Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

Abstract

Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺ and by addition of SB203580 (2 µM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca²⁺ entry, oxidative stress, and p38 kinase.

MDM2-Dependent Rewiring of Metabolomic and Lipidomic Profiles in Dedifferentiated Liposarcoma Models

“…In previous literature, 3-hydroxy-3-methyl-glutaryl-CoA reductase reductase inhibitor (statin) treatment was noted to result in ceramide elevation [14][15][16]. Given the importance of the ceramide metabolites in our findings, we wanted to explore whether induction of ceramides altered chemosensitivity.…”

Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis

“…100% hemolysate in hemolysis tests was prepared by mixing the erythrocyte solution with distilled water. As a measure of hemolysis, the percent of hemolysis in each tube is calculated by dividing the absorbance of the test tube by the absorbance of 100% hemolysis [ 25 ].…”

“…Flowcytometry was used to quantify the percent of Annexin V-PE positive erythrocyte and also to measure forward scatter [ 25 ]. In brief, after 24 hours of incubation, cells were washed in Ringer solution.…”

“…Intracellular Ca 2+ was measured using Fluo 3 /AM flowcytometry [ 25 ]. In brief, erythrocytes were washed in Ringer solution and then loaded with Fluo 3 /AM (Calbiochem; Bad Soden, Germany) in Ringer solution containing 5 mM CaCl 2 and 2 µ M Fluo 3 /AM.…”

“…2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA, Sigma) was used in order to determine the role of oxidative stress in Al-induced eryptosis fluorescent ROS assay [ 25 ]. In this assay, DCFDA was oxidized in the presence of ROS and becomes green fluorescent.…”