Abstract
Summary Statement
Survivin is a member of the inhibitor of apoptosis family, which may contribute to the progression of human MFH via inhibiting the mitochondrial apoptosis, and may be considered as a potent therapeutic target for the treatment of human MFH.
Introduction
Survivin is a member of the inhibitor of apoptosis (IAP) family, which usually expresses in the embryonic lung and fetal organs in the developmental stages, but is undetectable in normal adult tissues other than thymus, placenta, CD34+ stem cells, and basal colonic epitherial cells. However, several studies reported that survivin is highly expressed in various human malignancies, including sarcomas, and increased expression of survivin is an unfavorable prognostic marker correlating with decreased overall survival in cancer patients. We have previously reported that survivin was strongly expressed in human malignant fibrous histiocyoma (MFH), however, the roles of survivin in human MFH have not been studied. The aim of this study was to evaluate the effect of survivin inhibition on apoptotic activity in human MFH cells.
Methods
Nara-H, a human MFH cell line which expresses the high levels of survivin, was used in this study. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. To evaluate the effect of survivin inhibition on MFH cell apoptosis, cells were transfected with either a survivin specific siRNA (survivin-siRNA) or a non-specific control siRNA (control-siRNA) by lipofection method. After siRNA transfection, the efficiency of siRNA knockdown of survivin was assessed by quantitative real time PCR. Expressions of apoptosis-related proteins, such as caspase-3, caspase-9 and PARP, were assessed by immunoblot analysis, and the apoptotic activity was evaluated by flow cytometric analysis.
Results
Transfection of survivin-siRNA strongly suppressed the expression of survivin compared with control-siRNA. Immunoblot analyses revealed that expressions of cleaved forms of caspase-3, caspase-9 and PARP were increased in survivin-siRNA transfected cells, while the expressions were barely detected in control cells. In flow cytometric analysis, the number of apoptotic cells was significantly increased in survivin-siRNA transfected cells compared with that in control cells.
Discussion/Conclusion
Previous studies revealed that survivin regulates the mitochondrial apoptotic pathway, and that overexpression of survivin is associated with tumor growth, progression, and resistance to conventional targeted anticancer agents in various human malignancies. In the current study, we demonstrated that siRNA knockdown of survivin induced the cleavage of caspase-3, caspase-9 and PARP, and increased the apoptotic activity in human MFH cells. The findings in this study strongly suggest that survivin may contribute to the progression of human MFH via inhibiting the mitochondrial apoptosis in human MFH, and may be considered as a potent therapeutic target for the treatment of human MFH.
