A novel combination of an AKT activator with an AKT inhibitor exacerbates hydrogen peroxide-induced death of human cardiac microvascular endothelial cells

Abstract

<b>Abstract ID 28208</b> <b>Poster Board 560</b> Ischemic heart disease is a leading cause of death worldwide. Oxidative stress-associated cell death represents a significant contributor to myocardial damage in patients with acute myocardial infarction. Cardiac microvascular endothelial cells play an essential role in cell survival and cardiac functions. Activation of AKT/protein kInase B is usually associated with cell survival. However, the combination of AKT activation with AKT inhibition on cell survival remains unknown. In the presented study, human cardiac microvascular endothelial cells (HCMECs) were cultured in endothelial cell medium supplemented with 5% fetal bovine serum and endothelial cell growth supplement in 5% CO₂ incubator. HCMECs from passage 4 to 7 were sub-cultured in 96-well plates coated with collagen I. Cells were pretreated with SC-79 (an AKT activator) at 4 μg/mL and MK-2206 hydrochloride (an AKT inhibitor) at 5 μM respectively or in combination for one hour followed by insult of hydrogen peroxide (H₂O₂, 5 mM for one hour). Cell viability was determined with measurement of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase (LDH) release into medium. H₂O₂ formation from HCMECs was measured by using fluorometric assay kit. Western blot was used to determine the protein expression of total AKT and phospho-AKT (Ser473) in HCMECs. Cell treated with H₂O₂ reduced formation of MTT and increased release of LDH as well as formation of H₂O₂ from HCMECs. The cell death induced by H₂O₂was not affected with pretreatment of either SC-79 or MK-2206 alone. Nevertheless, a combination of SC-79 and MK-2206 enhanced cell death (P&lt;0.003 vs H₂O₂ group) without alteration of H₂O₂ formation from HCMECs. Our results clearly indicated that a combination of AKT activator with AKT inhibitor created a novel cell death pathway. This study was supported by a Seed Grant Award from California Northstate University College of Pharmacy.