Oxidative stress induced by low-dose doxorubicin promotes the invasiveness of osteosarcoma cell line U2OS in vitro

Reactive oxygen species (ROS) are known to mediate doxorubicin (DOX)-induced apoptosis and are the major cause of DOX toxicity. We introduce a novel in vitro phenomenon of osteosarcoma (OS) cell line caused by low-dose DOX-induced oxidative stress. Human osteosarcoma cell line U2OS was used for the experiments. Hydrogen peroxide (H2O2) and the antioxidant compound N-acetylcysteine (NAC) were used to investigate the involvement of oxidative stress. In proliferation assays, low dose of DOX (below 200 nM) did not affect U2OS proliferation significantly for up to 48 h. In MatrigelTM invasion assay, DOX increased the invasiveness of U2OS at around 100 nM, which is a subclinical concentration. Quantitative real-time polymerase chain reaction and gelatin zymography showed increased MMP-9 expression and increased MMP-9 enzymatic activity, respectively, in the presence of DOX doses that increased the invasiveness of U2OS. H2O2, a representative source of ROS, also increased the invasiveness of U2OS as DOX did, with similar patterns. However, when the cells were pre-treated with NAC, no DOX- or H2O2-mediated increase of invasiveness or MMP-9 expression was evident. The results suggest that oxidative stress induced by low-dose DOX promotes the invasiveness of osteosarcoma cell line U2OS in vitro, through MMP-9 induction.