Functional and biological analysis of Bcl-xL expression in human osteosarcoma

Abstract

Bcl-xL, a member of Bcl-2 protein family functioned as dominant regulators of apoptotic cell death, has been reported to play important roles in malignant transformation and tumor development. In the present study, our aim was to explore the roles of Bcl-xL overexpression and determine its possibility as a therapeutic target in human osteosarcoma. Real-time quantitative RT-PCR and Western blot or immunohistochemistry assays were performed to detect the expression of Bcl-xL mRNA and protein in human osteosarcoma cell lines or tissue samples. The expression of other Bcl-2 family proteins (Bcl-2, Mcl-1, Bim and Bik) in osteosarcoma tissues was also detected by immunohistochemistry. The associations of Bcl-xL mRNA expression with clinicopathologic factors and prognosis of osteosarcoma patients were evaluated. RNA interference or gene overexpression technologies were employed to downregulate or upregulate endogenous Bcl-xL expression in osteosarcoma cells and the effects of Bcl-xL downregulation or upregulation on phenotypes and chemo- or radiosensitivity of human osteosarcoma cells were analyzed. Finally, the mechanism of synergistic effects of Bcl-xL downregulation and chemo- or radiotherapy was explored by detecting the activity of caspase-3. The expression levels of Bcl-xL mRNA and protein in high metastatic osteosarcoma cells showed higher than those in low metastatic osteosarcoma cells. Moreover, the levels of Bcl-xL mRNA expression were significantly higher in osteosarcoma tissues than those in chondroma or corresponding non-tumor tissues (P<0.01), and osteosarcoma tissues showed stronger immunostaining of Bcl-xL protein than non-tumor tissues. The stronger staining of Bcl-2 and Mcl-1 proteins was also observed, while the staining of pro-apoptotic proteins (Bim and Bik) was significantly weaker or not detected in osteosarcoma tissues. The higher levels of Bcl-xL mRNA expression were significantly correlated with advanced clinical stage (P=0.005) or hematogenous metastasis (P=0.001) of osteosarcoma patients. Osteosarcoma patients with higher Bcl-xL mRNA expression showed a poorer survival compared with those with lower expression (P=0.039). Bcl-xL downregulation or upregulation could significantly reduce or increase the proliferation capacity of osteosarcoma cells. Furthermore, Bcl-xL downregulation could significantly enhance in vitro chemo- or radiosensitivity of osteosarcoma cells, which might be associated with elevated activity of caspase-3. Taken together, overexpression of Bcl-xL may play important roles in osteosarcoma progression and this molecule will be a potential chemo- or radiotherapeutic molecular target for osteosarcoma therapy.