Mechanisms of AM404-induced [Ca²⁺]_i rise and death in human osteosarcoma cells

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• 作者：Hong-Tai Chang ; Chorng-Chih Huang ; He-Hsiung Cheng ; Jue-Long Wang ; Ko-Long Lin ; Pei-Te Hsu ; Jeng-Yu Tsai ; Wei-Chuan Liao ; Yih-Chau Lu ; Jong-Khing Huang ; Chung-Ren Jan
• 关键词：AM404 ; Apoptosis ; Ca²⁺ ; Fura-2 ; Osteosarcom cells
• 刊名：Toxicology Letters
• 出版年：2008
• 期刊代码：49_03784274
• 类别：pha
• 出版时间：10 June 2008
• 卷：179
• 期：1
• 页码：53-58
• 文件大小：549 K

摘要

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 60 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. AM404 induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, Ni²⁺, nifedipine and verapamil. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), AM404-induced [Ca²⁺]_i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca²⁺]_i rise. At concentrations between 10 and 200 μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 μM AM404 was partly reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca²⁺]_i rise by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via L-type Ca²⁺ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.