Effects of osteopontin inhibition on radiosensitivity of MDA-MB-231 breast cancer cells.
BACKGROUND
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. 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.


METHODS
MDA-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.


RESULTS
Treatment 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.


CONCLUSIONS
Our 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.
Hahnel
Antje
A
Department of Radiotherapy, Martin-Luther-University Halle-Wittenberg, Dryanderstr 4, 06110 Halle, Germany. antje.hahnel@medizin.uni-halle.de
Wichmann
Henri
H
Kappler
Matthias
M
Kotzsch
Matthias
M
Vordermark
Dirk
D
Taubert
Helge
H
Bache
Matthias
M
D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000528:radiation effects; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D011836:Radiation Tolerance / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D034741:RNA, Small Interfering; D053495:Osteopontin