Safrole-induced cellular Ca 2+ increases and death in human osteosarcoma cells

Abstract

The effect of the carcinogen safrole on intracellular Ca 2+ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca 2+ handling and viability in MG63 human osteosarcoma cells. Cytosolic free Ca 2+ levels ([Ca 2+] i) in populations of cells were measured using fura-2 as a fluorescent Ca 2+ probe. Safrole at concentrations above 130 μM increased [Ca 2+] i in a concentration-dependent manner with an EC 50 value of 450 μM. The Ca 2+ signal was reduced by 30% by removing extracellular Ca 2+. Addition of Ca 2+ after safrole had depleted intracellular Ca 2+ induced Ca 2+ influx, suggesting that safrole caused Ca 2+ entry. In Ca 2+-free medium, after pretreatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) failed to release more Ca 2+; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca 2+] i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca 2+ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca 2+] i increase. Trypan exclusion assays revealed that incubation with 65 μM safrole for 30 min did not kill cells, but incubation with 650 μM safrole for 10–30 min nearly killed all cells. Flow cytometry demonstrated that safrole evoked apoptosis in a concentration-dependent manner. Safrole-induced cytotoxicity was not reversed by chelation of Ca 2+ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca 2+] i increase by causing Ca 2+ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca 2+ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca 2+-independent manner.