The apoptosis of HUVCE cell line induced by L-amino acid oxidase from snake of bungarus fasciatus / 中国病理生理杂志

ABSTRACT

AIM: To investigate the effect of purifried L-amino acid oxidase (LAO) from bungarus fasciatus snake venom on apoptosis and growth of HUVCE cell line. METHODS: The L-amino acid oxidase was purified by SP-sepharose HP column followed by Heperin-Sepharose (FF) column. The homogeneity of the preparation was examined by SDS-PAGE and the molecular weight of LAO was determined by SDS-PAGE and high performance liquid chromatography (HPLC) gel-filtration. The MTT assay was used to detect the viability of cells. Flow cytometry and laser confocal microscopy were used to identiyfy the cell cycle and apoptotic morphology after cells treated with LAO. RESULTS: An L-amino acid oxidase (BF-LAO) was successfully purified from the venom of bungarus fasciatus. It showed a single band in SDS-PAGE under both reduced and non-reduced conditions. The apparent molecular weight was determined to be 60 kD by SDS-PAGE and 70 kD by HPLC gel filtration. LAO inhibited growth and induced apoptosis of HUVCE cell line in a dose-dependent manner after 12 h incubation, with the 50% inhibitory concentration (IC_ 50 ) being of 2.8 mg/L. Flow cytometry and laser confocal microscope showed a typical apoptotic peak and morphological changes of these cells. CONCLUSION: The L-amino acid oxidase from bungarus fasciatus snake venom could inhibit the HUEVC cell growth and induce the cell apoptosis.