Ca2+ mobilization induced by W-7 in MG63 human osteosarcoma cells

Abstract

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a widely used calmodulin inhibitor, on intracellular free Ca2+levels ([Ca2+]i) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+probe. W-7 (20–1000 μ m) induced an increase in [Ca2+]iin a dose-dependent manner, with an EC50of 100 μ m. The [Ca2+]isignal comprised an initial rise and a sustained plateau without significant decay within 5 min. External Ca2+removal decreased the Ca2+signals by reducing the peak and sustained phase, indicating W-7-activated intracellular Ca2+release and extracellular Ca2+influx. W-7 (500 μ m) failed to induce a [Ca2+]iincrease in a Ca2+-free medium after pre-treatment with thapsigargin (1 μ m), an endoplasmic reticulum Ca2+pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca2+release induced by thapsigargin. This suggests that W-7 (500 μ m) released internal Ca2+mainly from the endoplasmic reticulum. The addition of 3 mm Ca2+increased [Ca2+]idose-dependently after preincubation with 20–1000 μ m W-7 in a Ca2+-free medium, implying that W-7 induced capacitative Ca2+entry. W-7-induced Ca2+release was not altered by inhibiting phospholipase C with 2 μ m 1-(6-((17β - 3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Tryphan blue assay demonstrated that W-7 (200 μ m) caused gradual cell death within 30 min of the initial drug exposure. Together, it was found that W-7 induced [Ca2+]iincreases in human osteosarcoma cells by releasing internal Ca2+from the endoplasmic reticulum, and also by triggering Ca2+influx. W-7 may be cytotoxic to osteosarcoma cells.