[Selective inducement effect of bacterial redox protein azurin on apoptosis of human osteosarcoma cell line U2OS].
OBJECTIVE
Bacterial redox protein azurin can selectively inhibit proliferation, and induce apoptosis in various human cancer cells. These in vitro findings have been confirmed in vivo in nude mice bearing tumor xenograft. Furthermore, azurin could diminish tumor volume in nude mice bearing tumor xenograft with no evidence of toxicity. The mechanism of its action is unknown. This study was designed to explore the effects of azurin on apoptosis of osteosarcoma cell line U2OS, and its molecular mechanism.


METHODS
U2OS cells were cultured with different concentrations of azurin. Cell viability was detected by MTT assay. The 50% inhibitory concentration (IC(50)) of azurin was calculated by Bliss method. Apoptotic morphology and apoptotic body of U2OS cells were observed under fluorescent microscope, and transmission electron microscope. DNA ladder was observed through agarose gel electrophoresis. Cell apoptosis was determined by flow cytometry (FCM). Protein levels of Bax, Bcl-2, and Caspase-3 were quantified by Western blot.


RESULTS
Azurin inhibited growth and induced apoptosis of U2OS cells in a dose-dependent manner 48 h after treatment, with the IC(50) of(114.54+/-7.65) mg/L. Moreover, its inhibitory effect was significantly higher on U2OS cells than on MG63 cells or L-02 cells under the same concentrations (P<0.05). When treated with 100 or 200 mg/L of azurin for 24 h, U2OS cells showed typical apoptotic morphology, typical DNA ladder bands were observed. No apoptotic feature was observed in control cells. Sub-G1 peak and apoptotic peak of U2OS cells were observed when exposed to 200 mg/L of azurin for 48 h with the apoptosis index of 35.8%, cell cycle was arrested in G1 phase. Bcl-2 was down-regulated when U2OS cells were treated with azurin for 24 h, while Bax and caspase-3 were up-regulated.


CONCLUSIONS
Azurin could selectively induce apoptosis of human osteosarcoma U2OS cells. The induction of apoptosis by azurin may be closely associated with down-regulation of Bcl-2, up-regulation of Bax, and activation of Caspase-3.
Ye
Zhao-Ming
ZM
Department of Orthopedics, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310009, P.R.China.
Miao
Xu-Dong
XD
Yang
Di-Sheng
DS
Xu
Rong-Zhen
RZ
Huang
Xin
X
Ge
Fen-Fen
FF
D017209:Apoptosis / Q000187:drug effects*; D001400:Azurin / Q000008:administration & dosage / Q000494:pharmacology*; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D001400:Azurin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases