Ca(2+) mobilization induced by W-7 in MG63 human osteosarcoma cells.
The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a widely used calmodulin inhibitor, on intracellular free Ca(2+)levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+)probe. W-7 (20-1000 micro m) induced an increase in [Ca(2+)](i)in a dose-dependent manner, with an EC(50)of 100 microm. The [Ca(2+)](i)signal comprised an initial rise and a sustained plateau without significant decay within 5 min. External Ca(2+)removal decreased the Ca(2+)signals by reducing the peak and sustained phase, indicating W-7-activated intracellular Ca(2+)release and extracellular Ca(2+)influx. W-7 (500 microm) failed to induce a [Ca(2+)](i)increase in a Ca(2+)-free medium after pre-treatment with thapsigargin (1 microm), an endoplasmic reticulum Ca(2+)pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca(2+)release induced by thapsigargin. This suggests that W-7 (500 microm ) released internal Ca(2+)mainly from the endoplasmic reticulum. The addition of 3 mm Ca(2+)increased [Ca(2+)](i)dose-dependently after preincubation with 20-1000 microm W-7 in a Ca(2+)-free medium, implying that W-7 induced capacitative Ca(2+)entry. W-7-induced Ca(2+)release was not altered by inhibiting phospholipase C with 2 microm 1-(6-((17 beta - 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 microm) caused gradual cell death within 30 min of the initial drug exposure. Together, it was found that W-7 induced [Ca(2+)](i)increases in human osteosarcoma cells by releasing internal Ca(2+)from the endoplasmic reticulum, and also by triggering Ca(2+)influx. W-7 may be cytotoxic to osteosarcoma cells.
Jan
C R
CR
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Taiwan and Department of Biology and Institute of Life Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan. crjan@isca.vghks.gov.tw
Lu
C H
CH
Chen
Y C
YC
Cheng
J S
JS
Tseng
L L
LL
Jun-Wen
W
W
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001842:Bone and Bones / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D012516:Osteosarcoma; D013449:Sulfonamides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D013449:Sulfonamides; C017967:W 7; D002118:Calcium