Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis☆
Section snippets
Materials and methods
Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics were obtained from Gibco-BRL (Gland Island, NY). The MAPK inhibitors, SB203580, U0126, and SP600125 and the caspase inhibitors, z-Val-Ala-Asp-CH2F (zVAD-fmk) and z-ASP (OCH3)-Glu (OCH3)-Val-Asp-CH2F (zDEVD-fmk), were obtained from Calbiochem–Novabiochem (San Diego, CA). Anti-JNK, p38, ERK, c-Jun, phospho-JNK (Thr-183/Tyr-185), phospho-ERK (Thr-202/Tyr-204), phospho-c-Jun (Ser-63), and caspase-3
Cd induced apoptosis of neuronal cells through phosphorylation of JNK
To assess the toxic effects of Cd on human neuronal cell, SH-SY5Y cells were treated with Cd for 12 h at various concentrations (0–100 μM) and assayed for cell viability using the MTT assay, DAPI staining, and TUNEL staining. Loss of viability occurred in a dose-dependent manner in response to Cd treatment (Fig. 1A). At 25 μM Cd, the cell viability reduced to about 54.1 ± 3.4% of the untreated control cells. Phase-contrast microscopic observation showed the damaged cells which were more rounded in
Discussion
Cd-induced apoptosis was described previously in various cells including neuronal cell [5], [15], [23], [29] but its underlying molecular mechanism was not clearly elucidated. In the present study, we used SH-SY5Y cell, a human neuroblastoma cell line, in order to examine the intracellular signaling pathways involved in Cd-induced neuronal cell apoptosis. We found that Cd induced apoptosis as determined by TUNEL assay and DAPI staining, and that the apoptosis occurs through activation of the
Acknowledgments
The authors thank Dr. S.Y. Suh for her valuable comments and Mrs. S.Y. Hur for her assistance in preparing the manuscript. This study was supported by “The Eco-technopia 21 project” from Ministry of Environment for Drs. S.A. Jo and C.K. Moon.
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Abbreviations: Cd, cadmium; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p38, p38 MAP kinase; MKK4/7, MAP kinase kinase 4/7; ASK-1, apoptosis signal regulating kinase; DN, dominant negative.