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Stimulation of Suicidal Erythrocyte Death by Garcinol
Antonella Fazio1, Marilena Briglia, Caterina Faggio
1Department of Physiology, University of Tuebingen, Tuebingen, Germany.
Background/Aims:
The benzophenone garcinol from dried fruit rind of Garcinia indica counteracts malignancy, an effect at least in part due to stimulation of apoptosis. The proapototic effect of garcinol is attributed in part to inhibition of histone acetyltransferases and thus modification of gene expression. Moreover, garcinol triggers mitochondrial depolarisation. Erythrocytes lack gene expression and mitochondria but are nevertheless able to enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, energy depletion and Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how garcinol induces eryptosis.
Methods:
To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and cytosolic ATP levels utilizing a luciferin-luciferase-based assay.
Results:
A 24 hours exposure of human erythrocytes to garcinol (2.5 or 5 µM) significantly increased the percentage of annexin-V-binding cells. Garcinol decreased (at 1 µM and 2.5 µM) or increased (at 5 µM) forward scatter. Garcinol (5 µM) further increased Fluo3-fluorescence, increased DCFDA fluorescence, and decreased cytosolic ATP levels. The effect of garcinol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+.
Conclusions:
Garcinol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation, energy depletion and Ca2+ entry.
Insights
Garcinol, a compound from Garcinia indica, induces an apoptosis-like cell death in erythrocytes, known as eryptosis. This process involves cell shrinkage and membrane changes, driven by oxidative stress, energy depletion, and calcium influx.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Garcinol, a benzophenone from Garcinia indica, exhibits anticancer properties by inducing apoptosis.
- Apoptosis involves programmed cell death, characterized by specific cellular changes.
- Eryptosis is an apoptosis-like process in erythrocytes, triggered by factors like oxidative stress and energy depletion.
Purpose of the Study:
- To investigate whether garcinol induces eryptosis in human erythrocytes.
- To elucidate the mechanisms by which garcinol affects erythrocyte membrane and function.
Main Methods:
- Assessed phosphatidylserine exposure using annexin-V binding.
- Measured cell volume via forward scatter.
- Quantified intracellular calcium ([Ca2+]i) using Fluo3-fluorescence.
- Determined reactive oxygen species (ROS) formation with DCFDA.
- Analyzed cytosolic ATP levels using a luciferin-luciferase assay.
Main Results:
- Garcinol (2.5 or 5 µM) significantly increased annexin-V binding, indicating phosphatidylserine exposure.
- Garcinol altered cell volume (forward scatter) in a dose-dependent manner.
- High garcinol concentration (5 µM) elevated intracellular calcium and ROS formation, while decreasing ATP levels.
- The effect on annexin-V binding was partially dependent on extracellular calcium.
Conclusions:
- Garcinol induces eryptosis in human erythrocytes.
- Key mechanisms include cell shrinkage, phospholipid scrambling, ROS generation, energy depletion, and calcium influx.