evaluate the effect of the oral administration of Lactobacillus casei CRL 431 on platelet and endothelial activation mechanisms in a respiratory pneumococcal infection model in mice. S. pneumoniae induced an increase in platelet counts and enhanced the expression of P-selectin in control group, with higher endothelial activation in lung shown by the increase in von Willebrand factor (vWF) and vascular cell adhesion molecule 1 (VCAM-1) expression. Also, infection induced a decrease in CXCR-4 leukocytes, increased expression in annexinV and cell death at the pulmonary level and decreased antithrombin levels in bronchoalveolar lavage. In contrast, L. casei mice restored platelet counts, favoured faster P-selectin expression, lower vWF levels and VCAM-1 expression than control group. Also, L. casei induced higher levels of annexinV expression and lower cell death in the lung. Moreover, it was able to modulate antithrombin levels within the normal range, which would indicate lower coagulation activation and a protective effect locally exerted by L. casei. In this work, the ability of L. casei to favourably modulate platelet and endothelial functionality during a pulmonary infection with S. pneumoniae was demonstrated. Our findings offer a promising perspective for the use of this probiotic strain in the prevention of thrombotic complications associated with pneumococcal pneumonia, especially in at-risk patients. In addition, the use of L. casei would provide novel alternatives for the prevention and treatment of thrombosis associated with various diseases.

Microparticle Responses to Aerobic Exercise and Meal Consumption in Healthy Men.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

Apoptosis: A biomarker of high-risk phenotype in pediatric acute myeloid leukemia?

Dysregulation of apoptosis has been explored in acute myeloid leukemia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown. This study was aimed to analyze percentage of apoptosis and apoptosis mediated through the intrinsic pathway with clinical outcomes in patients with pediatric AML. This prospective study included pediatric AML patients enrolled from July 2013 to August 2016. Annexin-V (marker of total apoptosis) and caspase-9 expression (marker of intrinsic pathway) was determined in baseline bone marrow (BM) samples by flow cytometery and compared with controls (unaffected BM of solid tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and event-free survival (EFS) were compared using log-rank test. A total of 151 AML patients were enrolled, median age 10 (range: 0.7-18years). Annexin-V expression in blast cells was significantly high in AML patients as compared to BM of subjects with solid tumors (P=0.01) and PB of healthy subjects (P=0.04). Caspase-9 expression in blast cells was not significantly different. Median annexin-V expression was significantly higher in patients with WBC count ≥11000/mm3 (P=0.02), poor-risk cytogenetics (P=0.02), the absence of RUNX1-RUNX1T1 translocation (P=0.004), and the absence of NPM1 mutation (P=0.05). Patients with high annexin-V expression had significantly inferior OS (P=0.05) in univariate analysis but not in multivariate analysis (P=0.32). Apoptosis as a whole was found to be activated in baseline BM samples of AML patients. High apoptosis may be associated with high-risk phenotype in this disease.

Differential Effects of In Vitro Treatment with Cinobufotalin on Three Types of Ovarian Cancer Cells.

Cinobufotalin (CINO), a cardiotonic steroid, has been used as an anticancer agent. This study assessed the cell-specific effect of CINO on SK-OV-3, CRL-1978 and CRL-11731 ovarian cancer cells which differ in terms of their respective karyotypes. Cell cultures were treated with CINO (0.1, 1, 5 and 10 μM) for 24, 48, and 72 h. Cell proliferation, migration, and invasion were measured using CellTiter, Cytoselect, and FluoroBlock assays, respectively. Expression of proliferating cell nuclear antigen (PCNA) was evaluated by western blot analysis. Cell viability was determined by fluorescence-activated cell sorting. Immunofluorescence was performed using Annexin-V staining and fluorescein isothiocyanate (FITC). Mitochondrial membrane potential (MMP) was measured using MitoTracker™ Red. CINO at 0.5 μM inhibited SK-OV-3, CRL-1978, and CRL-11731 proliferation, migration, and invasion. Each cell type differed in response to CINO doses for PCNA, Annexin-V expression and MMP. The antineoplastic property of CINO is consistent, but its mode of action varies among cell lines.

The combination of a sphingosine kinase 2 inhibitor (ABC294640) and a Bcl-2 inhibitor (ABT-199) displays synergistic anti-myeloma effects in myeloma cells without a t(11;14) translocation.

Multiple myeloma (MM) remains an incurable disease in need of the development of novel therapeutic agents and drug combinations. ABT‐199 is a specific Bcl‐2 inhibitor in clinical trials for MM; however, its activity as a single agent was limited to myeloma patients with the t(11;14) translocation who acquire resistance due to co‐expression of Mcl‐1 and Bcl‐xL. These limitations preclude its use in a broader patient population. We have recently found that a sphingosine kinase 2‐specific inhibitor (ABC294640) induces apoptosis in primary human CD138+ cells and MM cell lines. ABC294640 is currently in phase I/II clinical trials for myeloma (clinicaltrials.gov: #NCT01410981). Interestingly, ABC294640 down‐regulates c‐Myc and Mcl‐1, but does not have any effects on Bcl‐2. We first evaluated the combinatorial anti‐myeloma effect of ABC294640 and ABT‐199 in vitro in 7 MM cell lines, all of which harbor no t(11;14) translocation. Combination index calculation demonstrated a synergistic anti‐myeloma effect of the combination of ABC294640 and ABT‐199. This synergistic anti‐myeloma effect was maintained even in the presence of bone marrow (BM) stromal cells. The combination of ABC294640 and ABT‐199 led to enhanced cleavage of PARP and caspase‐3/9 and increased Annexin‐V expression, consistent with the induction of apoptosis by the combination treatment. In addition, the combination of ABC294640 and ABT‐199 resulted in the down‐regulation of the anti‐apoptotic proteins Mcl‐1, Bcl‐2, and Bcl‐xL and the cleavage of Bax and Bid. The combination induced both the mitochondrial mediated‐ and caspase‐mediated apoptosis pathways. Finally, the combination of ABC294640 and ABT‐199 resulted in augmented anti‐myeloma effect in vivo in a mouse xenograft model. These findings demonstrate that the co‐administration of ABC294640 and ABT‐199 exhibits synergistic anti‐myeloma activity in vitro and in vivo, providing justification for a clinical study of this novel combination in patients with relapsed/refractory multiple myeloma.

Native High-Density Lipoprotein and Melatonin Improve Platelet Response Induced by Glycated Lipoproteins

Activated platelets and glycated lipoproteins are responsible for atherothrombosis in diabetics. Melatonin and native high-density lipoproteins are crucial in the preservation of pro/oxidant-antioxidant balance. The aim of the present study was to investigate the in vitro effects of native high-density lipoproteins and melatonin on altering the platelet response induced by glycated lipoproteins. Low-density lipoproteins and high-density lipoproteins were purified from plasma by ultracentrifugation and were glycated with glucose for three weeks. After incubation with or without melatonin/or native highdensity lipoproteins, low-density lipoproteins, glycated low-density lipoproteins/glycated high-density lipoproteins were added to ADP-induced platelets. Oxidative parameters, caspase-3/9 and nitric oxide levels were measured spectrophotometrically; CD62-P/ annexin-V expression was determined by flow cytometry. In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, platelet malondialdehyde/ protein carbonyl, P-selectin, annexin-V, caspase-3/9 levels were increased (ranging from P &lt; 0.001 to P &lt; 0.01); glutathione and nitric oxide levels were reduced (ranging from P &lt; 0.001 to P &lt; 0.01). In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, melatonin treatment reduced malondialdehyde, protein carbonyl, CD62-P, annexin-V and caspase-3/9 (P &lt; 0.001, P &lt; 0.01) levels and elevated nitric oxide (only glycated low-density lipoproteins). In glycated low-density lipoprotein/glycated high-density lipoprotein-treated groups, native high-density lipoprotein treatment reduced malondialdehyde, protein carbonyl, annexin-V, caspase-3/9 levels (P &lt; 0.001, P &lt; 0.01) and increased glutathione; nitric oxide levels (only with gly-HDL). Both melatonin and high-density lipoproteins should be regarded as novel promising mechanism-based potential therapeutic targets to prevent atherothrombosis in diabetics.

Activation of Caspase‐8/BID Pathway in Dexamethasone‐Induced Apoptosis in a Human Lens Epithelial Cell Line

BackgroundProlonged glucocorticoid steroid usage is capable of inducing changes to the transcription of genes in lens epithelial cells and is associated with the development of several ocular diseases, including cataract formation. Mechanisms of cataract formation, however, are not well known, although apoptosis has been implicated. We therefore investigated glucocorticoid dexamethasone‐induced apoptosis in a human lensepithelial cell line.MethodsHuman lens epithelial cells were exposed to dexamethasone and apoptosis was evaluated using flow cytometry for AnnexinV expression, chromogenic substrates for Caspase activity measurements, Western blotting for Bax andBCL‐2 expression as well as PARP cleavage, and agarose gel electrophoresis for detection of DNA fragmentation. Mitochondrial pathway activity was determined by measuring the levels of Cytochrome C release, Bax translocation to the mitochondria, and BID cleavage.ResultsA significant induction of early apoptosis was observed by the AnnexinV/PI dual staining flow cytometric analyses. Increased expression ratios of pro‐apoptotic protein Bax toanti‐apoptotic protein BCL‐2 following dexamethasone exposure demonstrated apro‐apoptotic tendency. Sequential activation of caspase‐8 and caspase‐3, and BID cleavage with Cytochrome C extrusion from mitochondria, indicated an extrinsic apoptotic pathway. Nuclear changes of DNA fragmentation and PARP cleavage, both indicative of late stage apoptosis, were detected by electrophoresis only after dexamethasone exposure, despite lack of increased late stage apoptotic detection using Flow cytometry.ConclusionResults suggest that Dexamethasone exposure induces an extrinsic apoptotic pathway in a human lensepithelial cell line which follows along the activation of caspase‐8/BIDpathway and supports the hypothesis that apoptosis may be an important mechanism involved in steroid‐induced cataract formation.Support or Funding InformationIntramural Funding from the Department of Defense

Abstract 3506: Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models

Abstract BACKGROUND: The proteosome is clinically validated as a target for cancer therapeutics. Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1/2 studies of CFZ reported signals of clinical activity in solid tumors. The aim of this study was to investigate the activity of CFZ in triple negative breast cancer models. METHODS: A diverse panel of human triple negative breast cancer cell lines was used to investigate the anti-tumor activity of CFZ. Cell viability following CFZ treatment was assessed using MTT assay. Proliferation was monitored both with IncuCyte Zoom real-time imaging system and 3D-ON-TOP clonogenic assay. Annexin-V flow cytometry was used to measure apoptosis. Apoptotic protein levels were measured with Western blot and proteome profiler array. MCL1 and Ki67 expression were also assessed using immunocytochemistry. RESULTS: CFZ had marked anti-proliferative activity in MDA-MD231, MDA-MB468, BT20, and SUM149 TNBC cell lines, with IC50 values following 96-hour exposure from 8 nM to 25 nM by MTT assay. The anti-proliferative activity of CFZ was observed by 3D-ON-TOP clonogenic assay. The anti-proliferative activity of CFZ was also demonstrated with IncuCyte proliferation assay. Proliferation was inhibited by low nanomolar concentrations in both MDA-MB468 and BT20 cells. Western blot analysis of CFZ-treated BT20, MDA-MB231 and MDA-MB468 cells showed cleavage of poly ADP ribose polymerase (PARP) and CASPASE-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. However, expression level of anti-apoptotic protein MCL1 was increased in RAS-RAF mutated cells (MDA-MB231) but not in PTEN-null MDA-MD468 cells following the treatment of CFZ. Double immune-fluorescence staining of MDA-MB231 cells following the treatment of CFZ showed higher expression of MCL1 but no change in Ki67 expression. Interestingly, a combination of CFZ and TOR kinase inhibitor (MLN0128) enhanced cleaved CASPASE3 and BIM expression with a concomitant increased induction of apoptosis (annexinV expression) in MDA-MB231 cells. CONCLUSIONS: CFZ demonstrated anti-proliferative activity in TNBC cell lines in vitro. Flow cytometric analysis of annexin V-positive cells and Western blot expression of cleaved CASPASE 3, cleaved PARP and BIM indicated that CRZ -induced TNBC cell death through an apoptosis-dependent manner. Citation Format: Mathew Larson, Jennifer H. Carlson, Yuliang Sun, Tonia Bucholz, Casey Williams, Nandini Dey, Brian Leyland-Jones, Pradip De. Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3506.

TGF-β1-induced regulatory T cells directly suppress B cells through non-cytotoxic manner (IRC11P.423)

Abstract Methods: Naïve CD4+ T cells and B cells were isolated from C57BL/6 mice. Peripherally induced CD4+Foxp3+ regulatory T cells (itregs) was induced with IL-2 and TGF-β. The thymus-derived naturally CD4+Foxp3+ regulatory T cells (ntregs) was obtained from C57BL/6 mice. iTregs and nTregs cells were cultured with B cells for 3-5 days in vitro. The activation marker, proliferation, Annexin-V and 7-AAD expression by B cells was determined by FACS. For in vivo experiment, iTregs and nTregs cells were transferred to endogenous T cell-deleted lupus prone mice. The IgG antibody production was measured by ELISA at the indicated time points. Results: We found that iTregs share similar functional characteristics with nTregs, both iTregs and nTegs can directly suppress the proliferation and production of autoantibodies of B cells. Unlike nTregs, iTregs suppressed B cells through non-cytotoxic manner. Moreover, the TGF-β signal is important for the effects of iTregs on B cells. We further showed that iTregs directly suppress B cells in vitro and in vivo in lupus prone mice. Conclusions: Our data implicate the role and advantage of iTregs in treating B cell-mediated autoimmune diseases.