Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: an in vivo and in vitro study.
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 Ca(2+)activity ([Ca(2+)](i)), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo.


METHODS
[Ca(2+)](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.
Department of Physiology, Tuebingen, Tübingen, Germany.
Adrian
A
Lupescu
Nazneen
N
Shaik
Kashif
K
Jilani
Christine
C
Zelenak
Elisabeth
E
Lang
Venkanna
V
Pasham
Mohanad
M
Zbidah
Ansgar
A
Plate
Michael
M
Bitzer
Michael
M
Föller
Syed M
SM
Qadri
Florian
F
Lang
D000255:Adenosine Triphosphate / Q000378:metabolism; D000970:Antineoplastic Agents / Q000009:adverse effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy; D016923:Cell Death / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy; D009536:Niacinamide / Q000009:adverse effects / Q000031:analogs & derivatives*; D018384:Oxidative Stress / Q000187:drug effects; D010671:Phenylurea Compounds / Q000009:adverse effects*; D010718:Phosphatidylserines / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000009:adverse effects*; D000077157:Sorafenib
D000970:Antineoplastic Agents; D002518:Ceramides; D010671:Phenylurea Compounds; D010718:Phosphatidylserines; D047428:Protein Kinase Inhibitors; D009536:Niacinamide; D000255:Adenosine Triphosphate; D000077157:Sorafenib