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KMID : 0893420130140030281
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Journal of Veterinary Science
2013 Volume.14 No. 3 p.281 ~ p.289
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Gossypol acetic acid induces apoptosis in RAW264.7 cells via a caspase-dependent mitochondrial signaling pathway
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Deng Sijun
Yuan Hui
Yi Jine
Lu Yin
Wei Qiang
Guo Chengzhi
Wu Jing
Yuan Liyun
He Zuping
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Abstract
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To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 μmol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (ΔΨm) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of ΔΨm in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
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KEYWORD
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apoptosis, gossypol acetic acid, mitochondrial signaling pathway
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