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Anti-pre-Lie algebras, Novikov algebras and commutative 2-cocycles on Lie algebrasWe introduce the notion of anti-pre-Lie algebras as the underlying algebraic structures of nondegenerate commutative 2-cocycles which are the "symmetric" version of symplectic forms on Lie algebras. They can be characterized as a class of Lie-admissible algebras whose negative left multiplication operators make representations of the commutator Lie algebras. We observe that there is a clear analogy between anti-pre-Lie algebras and pre-Lie algebras by comparing them in terms of several aspects. Furthermore, it is unexpected that a subclass of anti-pre-Lie algebras, namely admissible Novikov algebras, correspond to Novikov algebras in terms of $q$-algebras. Consequently, there is a construction of admissible Novikov algebras from commutative associative algebras with derivations or more generally, admissible pairs. The correspondence extends to the level of Poisson type structures, leading to the introduction of the notions of anti-pre-Lie Poisson algebras and admissible Novikov-Poisson algebras, whereas the latter correspond to Novikov-Poisson algebras., 29 pages

Background and aims Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like-2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. Approach and results In this study, we demonstrated that PLAGL2 was up-regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR-AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2-EGFR-HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells' response to the anti-EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. Conclusions This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.

Immunotherapy has revolutionized the treatment of cancer in recent years and achieved overall success and long-term clinical benefit in patients with a wide variety of cancer types. However, there is still a large proportion of patients exhibiting limited or no responses to immunotherapeutic strategy, some of which were even observed with hyperprogressive disease. One major obstacle restricting the efficacy is that tumor-reactive CD8+ T cells, which are central for tumor control, undergo exhaustion, and lose their ability to eliminate cancer cells after infiltrating into the strongly immunosuppressive tumor microenvironment. Thus, as a potential therapeutic rationale in the development of cancer immunotherapy, targeting or reinvigorating exhausted CD8+ T cells has been attracting much interest. Hitherto, both intrinsic and extrinsic mechanisms that govern CD8+ T-cell exhaustion have been explored. Specifically, the transcriptional and epigenetic landscapes have been depicted utilizing single-cell RNA sequencing or mass cytometry (CyTOF). In addition, cellular metabolism dictating the tumor-infiltrating CD8+ T-cell fate is currently under investigation. A series of clinical trials are being carried out to further establish the current strategies targeting CD8+ T-cell exhaustion. Taken together, despite the proven benefit of immunotherapy in cancer patients, additional efforts are still needed to fully circumvent limitations of exhausted T cells in the treatment. In this review, we will focus on the current cellular and molecular understanding of metabolic changes, epigenetic remodeling, and transcriptional regulation in CD8+ T-cell exhaustion and describe hypothetical treatment approaches based on immunotherapy aiming at reinvigorating exhausted CD8+ T cells.

BackgroundAs a novel class of noncoding RNAs, circRNAs have been recently identified to regulate tumorigenesis and aggressiveness. However, the function of circRNAs in colorectal cancer (CRC) metastasis remains unclear. We aimed to identify circRNAs that are upregulated in CRC tissues from patients and study their function in CRC metastasis.MethodsWe compared six pairs of CRC tissues and their matched adjacent non-tumor tissues by using circRNA microarray. We first evaluated the expression of circPTK2 (hsa_circ_0005273) in fresh tissues from CRC tumors and corresponding adjacent tissues by qPCR analysis. CircPTK2 expression levels in the tissue microarray with 5 years of survival information were determined by RNA-ISH analysis. Meanwhile, the expression levels of circulating circPTK2 were further analyzed according to the patients’ clinical features. We analyzed cell apoptosis, colony formation, migration, and invasion in CRC cells. To further elucidate the effect of circPTK2 in CRC metastasis, we also conducted a colon cancer hepatic and pulmonary metastasis experiment. We used RNA biotin-labeled pull down and mass spectrometry to identify the target of circPTK2. We established a PDTX model to evaluate the effect of shRNA specifically targeting circPTK2 on tumor metastasis.ResultsWe identified a novel circRNA, circPTK2, which is back-spliced of three exons (exons 27, 28 and 29) of PTK2 by using circRNA microarray, bioinformatics and functional studies. CircPTK2 was elevated in CRC tissues and positively associated with tumor growth and metastasis. CRC patients with increased circPTK2 expression were positively correlated with poorer survival rates. Furthermore, our studies showed that circPTK2 could promote EMT of CRC cells in vitro and in vivo by binding to vimentin protein on sites Ser38, Ser55 and Ser82. We further demonstrated the interaction of circPTK2 and vimentin mediated the regulation of CRC by knockdown or overexpression of vimentin. In addition, we revealed that tail vein injection of shRNA specifically targeting circPTK2 blunt tumor metastasis in a patient-derived CRC xenograft model.ConclusionsCollectively, these results demonstrate that circPTK2 exerts critical roles in CRC growth and metastasis and may serve as a potential therapeutic target for CRC metastasis, and also a promising biomarker for early diagnosis of metastasis.

Additional file 2: Figure S2. CircPTK2 promoted aggressive phenotypes of CRC cells in vitro. Cells were transfected with circPTK2 siRNA or circPTK2-overexpressing plasmid for 48 h. Then cells were harvested for annexin-V and PI staining. Cell apoptosis was detected with flow cytometry analysis. The cells were stained by crystal violet to evaluate cell proliferation, migration, and invasion capability. (A, B) The effect of circPTK2 knockdown on SW480 apoptosis. (C, D) The effect of circPTK2 overexpression on SW620 apoptosis. (E-H) Representative images and quantification results of the colony formation of cells. (I-L) Representative images and quantification results of the migration and invasion of cells. The data are the mean ± SEM. *P

Circulating tumor cells (CTCs) are reported as the precursor of tumor metastases, implying that stifling CTCs would be beneficial for metastasis prevention. However, challenges remain for the application of therapies that aim at CTCs due to lack of effective CTC-targeting strategy and sensitive therapeutic agents. Herein, we propose a general CTC-intervention strategy based on neutrophil cyto-pharmaceuticals for suppressing CTC colonization and metastasis formation. Breast cancer 4T1 cells were infused as the mimic CTCs, and we first elucidate 4T1 cells trapped in neutrophil extracellular traps (NETs) expressing high levels of hypoxia-inducible factor-1α (HIF-1α) due to NET formation and thus promoting tumor cell colonization through enhanced migration, invasion and stemness. After verifying HIF-1α as a potential target for metastasis prevention, living neutrophil cyto-pharmaceuticals (CytPNEs) loaded with HIF-1α inhibitor are fabricated to therapeutically inhibit HIF-1α. We demonstrate that CytPNEs can specially convey the HIF-1α inhibitor to 4T1 cells according to the inflammatory chemotaxis of neutrophils and down-regulate HIF-1α, thereby inhibiting metastasis and prolonging the median survival of mice bearing breast cancer lung metastasis. Our research offers a new perspective for understanding the mechanism of CTC colonization, and puts forward the strategy of targeted intervention of CTCs as a meaningful treatment for tumor metastasis. This article is protected by copyright. All rights reserved.

Immunotherapy has revolutionized the treatment of cancer in recent years and achieved overall success and long-term clinical benefit in patients with a wide variety of cancer types. However, there is still a large proportion of patients exhibiting limited or no responses to immunotherapeutic strategy, some of which were even observed with hyperprogressive disease. One major obstacle restricting the efficacy is that tumor-reactive CD8

Background/Aims: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.

SummaryCD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44-/- ) were compared to platelets from corresponding wild-type mice (cd44+/+ ). In resting platelets, P-selectin abundance, αllbβ3 inte-grin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44-/- and cd44+/+ platelets. Platelet degranulation and αllbβ3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca2+-store depletion with thapsigargin (1 µM), effects more pronounced in cd44-/- than in cd44+/+ platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44-/- than in cd44+/+ platelets. Thrombin further decreased forward scatter in cd44-/- and cd44+/+ platelets, an effect which tended to be again more pronounced in cd44-/- than in cd44+/+ platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s-1) were significantly augmented in cd44-/- mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and prothrombotic potential of platelets.

Background/Aims: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca2+-concentration ([Ca2+]i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. Methods: Blood was drawn from healthy young (n=11, age 31.3±1.7 years) and elderly (n=16, age 88.6±0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. Results: Elderly individuals suffered from severe anemia (hemoglobin 10.5±0.3 g/100 ml) despite enhanced number of reticulocytes (2.3±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5±0.2%) than in the young volunteers (1.3±0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca2+]i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. Conclusions: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca2+ influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease. (Hepatology 2015;61:275–284)

Recently we have reported potent anti-cancer actions of various steroidal Na + /K + ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na + /K + ATPase inhibitor induced late de-phosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na + /K + ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, up-regulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.

Background: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. Methods: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). Results: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. Conclusions: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

Fil: Bissinger, Rosi. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Lang, Elisabeth. University of Duesseldorf. Hepatology and Infectious Diseases. Department of Gastroenterology; Alemania Fil: Ghashghaeinia, Mehrdad. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Zelenak, Christine. Charité Medical University. Department of Internal Medicine; Alemania Fil: Fehrenbacher, Birgit. University of Tuebingen. Department of Dermatology; Alemania Fil: Honisch, Sabina. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Chen, Hong. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Fakhri, Hajar. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Umbach, Anja T. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Liu, Guilai. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Rexhepaj, Rexhep. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Rexhepaj, Rexhep. University of Bonn. Institute of Biochemistry and Molecular Biology; Alemania Fil: Liu, Guoxing. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Schaller, Martin. University of Tuebingen. Department of Dermatology; Alemania Fil: Mack, Andreas F. University of Tuebingen. Institute of Anatomy; Alemania Fil: Lupescu, Adrian. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. National Institute of Health. Neurobiology Laboratory; Estados Unidos Fil: Lang, Florian. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Qadri, Syed M. University of Tuebingen. Vascular Medicine and Physiology. Institute of Cardiology; Alemania Fil: Qadri, Syed M. McMaster University. Department of Pathology and Molecular Medicine; Canadá Abstract: Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca(2+) activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2(-/-)) and corresponding wild-type mice (Gαi2(+/+)). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2(-/-) and Gαi2(+/+) mice but the mean corpuscular volume was significantly larger in Gαi2(-/-) mice. Spontaneous PS exposure of circulating Gαi2(-/-) erythrocytes was significantly lower than that of circulating Gαi2(+/+) erythrocytes. PS exposure was significantly lower in Gαi2(-/-) than in Gαi2(+/+) erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca(2+) activity and cell shrinkage. Moreover, Gαi2(-/-) erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2(+/+) erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.

Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i), which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE) through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP), which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i) in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml) and CRP (5ug/ml) within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM) and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM). However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM). Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

Background: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+-activity ([Ca2+]i), and ceramide. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from specific antibody binding. Results: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased ROS abundance (5 µg/ml), significantly increased [Ca2+]i (7.5 µg/ml) and significantly increased ceramide abundance (10 µg/ml). The up-regulation of annexin-V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca2+. Even in the absence of extracellular Ca2+, mefloquine significantly increased annexin-V-binding. Conclusions: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance.

Background/Aims: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. Methods: Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. Results: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. Conclusions: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.

// Sabina Honisch 1 , Willi Yu 1 , Guilai Liu 1 , Ioana Alesutan 1 , Syeda T. Towhid 1 , Anna Tsapara 2 , Sabine Schleicher 3 , Rupert Handgretinger 3 , Christos Stournaras 1,2,* and Florian Lang 1,* 1 Department of Physiology, University of Tubingen, Tubingen, Germany 2 Department of Biochemistry, University of Crete Medical School, Heraklion, Greece 3 Department of Hematology and Oncology, Children’s Hospital, University Hospital of Tuebingen, Tubingen, Germany * These authors are contributed equally and thus share last authorship Correspondence to: Florian Lang, email: // Keywords : cancer, apoptosis, PI-3K, BCL-2, bax Received : July 31, 2014 Accepted : February 13, 2015 Published : March 14, 2015 Abstract Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis. Chorein fosters neuronal cell survival, cortical actin polymerization and cell stiffness. In view of its anti-apoptotic effect in neurons, we explored whether chorein is expressed in cancer cells and influences cancer cell survival. RT-PCR was employed to determine transcript levels, specific siRNA to silence chorein, FACS analysis to follow apoptosis and Western blotting to quantify protein abundance. Chorein transcripts were detected in various cancer cell types. The mRNA coding for chorein and chorein protein were most abundant in drug resistant, poorly differentiated human rhabdomyosarcoma cells. Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule. Moreover, chorein silencing diminished transcript levels and protein expression of anti-apoptotic BCL-2 and enhanced transcript levels of pro-apoptotic Bax. Silencing of chorein in rhabdomyosarcoma cells was followed by mitochondrial depolarization, caspase 3 activation and stimulation of early and late apoptosis. In conclusion, chorein is expressed in various cancer cells. In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

// Sebastian Schmidt 1 , Guoxing Liu 1 , Guilai Liu 1 , Wenting Yang 1 , Sabina Honisch 1 , Stavros Pantelakos 1,2 , Christos Stournaras 1,2 , Arnd Honig 3 , Florian Lang 1 1 Department of Physiology, University of Tubingen, D72076 Tubingen, Germany 2 University of Crete Medical School, Department of Biochemistry, Voutes, Heraklion, 71110, Greece 3 Department of Gynecology, University Hospital of Wurzburg, D97080 Wurzburg, Germany Correspondence: Florian Lang, email: // Keywords : Ca2+ release activated Ca2+ channel, SOCE, Akt, SH-6, 2-APB, apoptosis Received : April 22, 2014 Accepted : May 28, 2014 Published : May 28, 2014 Abstract Mechanisms underlying therapy resistance of tumor cells include protein kinase Akt. Putative Akt targets include store-operated Ca 2+ -entry (SOCE) accomplished by pore forming ion channel unit Orai1 and its regulator STIM1. We explored whether therapy resistant (A2780cis) differ from therapy sensitive (A2780) ovary carcinoma cells in Akt, Orai1, and STIM1 expression, Ca 2+ -signaling and cell survival following cisplatin (100µM) treatment. Transcript levels were quantified with RT-PCR, protein abundance with Western blotting, cytosolic Ca 2+ -activity ([Ca 2 + ]i) with Fura-2-fluorescence, SOCE from increase of [Ca 2 + ]i following Ca 2+ -readdition after Ca 2+ -store depletion, and apoptosis utilizing flow cytometry. Transcript levels of Orai1 and STIM1, protein expression of Orai1, STIM1, and phosphorylated Akt, as well as SOCE were significantly higher in A2780cis than A2780 cells. SOCE was decreased by Akt inhibitor III (SH-6, 10µM) in A2780cis but not A2780 cells and decreased in both cell lines by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-ABP, 50µM). Phosphatidylserine exposure and late apoptosis following cisplatin treatment were significantly lower in A2780cis than A2780 cells, a difference virtually abolished by SH-6 or 2-ABP. In conclusion, Orai1/STIM1 expression and function are increased in therapy resistant ovary carcinoma cells, a property at least in part due to enhanced Akt activity and contributing to therapy resistance in those cells.

Background/Aims: Ipratropium bromide, an anticholinergic agent widely used in obstructive lung disease, has previously been shown to trigger suicidal death of nucleated cells or apoptosis. Despite their lack of mitochondria and nuclei, key organelles in the execution of apoptosis, erythrocytes may similarly undergo suicidal cell death, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether ipratropium bromide triggers eryptosis. Methods: [Ca Ca2+]i was estimated utilizing Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. Results: A 48 h exposure to ipratropium bromide (1 nM) significantly increased [Ca2+]i, decreased forward scatter and increased annexin-V-binding. Ipratropium bromide treatment was followed by slight but significant increase of hemolysis. Removal of extracellular Ca2+ or inhibition of Ca2+ permeable cation channels with amiloride (1 mM) virtually abolished cell membrane scrambling. Ca2+ ionophore ionomycin (1 µM, 30 min) increased the percentage of phosphatidylserine exposing erythrocytes to similarly high levels in the absence and presence of ipratropium bromide (1 nM). Conclusions: Ipratropium bromide triggers suicidal erythrocyte death or eryptosis, an effect mainly due to stimulation of Ca2+-entry.

The Na+-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of Ig, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.