Abstract
BackgroundOsteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. Since little is known about the relationship between OPN expression and radiosensitivity, we investigated the cellular and radiation induced effects of OPN siRNA in human MDA-MB-231 breast cancer cells.MethodsMDA-MB-231 cells were transfected with OPN-specific siRNAs and irradiated after 24 h. To verify the OPN knockdown, we measured the OPN mRNA and protein levels using qRT-PCR and Western blot analysis. Furthermore, the functional effects of OPN siRNAs were studied by assays to assess clonogenic survival, migration and induction of apoptosis.ResultsTreatment of MDA-MB-231 cells with OPN siRNAs resulted in an 80% decrease in the OPN mRNA level and in a decrease in extracellular OPN protein level. Transfection reduced clonogenic survival to 42% (p = 0.008), decreased the migration rate to 60% (p = 0.15) and increased apoptosis from 0.3% to 1.7% (p = 0.04). Combination of OPN siRNA and irradiation at 2 Gy resulted in a further reduction of clonogenic survival to 27% (p < 0.001), decreased the migration rate to 40% (p = 0.03) and increased apoptosis to 4% (p < 0.005). Furthermore, OPN knockdown caused a weak radiosensitization with an enhancement factor of 1.5 at 6 Gy (p = 0.09) and a dose modifying factor (DMF10) of 1.1.ConclusionOur results suggest that an OPN knockdown improves radiobiological effects in MDA-MB-231 cells. Therefore, OPN seems to be an attractive target to improve the effectiveness of radiotherapy.
Highlights
Osteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients
Effects of OPN siRNA constructs on mRNA and protein levels with or without irradiation At 24 h and 72 h after transfection, the OPN mRNA level in cells treated with OPN-specific siRNAs (Mix, OpnS) was approximately 20% compared to that in cells treated with control siRNA (Fig. 1A.)
We found that irradiation alone had no effect on OPN mRNA levels
Summary
Osteopontin (OPN) is a secreted glycophosphoprotein that is overexpressed in various tumors, and high levels of OPN have been associated with poor prognosis of cancer patients. In patients with head and neck cancer, high OPN plasma levels have been associated with poor prognosis following radiotherapy. OPN is a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) protein family and contains a characteristic RGD-motif that mediates the binding to aνb-integrin receptors and a thrombin cleavage side, which releases a CD44-binding domain. Le and co-workers reported that high OPN plasma levels are associated with tumor hypoxia in head and neck squamous cell carcinomas and correlate with poor clinical outcome [12]. A clinical study by Overgaard and co-workers [13] found that high OPN plasma concentrations are associated with a poor prognosis after radiotherapy for patients with head and neck cancer. It is important to investigate OPN and its role in cancer progression to improve the opportunities of cancer therapy, especially the effectiveness of radiotherapy
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