Journal of Veterinary Science  2013;14(3):281-289

doi:10.4142/jvs.2013.14.3.281

Gossypol acetic acid induces apoptosis in RAW264.7 cells via a caspase-dependent mitochondrial signaling pathway.

Sijun DENG 1 2 ; Hui YUAN ; Jine YI ; Yin LU ; Qiang WEI ; Chengzhi GUO ; Jing WU ; Liyun YUAN ; Zuping HE

Affiliations

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Keywords

apoptosis; gossypol acetic acid; mitochondrial signaling pathway

Country

Republic of Korea

Language

English

Abstract

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 micromol/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 (DeltaPsi(m)) 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 DeltaPsi(m) 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.