Molecular and Cellular Reports A Pharmacological Study of the Action of Esculetin on Human Prostate Cancer Cells

Abstract

Esculetin is a derivative of coumarin, and is the dominant, vigorous component of the conventional Chinese medicine Cortex Fraxini. Recently, the molecular pathway study and clinical use of Cortex Fraxini and esculetin are becoming intensive. In vitro, esculetin has been shown to provoke apoptotic responses via mitochondrial routes and other cellular responses in diverse cell types. The action of esculetin on cytosolic Ca2+ concentration ([Ca2+]i) in prostate cells is unknown. [Ca2+]i were assayed by applying fura-2, a fluorescent Ca2+-sensitive probe. WST-1 was used to measure cell death. Esculetin at doses of 25–100 µM provoked [Ca2+]i raises. Removing external Ca2+ decreased the response by 15%. Esculetin (100 µM) provoked Mn2+ entry implying Ca2+ influx. Esculetin-provoked Ca2+ influx was suppressed by half by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X); and by three inhibitors of store-operated Ca2+ channels: nifedipine, econazole and SKF96365. In the absence of Ca2+, pretreatment with the endoplasmic reticulum (ER) Ca2+ pump suppressor thapsigargin completely suppressed esculetin-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122 eliminated esculetin-provoked [Ca2+]i raises. Esculetin at 20–70 µM caused death of cells, which was not prevented by incubation with the Ca2+ binder 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). In sum, in PC3 prostate cells, esculetin provoked [Ca2+]i raises by provoking PLC-associated Ca2+ discharge from ER and Ca2+ influx via PKC-sensitive store-operated Ca2+ influx. Additionally, esculetin provoked Ca2+-dissociated cell death.