No abstract

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca(2+) activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca(2+)-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin-luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca(2+). The present observations disclose a novel effect of chromium (VI), i.e. Ca(2+) entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.

Background: Sorafenib (Nexavar^®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar^® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca²⁺activity ([Ca²⁺]_i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca²⁺]_i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar^® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar^®-based chemotherapy.

Naringin is a dietary flavonoid from citrus fruits with antioxidant and antiapoptotic activity. Similar to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increased cytosolic Ca²⁺ activity, e.g. following energy depletion or oxidative stress. The present study thus explored whether naringin interferes with eryptosis. To this end, the cytosolic Ca²⁺ concentration was estimated from Fluo3 fluorescence, phosphatidylserine exposure from annexin-V-binding and cell volume from forward scatter in FACS analysis. As a result, energy depletion (48 h glucose removal) and oxidative stress (30 min exposure to 0.3 mM tert-butylhydroperoxide) increased Fluo-3 fluorescence, decreased the erythrocyte forward scatter and enhanced the percentage of annexin-V-binding erythrocytes. Naringin (up to 40 µM) did not significantly modify Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding in the presence of glucose and absence of oxidative stress. Naringin, however, significantly blunted the effect of glucose depletion and oxidative stress on Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding. In conclusion, naringin blunts the increase of cytosolic Ca²⁺ concentration, the shrinkage, the cell membrane scrambling and thus the suicidal death of erythrocytes following energy depletion or oxidative stress.

Fumagillin, a cyclohexane isolated from fungus Aspergillus fumigatus, has anti-infective and anti-cancer potency. Fumagillin is at least partially effective by inducing suicidal death or apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca 2+ -activity ([Ca 2+ ] i ) and ceramide. The present study explored whether fumagillin (5-100 lM) could stimulate eryptosis. To this end, [Ca 2+ ] i was estimated from Fluo3 fluorescence, ceramide by utilizing specific antibodies, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding and haemolysis from haemoglobin release. As a result, a 48-hr exposure to fumagillin significantly increased [Ca 2+ ] i ( ! 10 lM), enhanced ceramide abundance (100 lM), triggered annexin V binding ( ! 10 lM) and decreased forward scatter ( ! 10 lM). Fumagillin exposure was followed by slight but significant increase of haemolysis. Removal of extracellular Ca . The effect of fumagillin or of its synthetic derivative TNP-470 in cancer is considered to result in part from its anti-angiogenic potency, resulting at least in part from its inhibitory effect on methionine aminopeptidases (MetAP) [1,3,[5][6][7][8][9][10][11][12]. Beyond that, fumagillin has been shown to trigger suicidal cell death or apoptosis [8,13,14].In analogy to the apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling and cell shrinkage [15] The present study explored whether treatment of erythrocytes with fumagillin (5-100 lM) influences cytosolic Ca 2+ activity, stimulates cell membrane scrambling and modifies cell volume. Materials and MethodsErythrocytes, solutions and chemicals. Leucocyte-depleted erythrocytes were kindly provided by the blood bank of the University of T€ ubingen. The study was approved by the ethics committee of the University of T€ ubingen (184/2003V). Erythrocytes were incubated in vitro at a haematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose and 1 CaCl 2 ; pH 7.4 at 37°C for 48 hr. Where indicated, erythrocytes were exposed to fumagillin (5-100 lM; Enzo, L€ orrach, Germany) at the indicated concentrations. In Ca 2+ -free Ringer solution, 1 mM CaCl 2 was substituted by 1 mM glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA).FACS analysis of annexin V binding and forward scatter. After incubation under the respective experimental condition, 50 ll cell suspension was washed in Ringer solution containing 5 mM CaCl 2 and then stained with Annexin V FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 20 min. under protection from light. In the following, the forward scatter (FSC) of the cells was determined,...