Stimulation of Hemolysis and Eryptosis by α-Mangostin through Rac1 GTPase and Oxidative Injury in Human Red Blood Cells

Abstract

Background: Chemotherapy-related anemia is prevalent in up to 75% of patients, which may arise due to hemolysis and eryptosis. Alpha-mangostin (α-MG) is a polyphenolic xanthonoid found in the mangosteen tree (Garcinia mangostana) whose antitumor medicinal properties are well-established. Nevertheless, the potential toxic effects of α-MG on red blood cells (RBCs) have, as of yet, not been as well studied. Methods: RBCs were exposed to 1–40 μM of α-MG for 24 h at 37 °C. Hemolysis and related markers were measured using colorimetric assays, eryptotic cells were identified through Annexin-V-FITC, Ca2+ was detected with Fluo4/AM, and oxidative stress was assessed through H2DCFDA using flow cytometry. The toxicity of α-MG was also examined in the presence of specific signal transduction inhibitors and in whole blood. Results: α-MG at 10–40 μM caused dose-dependent hemolysis with concurrent significant elevation in K+, Mg2+, and LDH leakage, but at 2.5 μM it significantly increased the osmotic resistance of cells. A significant increase was also noted in Annexin-V-binding cells, along with intracellular Ca2+, oxidative stress, and cell shrinkage. Moreover, acetylcholinesterase activity was significantly inhibited by α-MG, whose hemolytic potential was significantly ameliorated by the presence of BAPTA-AM, vitamin C, NSC23766, and isosmotic sucrose but not urea. In whole blood, α-MG significantly depleted intracellular hemoglobin stores and was selectively toxic to platelets and monocytes. Conclusions: α-MG possesses hemolytic and eryptotic activities mediated through Ca2+ signaling, Rac1 GTPase activity, and oxidative injury. Also, α-MG leads to accelerated cellular aging and specifically targets platelet and monocyte populations in a whole blood milieu.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC

“…Prevention of hypotonic lysis, on the other hand, suggests that RA intercalates into the lipid bilayer and expands the membrane, allowing the cell to withstand more water influx relative to untreated cells before hemolysis ensues. Counteracting hemolysis ostensibly by increasing cellular volume has previously been observed with Ginkgo biloba leaf extract [25] and mangostin [15].…”

“…To prepare a 50 mM stock solution of RA, 10 mg were dissolved in 555 µL of DMSO, and aliquots were stored at −80 • C. Standard Ringer solution was composed of 125 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 32 mM HEPES, 5 mM glucose, and 1 mM CaCl 2 with a pH of 7.4. Several experiments were conducted to manipulate the extracellular environment by eliminating extracellular Ca 2+ , substituting NaCl and KCl with 125 mM KCl, substituting NaCl with 250 mM sucrose, or adding 10% w/v PEG 8000 [15].…”

“…Control and experimental (800 µM) cells were stained with Giemsa stain as per standard protocols. For SEM analysis, cells fixed in 2.5% glutaraldehyde were stained with 1% osmium tetraoxide, dried in 50-100% ethanol, and visualized using the JSM-7610F ultrahigh-resolution Schottky field emission scanning electron microscope at 15.0 kV (JEOL Co., Ltd., Akishima, Tokyo, Japan) [15].…”

Eriocitrin Disrupts Erythrocyte Membrane Asymmetry through Oxidative Stress and Calcium Signaling and the Activation of Casein Kinase 1α and Rac1 GTPase

“…Inhibiting Rac1 GTPase activity with NSC23766 significantly reduced ERN-mediated hemolysis (Figure 7), suggesting that Rac1 GTPase participates in ERN toxicity to RBCs, most likely by promoting ROS formation [40]. Notably, oxysterols [41] and α-mangostin [42] have recently been reported as modulators of Rac1 GTPase in RBCs.…”

“…A CBC was performed on whole blood diluted 1:2 with PBS following incubation for 24 h at 37 • C with and without 100 µM of ERN using a BC-6200 hematology analyzer (Mindray Medical International Limited, Shenzhen, China) [42].…”