Safrole-induced cellular Ca²⁺ increases and death in human osteosarcoma cells

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• 作者：Hsueh-Chi Lin ; He-Hsiung Cheng ; Chun-Jen Huang ; Wei-Chuan Chen ; I-Shu Chen ; Shiuh-Inn Liu ; Shu-Shong Hsu ; Hong-Tai Chang ; Jong-Khing Huang ; Jin-Shyr Chen et al.
• 关键词：Ca²⁺ ; Fura-2 ; MG63 cells ; Osteosarcoma cells ; Safrole ; Thapsigargin
• 刊名：Pharmacological Research
• 出版年：2006
• 期刊代码：54_10436618
• 类别：pha
• 出版时间：August 2006
• 卷：54
• 期：2
• 页码：103-110
• 文件大小：400 K

摘要

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 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 450 μM. 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 μM safrole, 1 μM 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 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 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.