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Triggering of Suicidal Erythrocyte Death by Pazopanib
Elena Signoretto1, Jens Zierle, Rosi Bissinger
1Department of Cardiology & Vascular Medicine, University of Tuebingen, Tuebingen, Germany.
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.
Insights
Pazopanib, a cancer drug, induces eryptosis, a form of programmed red blood cell death. This process involves calcium influx, oxidative stress, and increased ceramide levels, contributing to cell shrinkage and membrane changes.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Pazopanib is a multi-targeted kinase inhibitor used in cancer therapy.
- Eryptosis, or "suicidal death" of erythrocytes, shares mechanisms with apoptosis.
- Eryptosis is characterized by cell shrinkage and phosphatidylserine translocation.
Purpose of the Study:
- To investigate if pazopanib induces eryptosis in human erythrocytes.
- To determine the role of calcium (Ca2+) entry, oxidative stress, and ceramide in pazopanib-induced eryptosis.
Main Methods:
- Assessed phosphatidylserine exposure using annexin-V binding.
- Measured cell volume via forward scatter.
- Quantified reactive oxygen species (ROS) using DCF fluorescence.
- Determined ceramide abundance with specific antibodies.
Main Results:
- Pazopanib exposure increased annexin-V binding and erythrocyte shrinkage.
- Significant hemolysis was observed following pazopanib treatment.
- The effect on annexin-V binding was partially dependent on extracellular Ca2+.
- Pazopanib elevated ROS production and ceramide levels.
Conclusions:
- Pazopanib effectively triggers eryptosis in human erythrocytes.
- The induction of eryptosis by pazopanib involves Ca2+ entry, oxidative stress, and ceramide accumulation.