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Background/aims: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma., Methods: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system., Results: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 - an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation., Conclusions: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2+]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis., Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine., Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca2+]i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration., Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment., Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry., Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB., Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE., Methods: In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin., Results: The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h)., Conclusion: Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis., Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy., Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin., Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: Arteritis is an inflammatory disease of the vascular wall leading to ischemia and vascular occlusion. Complications of arteritis include anemia, which could, at least in theory, result from suicidal erythrocyte death or eryptosis, which is characterized by erythrocyte shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include increased cytosolic Ca2+-concentration ([Ca2+]i), oxidative stress and ceramide formation. The present study explored whether and how arteritis influences eryptosis., Methods: Blood was drawn from patients suffering from arteritis (n=17) and from healthy volunteers (n=21). PS exposure was estimated from annexin V-binding, erythrocyte volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCFDA fluorescence and ceramide abundance from FITC-conjugated antibody binding in flow cytometry. The patients suffered from anemia despite 2.8±0.4% reticulocytes., Results: The percentage of PS-exposing erythrocytes was significantly higher in patients (1.1±0.1%) than in healthy volunteers (0.3±0.1%). The increase in PS exposure was paralleled by increase in oxidative stress and [Ca2+]i but not by significant changes of ceramide abundance. Erythrocyte PS exposure and ROS production were significantly enhanced in erythrocytes exposed to patient plasma as compared to exposure to plasma from healthy volunteers., Conclusion: Arteritis is associated with enhanced eryptosis due to increased [Ca2+]i and oxidative stress. The eryptosis contributes to or even accounts for the anemia in those patients. As eryptotic erythrocytes adhere to endothelial cells of the vascular wall, they could impede microcirculation and thus contribute to vascular occlusion., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: Chorein is a protein expressed in various cell types. Loss of function mutations of the chorein encoding gene VPS13A lead to chorea-acanthocytosis, an autosomal recessive genetic disease characterized by movement disorder and behavioral abnormalities. Recent observations revealed that chorein is a powerful regulator of actin cytoskeleton in erythrocytes, platelets, K562 and endothelial HUVEC cells., Methods: In the present study we have used Western blotting to study actin polymerization dynamics, laser scanning microscopy to evaluate in detail the role of chorein in microfilaments, microtubules and intermediate filaments cytoskeleton architecture and RT-PCR to assess gene transcription of the cytoskeletal proteins., Results: We report here powerful depolymerization of actin microfilaments both, in erythrocytes and fibroblasts isolated from chorea-acanthocytosis patients. Along those lines, morphological analysis of fibroblasts from chorea-acanthocytosis patients showed disarranged microtubular network, when compared to fibroblasts from healthy donors. Similarly, the intermediate filament networks of desmin and cytokeratins showed significantly disordered organization with clearly diminished staining in patient's fibroblasts. In line with this, RT-PCR analysis revealed significant downregulation of desmin and cytokeratin gene transcripts., Conclusion: Our results provide for the first time evidence that defective chorein is accompanied by significant structural disorganization of all cytoskeletal structures in human fibroblasts from chorea-acanthocytosis patients., (© 2015 S. Karger AG, Basel.)

Background/aims: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na(+)/K(+)-ATPase. The present study explored whether JAK3 regulates Na(+)/K(+)-ATPase in dendritic cells (DCs)., Methods: Ouabain (100 µM)-sensitive (Iouabain) and K(+)-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na(+)/K(+)-ATPase α1-subunit mRNA levels by RT-PCR, Na(+)/K(+)-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active (A568V)JAK3 or inactive (K851A)JAK3., Results: Na(+)/K(+)-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3(-/-)DCs than in jak3(+/+)DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3(+/+) DCs but not in jak3(-/-)DCs. Cellular ATP was significantly lower in jak3(-/-)DCs than in jak3(+/+)DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3(-/-)DCs than in jak3(+/+)DCs and strongly blunted by ouabain in both jak3(+/+) and jak3(-/-)DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of (A568V)JAK3 but not of (K851A)JAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between (A568V)JAK3 and (K851A)JAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM)., Conclusions: JAK3 down-regulates Na(+)/K(+)-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption., (© 2015 S. Karger AG, Basel.)

Background/aims: Janus kinase 3 (JAK3), a tyrosine kinase mainly expressed in hematopoietic cells, participates in the signaling stimulating cell proliferation. The kinase is expressed in dendritic cells (DCs), antigen presenting cells involved in the initiation and regulation of antigen-specific T-cell responses. Dendritic cell function is regulated by cytosolic Ca(2+) activity ([Ca(2+)]i). Mediators increasing [Ca(2+)]i in DCs include ATP and the chemokine receptor CXCR4 ligand CXCL12. The present study explored, whether JAK3 participates in the regulation of [Ca(2+)]i in DCs., Methods: Fura-2 fluorescence was employed to determine [Ca(2+)]i, and whole cell patch clamp to decipher electrogenic transport in immature DCs isolated from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+))., Results: Without treatment, [Ca(2+)]i was similar in jak3(-/-) and jak3(+/+) DCs. Addition of ATP (100 µM) was followed by transient increase of [Ca(2+)]i reflecting Ca(2+) release from intracellular stores, an effect significantly less pronounced in jak3(-/-) DCs than in jak3(+/+) DCs. CXCL12 administration was followed by a sustained increase of [Ca(2+)]i reflecting receptor operated Ca(2+) entry, an effect significantly less rapid in jak3(-/-) DCs than in jak3(+/+) DCs. In addition, the Ca(2+) release-activated Ca(2+) channel (CRAC) current triggered by IP3-induced Ca(2+) store depletion and CXCL12 was significantly higher in DCs from jak3(+/+) mice than in jak3(-/-) mice. Inhibition of sarcoendoplasmatic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (1 µM) in the absence of extracellular Ca(2+) was followed by a transient increase of [Ca(2+)]i reflecting Ca(2+) release from intracellular stores, and subsequent readdition of extracellular Ca(2+) in the continued presence of thapsigargin was followed by a sustained increase of [Ca(2+)]i reflecting store operated Ca(2+) entry (SOCE). Both, Ca(2+) release from intracellular stores and SOCE were again significantly lower in jak3(-/-) DCs than in jak3(+/+) DCs. Pretreatment of jak3(+/+) DCs with JAK inhibitor WHI-P154 (22 µM, 10 minutes or 24 hours) significantly blunted both thapsigargin induced Ca(2+) release and subsequent SOCE. Abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular bath by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular bath increased [Ca(2+)]i reflecting Na(+)/Ca(2+) exchanger activity, an effect again significantly less pronounced in jak3(-/-) DCs than in jak3(+/+) DCs., Conclusions: JAK3 deficiency is followed by down-regulation of cytosolic Ca(2+) release, receptor and store operated Ca(2+) entry and Na(+)/Ca(2+) exchanger activity in DCs., (© 2015 S. Karger AG, Basel.)

Background/aims: Functional membrane androgen receptors (mARs) have recently been described in colon tumor cells and tissues. Their activation by specific testosterone albumin conjugates (TAC) down-regulates the PI-3K/Akt pro-survival signaling and triggers potent pro-apoptotic responses both, in vitro and in vivo. The present study explored the mAR-induced regulation of gene products implicated in the tumorigenic activity of Caco2 colon cancer cells., Methods: In Caco2 human colon cancer cells transcript levels were determined by RT-PCR, protein abundance and phosphorylation by Western blotting and confocal microscopy, as well as cytoskeletal architecture by confocal microscopy., Results: We report time dependent significant decrease in Tyr-416 phosphorylation of c-Src upon mAR activation. In line with the reported late down-regulation of the PI-3K/Akt pathway in testosterone-treated colon tumors, GSK-3beta was phosphorylated at Tyr-216 after long term stimulation of the cells with TAC, a finding supporting the role of this kinase to promote apoptosis. PCR analysis revealed significant decrease of beta-catenin and cyclin D1 transcript levels following TAC treatment. Moreover, confocal laser scanning microscopic analysis disclosed co-localization of beta-catenin with actin cytoskeleton. It is thus conceivable that beta-catenin may participate in the reported modulation of cytoskeletal dynamics in mAR stimulated Caco2 cells., Conclusions: Our results provide strong evidence that mAR activation regulates late expression and/or activity of the tumorigenic gene products c-Src, GSK-3beta, and beta-catenin thus facilitating the pro-apoptotic response in colon tumor cells., (© 2014 S. Karger AG, Basel.)