Cytotoxicity and apoptosis of human osteosarcoma U2OS cells induced by recombinant soluble AZURIN

Abstract

To investigate the effects of recombinant AZURIN protein of P. aeruginosa on growth and apoptosis of U2OS cells. The AZURIN gene was amplified from the genome of P.aeruginosa by PCR, and cloned into prokaryotic expression vector pQE30. The soluble AZURIN protein was expressed in E. coli cells M15, then purified and refolded. After treatment of AZURIN, the cell cycle, proliferation and apoptosis were determined by morphological observation, MTT assay, flow cytometry(FCM) and DNA fragmentation assay. Mitochondrial membrane potential(DeltaPsim) was measured by FCM. The purity of recombinant protein AZURIN reached to 99.1%. Proliferation of U2OS cells were significantly inhibited 12 h after AZURIN (100-200 mg/L) treatment. Apoptosis peak and DNA ladder were observed. Mitochondrial membrane potential decreased gradually from 12 h to 72 h after AZURIN treatment. The recombinant AZURIN inhibit the growth of the human osteosarcoma U2OS cells and inducs apoptosis in vitroìwhich may be associated with the decrease of mitochondrial membrane potential.