Low dose doxorubicin increases the invasiveness of osteosarcoma through ROS/MMP-9 pathway in vitro

Abstract

Introduction: Doxorubicin (DOX or adriamycin) is a major chemotherapeutic agent for osteosarcoma (OS). It expectedly eliminates micro-metastasis, but suffers from cardiotoxicity that can be lethal. Trials have sought to reduce the cardiotoxicity by reducing the peak plasma levels of DOX, but results have been controversial. Besides, these attempts involving low dose DOX may produce still other adverse effects. The purpose of this study was to evaluate the effect of low concentrations of DOX on the invasiveness of OS, and the involvement of the reactive oxygen species/matrix metalloproteinase-9 (ROS/MMP-9) pathway.

Methods: The human OS 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 ROS signaling. The effects of DOX, H2O2, and NAC on U2OS proliferation was evaluated by the MTT assay. Invasiveness of U2OS was evaluated by MatrigelTM invasion assay and gelatin zymography in the presence of various doses of DOX or H2O2, with or without NAC pre-treatment. In each experimental condition, MMP-9 expression was measured using quantitative real time polymerase chain reaction (qPCR).

Results: In proliferation assays, DOX (up to 500 nM), H2O2 (up to 50 μM), and NAC (up to 10 mM) did not affect U2OS proliferation significantly for up to 24 h. MatrigelTM invasion assay and gelatin zymography revealed increased invasiveness of U2OS at around 100 nM of DOX, which is a subclinical concentration. In qPCR, MMP-9 expression was increased in the presence of DOX doses that increased the invasiveness of U2OS. H2O2, a representative source of ROS, also increased the invasiveness of U2OS. No DOX- or H2O2-mediated increased invasiveness or MMP-9 expression was evident when pre-treated with NAC.

Conclusions: Low-dose DOX increased U2OS invasiveness through ROS activation and MMP-9 induction in vitro. DOX may have oncologic adverse effects at low doses, which should be further investigated. The ROS/MMP-9 pathway could be a novel target for future research and targeted therapies for OS.