title: Safrole-induced cellular Ca2+ increases and death in human osteosarcoma cells.
abstract: The effect of the carcinogen safrole on intracellular Ca2+ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca2+ handling and viability in MG63 human osteosarcoma cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 130 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 450 microM. The Ca2+ signal was reduced by 30% by removing extracellular Ca2+. Addition of Ca2+ after safrole had depleted intracellular Ca2+ induced Ca2+ influx, suggesting that safrole caused Ca2+ entry. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca2+; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca2+]i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca2+ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca2+]i increase. Trypan exclusion assays revealed that incubation with 65 microM safrole for 30 min did not kill cells, but incubation with 650 microM 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 Ca2+ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca2+ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca2+-independent manner.
authors.affiliation: Department of Physical Medicine and Rehabilitation, St. Joseph Hospital, Kaohsiung 802, Taiwan.
authors.forename: Hsueh-Chi
authors.initials: HC
authors.lastname: Lin
authors.forename: He-Hsiung
authors.initials: HH
authors.lastname: Cheng
authors.forename: Chun-Jen
authors.initials: CJ
authors.lastname: Huang
authors.forename: Wei-Chuan
authors.initials: WC
authors.lastname: Chen
authors.forename: I-Shu
authors.initials: IS
authors.lastname: Chen
authors.forename: Shiuh-Inn
authors.initials: SI
authors.lastname: Liu
authors.forename: Shu-Shong
authors.initials: SS
authors.lastname: Hsu
authors.forename: Hong-Tai
authors.initials: HT
authors.lastname: Chang
authors.forename: Jong-Khing
authors.initials: JK
authors.lastname: Huang
authors.forename: Jin-Shyr
authors.initials: JS
authors.lastname: Chen
authors.forename: Yih-Chau
authors.initials: YC
authors.lastname: Lu
authors.forename: Chung-Ren
authors.initials: CR
authors.lastname: Jan
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002273:Carcinogens / Q000633:toxicity*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D012451:Safrole / Q000633:toxicity*; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D002273:Carcinogens; D002614:Chelating Agents; D005456:Fluorescent Dyes; D010703:Phorbol Esters; C119222:phorbol-12-myristate; D004533:Egtazic Acid; D019284:Thapsigargin; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D012451:Safrole; D002118:Calcium; D016257:Fura-2