Triggering of Suicidal Erythrocyte Death by Pazopanib

Background/aims: The multi-targeted kinase inhibitor pazopanib, a drug employed for the treatment of a wide variety of malignancies, has previously been shown to trigger Apoptosis. Similar to Apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms involved in the triggering of eryptosis include Ca2+ entry, oxidative stress and ceramide. The present study explored, whether pazopanib induces eryptosis and, if so, whether it is effective by Ca2+ entry, oxidative stress and/or ceramide.

Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, Reactive Oxygen Species (ROS) formation from DCF dependent fluorescence, and ceramide abundance utilizing specific Antibodies.

Results: A 48 hours exposure of human erythrocytes to pazopanib significantly increased the percentage of annexin-V-binding (≥ 25 µg/ml) and of shrunken erythrocytes (≥ 50 µg/ml). Pazopanib treatment further resulted in significant hemolysis (≥ 25 µg/ml). The effect of pazopanib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Pazopanib significantly increased DCF fluorescence (50 µg/ml) and ceramide abundance (50 µg/ml).

Conclusions: Pazopanib triggers eryptosis, an effect involving Ca2+ entry, oxidative stress and ceramide.