Target and mechanism of cardiotoxicity of recombinant human endostatin

Objective To explore the target and mechanisms of the cardiotoxicity of recombinant human endostatin(endostar).Methods The myocardial cell line H9c2 was used as subjects and the following tests were performed.(1) H9c2 cells were divided into the control group(without drug intervention),intervened groups including 100,200,and 400 μg/ml endostar co-cultured for 24 h or 48 h,respectively.The apoptosis rate of H9c2 cells in different groups were detected by flow cytometry.(2) The H9c2 cells were divided into the control group and the intervened group of 400 μg/ml endostar co-cultured for24 h.The changes of ultrastructure of cells were observed by transmission electron microscope.(3) The H9c2cells were divided into the control group and the intervened groups including 100,200,and 400 μg/ml endostar co-cultured for 18 h,respectively.The mitochondrial membrane potential was recorded by JC-1fluorescence probe.(4) The H9c2 cells were divided into the control group and the intervened group of400 μg/ml endostar co-cultured for 24 h.The release condition of cytochrome C were observed by method of immunocytochemistry.(5) The H9c2 cells were divided into the control group and the intervened groups including 100,200,and 400 μg/ml endostar co-cultured for 24 h,respectively.The ADP/ATP ratio was detected by the method of chemiluminescence.Results(1) The apoptotic rate of 200 μg/ml endostar cocultured for 24 h group and the 400 μg/ml endostar co-cultured for 24 h and 48 h groups were higher than that in the control group[24 h:(16.34 ± 3.72)%,(27.03 ±3.91)%vs.(6.99 ± 1.72)%;48 h:(24.89 ±4.77)%vs.(6.44 ± 1.81)%;all P 0.01].The apoptotic rate of the 200 μg/ml endostar co-cultured for24 h group was higher than that in the endostar co-cultured for 48 h group[(16.34 + 3.72)%vs.(11.34 ±3.09)%,P0.01].(2) The H9c2 cells' ultrastructure of control group presented normal.The H9c2 cells in the group of 400 μg/ml endostar co-cultured for 24 h showed pyknotic and fragmental in nuclei,the massed chromatin,intracellular vacuoles,dilation of endoplasmic reticulum,mitochondrial swelling,and appearance of apoptotic body.(3) Compared with the control group,the degree of depolarization of mitochondrial transmembrane potential decreased following of the increasing concentration of endostar in 100,200,and400 μg/ml endostar intervened groups.(4) Cytochrome C of the H9c2 cells in the control group mainly distributed in the mitochondria.Cytochrome C of the H9c2 cells in the group of 400 μg/ml endostar cocultured for 24 h were released from mitochondria to the cytoplasm.(5) ADP/ATP ratio of H9c2 cells in the200 μg and 400 u,g/ml endostar groups were significantly higher than those in the control group(1.14 ±0.11,1.31 ±0.18 vs.0.98 ±0.09,all P 0.01).Conclusions Cardiomyocyte mitochondria may be the target of the cardiotoxicity of endostar.The cardiomyocyte apoptosis evoked via the mitochondrial-dependent pathway is one of the mechanism of myocardial damage.