Influence of osteopontin silencing on survival and migration of lung cancer cells
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Abstract
Background and purpose
Osteopontin (OPN) is a multifunctional protein overexpressed in many cancers and is involved in tumor progression and metastasis. In lung cancer, elevated OPN expression is associated with an unfavorable prognosis. Therefore, inhibition of OPN is an attractive approach for improving survival.
Materials and methods
We used siRNA to specifically downregulate OPN expression in A549 lung cancer cells. OPN silencing was evaluated with quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA levels and with Western blotting for protein levels. Effects on cell proliferation were measured by cell counting. The influence on tumor cell migration was detected using a modified Boyden chamber. Changes in cell cycle distribution were assessed by flow cytometry. Using the colony formation assay, we determined changes in radiosensitivity.
Results
A specific and effective downregulation of OPN expression was detected in both RNA and protein levels. Cell proliferation and cell migration were significantly reduced by OPN silencing after 24 h and the effects were further increased by the addition of irradiation. The cell cycle distribution showed a reduction in S phase and an increase in cells arrested in both G0/G1 and G2/M phases. Specific enhancement of radiosensitivity was clearly shown after OPN knockdown.
Conclusion
The combination of OPN silencing and irradiation showed a synergistic effect leading to reduced cell survival.
Keywords
Radiosensitivity Osteopontin Lung cancer Migration SurvivalEinfluss der Osteopontinhemmung auf Überleben und Migration von Lungenkarzinomzellen
Zusammenfassung
Hintergrund und Ziel
Osteopontin (OPN) ist ein multifunktionelles Protein, welches in vielen Tumoren überexprimiert wird und an zahlreichen Schritten der Tumorprogression und Metastasierung beteiligt ist. Beim Bronchialkarzinom ist eine gesteigerte Osteopontinexpression mit einer schlechteren Prognose assoziiert. Daher stellt die Hemmung der Osteopontinexpression einen vielversprechenden Ansatz zur Verbesserung des Überlebens dar.
Material und Methoden
In der A549-Bronchialkarzinom-Zelllinie wurden zur Osteopontinhemmung spezifische siRNA eingesetzt. Die Expressionshemmung wurde auf RNA-Ebene mittels quantitativer RT-PCR und auf Proteinebene mithilfe der Western-Blot-Analyse ausgewertet. Auswirkungen auf die Zellproliferation wurden durch Zellzählung ermittelt. Eine Beeinflussung der Zellmigration wurde mittels modifizierter Boyden-Kammer bestimmt. Mit der Durchflusszytometrie ermittelten wir Änderungen im Zellzyklus. Eine Änderung der Strahlensensitivität wurde durch das klonogene Zellüberleben im Koloniebildungstest erfasst.
Ergebnisse
Eine spezifische und effektive Downregulation der Osteopontinexpression wurde sowohl auf RNA- als auch auf Proteinebene gezeigt. Hierdurch verringerten sich nach 24 h die Zellproliferation und die Migration signifikant, und durch die zusätzliche Bestrahlung wurden diese Effekte weiter verstärkt. In der Zellzyklusverteilung zeigte sich eine Abnahme in der S-Phase und eine Zunahme der Zellen in der G0/G1- und G2/M-Phase. Schließlich wurde nach OPN-Hemmung eine spezifische Verstärkung der Radiosensitivität im Kolonietest nachgewiesen.
Schlussfolgerung
Die Kombination der OPN-Downregulation mit einer Strahlentherapie zeigte eine synergistische Wirkung, die zu einem verringerten Zellüberleben führte.
Schlüsselwörter
Strahlensensitivität Osteopontin Lungenkarzinom Migration ÜberlebenNotes
Acknowledgments
We thank Dr. Matthias Kappler for advice with osteopontin detection and Natalia Niewidok and Sebastian Kuger for critical discussion.
Conflict of interest
On behalf of all authors, the corresponding author states that there are no conflicts of interest.
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