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

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.

Effect of amino acids on inhibition of lactate dehydrogenase-X by gossypol

Gossypol acetic acid (GAA) has been shown to have male antifertility effects, but there are pronounced differences among animal species. In the search of endogenous effector molecules, which interfere with the functions of GAA, we have studied the in vitro effect of various amino acids on the inhibition of the purified LDH-X by GAA. Histidine, cysteine and glycine were shown to block the effect of GAA. The effects of these amino acids were concentration dependent. Histidine and glycine protection was found to be complex type in which both the Km and Vmax were decreased compared to control. Arginine, glutamic acid, phenylalanine and valine were found to be ineffective against the inhibitory action of GAA.

Effects of gossypol on mice infected with Trypanosoma cruzi

Ratones albinos suizos adultos, inoculados intraperitonealmente con 1 000 ó 5 000 tripomastigotes (Trypanosoma cruzi, cepa Tulahuén), fueron tratados con gossypol-ácido acético diariamente, por vía oral, con dosis de 10 mg/kg/día, comenzando el día de la inoculación o 10 días antes y continuando hasta la muerte de los animales. Los niveles de parasitemia y el tiempo de sobrevida no fueron modificados por el tratamiento. Ratones inoculados con 100 tripomastigotes recibieron, por vía oral, 10 mg/kg/día de gossypolácido acético desde el día de la infección (grupo 1), o desde 30 días antes (grupo 2), hasta el 100§ día post-inoculación. Los animales de ambos grupos tratados mostraron niveles de parasitemia más bajos que los controles. La mortalidad, a los 24 días post-infección, fue significativamente más baja en ratones del grupo 2 que en los controles. En la casi totalidad de los animales tratados se observaban tripomastigotes circulantes al final del tratamiento. Se concluye que el glossypol, a la dosis de 10 mg/kg/día, tiene cierta acción favorable en ratones infectados con 100 tripomastigotes, disminuyendo la parasitemia y la tasa de mortalidad, pero no los cura ni previene la infección. Sangre total de ratones infectados fue adicionada con gossypol-ácido acético a concentraciones finales 5 micronM y 10 micronM y conservada durante 5 días en las condiciones habituales en bancos de sangre. Hubo reducción del número de tripomastigotes, per los parásitos remanentes conservaban su...