Ouabain Induces DNA Damage in Human Osteosarcoma U-2 OS Cells and Alters the Expression of DNA Damage and DNA Repair-associated Proteins.

Abstract

Ouabain, isolated from natural plants, exhibits anticancer activities; however, no report has presented its mechanism of DNA damage induction in human osteosarcoma cancer cells in vitro. The aim of this study was to investigate whether ouabain induces DNA damage and repair, accompanied with molecular pathways in human osteosarcoma cancer U-2 OS cells in vitro. The percentage of viable cell number was measured by flow cytometric assay; DNA damage was assayed by DAPI staining, comet assay, and agarose gel electrophoresis. DNA damage and repair associated protein expressions were assayed by western blotting assays. Ouabain reduced total cell viability, induced chromatin condensation, DNA fragmentation, and DNA damage in U-2 OS cells. Ouabain increased p-ATMSer1981, p-ATRSer428, and p53 at 2.5-10 μM, increased p-p53Ser15 at 10 μM; however, it decreased p-MDM2Ser166 at 2.5-10 μM. Ouabain increased p-H2A.XSer139, MDC-1, and PARP at 2.5-10 μM and BRCA1 at 5-10 μM; however, it decreased DNA-PK and MGMT at 2.5-10 μM in U-2 OS cells at 48 h treatment. Ouabain promoted expression and nuclear translocation of p-H2A.XSer139 in U-2 OS cells and this was confirmed by confocal laser microscopy. Ouabain reduced total viable cell number through triggering DNA damage and altering the protein expression of DNA damage and repair system in U-2 OS cells in vitro.