Mechanisms of AM404-induced [Ca]i rise and death in human osteosarcoma cells |
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Authors: | 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 |
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Affiliation: | 1. Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan;2. Department of Nursery, Tzu Hui Institute of Technology, Pingtung 926, Taiwan;3. Section of Allergy, Immunology & Rheumatology, Chi-Mei Medical Center, Tainan 710, Taiwan;4. Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan;5. Department of Orthopaedic Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan;6. Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan |
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Abstract: | 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. |
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Keywords: | AM404 Apoptosis Ca2+ Fura-2 Osteosarcom cells |
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