Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca2+ level ([Ca2+]i) and viability in human glioblastoma cells.Main methodsThe Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400-1000 μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Carvacrol-induced Ca2+ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca2+]i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca2+]i rise. At concentrations of 200-800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N--tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca2+]i rise by inducing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive, non store-operated Ca2+ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.