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Triggering of Suicidal Erythrocyte Death by Gefitinib
Abdulla Al Mamun Bhuyan1, Teresa Wagner1, Hang Cao1
1Department of Internal Medicine III, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Background/Aims:
The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress.
Methods:
Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence.
Results:
A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence.
Conclusions:
Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.
Insights
Gefitinib, used to treat lung cancer, triggers suicidal erythrocyte death (eryptosis) by causing cell shrinkage and membrane changes. This process involves extracellular calcium and oxidative stress, potentially explaining gefitinib-induced anemia.
Area of Science:
- Hematology
- Oncology
- Cell Biology
Background:
- Gefitinib is an epidermal growth factor receptor-tyrosine kinase inhibitor effective in non-small cell lung cancer.
- Gefitinib's anti-cancer effects involve apoptosis, but anemia is a known side effect.
- Anemia may arise from eryptosis, a form of suicidal erythrocyte death.
Purpose of the Study:
- To investigate if gefitinib induces eryptosis in human erythrocytes.
- To determine if gefitinib-induced eryptosis involves calcium influx and/or oxidative stress.
Main Methods:
- Flow cytometry was used to analyze erythrocyte volume (forward scatter).
- Annexin-V binding assessed phosphatidylserine exposure on the cell membrane.
- Intracellular calcium ([Ca2+]i) and reactive oxygen species (ROS) were measured using fluorescent indicators.
Main Results:
- Gefitinib exposure (≥ 2 µg/ml for 48 hours) reduced erythrocyte forward scatter, indicating shrinkage.
- Gefitinib significantly increased annexin-V binding, signifying phosphatidylserine exposure.
- While intracellular calcium did not significantly increase, blocking extracellular calcium blunted the annexin-V binding effect.
- Gefitinib significantly increased reactive oxygen species (ROS) production.
Conclusions:
- Gefitinib induces erythrocyte shrinkage and membrane scrambling, characteristic of eryptosis.
- This eryptosis is partly dependent on extracellular calcium.
- Gefitinib-induced eryptosis is accompanied by oxidative stress.