[Cytotoxicity and apoptosis of human osteosarcoma U2OS cells induced by recombinant soluble AZURIN].
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.
Miao
Xu-dong
XD
Department of Orthopedics, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China.
Ye
Zhao-ming
ZM
Yang
Di-sheng
DS
Xu
Rong-zhen
RZ
Li
Wei-xu
WX
Tao
Hui-ming
HM
D017209:Apoptosis / Q000187:drug effects*; D001400:Azurin / Q000235:genetics / Q000494:pharmacology*; D001859:Bone Neoplasms / Q000473:pathology* / Q000628:therapy; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D012516:Osteosarcoma / Q000473:pathology* / Q000628:therapy; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D001400:Azurin