Bioymifi, a novel mimetic of TNF-related apoptosis-induced ligand (TRAIL), stimulates eryptosis.

Abstract

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a cytokine that initiates apoptosis upon binding to death receptor 5 (DR5) on cancer cells. Small molecule TRAIL mimetics have therefore been investigated as promising chemotherapeutic agents. Since anemia of chemotherapy is common, our goal is to investigate the hemolytic and eryptotic properties of novel DR5 agonist bioymifi (BMF) and identify the underlying molecular mechanisms. Whole blood (WB) was stimulated with 100μM of BMF, whereas red blood cells (RBCs) were treated with 10-100μM of BMF for 24h at 37°C. WB was analyzed for RBC, leukocyte, and platelet indices, while RBCs were examined for hemolysis by light absorbance of free hemoglobin, membrane scrambling by Annexin V-FITC, calcium by Fluo4/AM, cellular morphology by light scatter, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) using flow cytometry. Caspase inhibitor Z-VAD-FMK, p38 inhibitor SB203580, casein kinase 1α inhibitor D4476, receptor-interacting protein 1 inhibitor necrostatin-2, reduced glutathione, or cyclooxygenase (COX) inhibitor aspirin were added accordingly. BMF exerted dose-responsive, calcium-independent hemolysis, reduced RBC hemoglobin, significantly increased Annexin V-, Fluo4-, and DCF-positive cells, along with a dual effect on forward and side light scatter. Notably, the cytotoxic potential of BMF was significantly mitigated upon pharmacological inhibition of p38. Furthermore, BMF exhibited selective toxicity to eosinophils and significantly diminished reticulocyte hemoglobin content. Altogether, these novel findings highlight the adverse outcomes of BMF exposure on RBC physiology and provide the first toxicological assessment of BMF as an antitumor agent.