Inhibition of NADPH oxidase 2 induces apoptosis in osteosarcoma: The role of reactive oxygen species in cell proliferation.

Osteosarcomas (OS) are aggressive tumors that are characterized by dysregulated growth and resistance to apoptosis. Reactive oxygen species (ROS) are thought to be important signal transduction molecules in the regulation of cell growth. ROS-generating nicotinamide adenine dinucleotide phosphate oxidase () family enzymes have previously been suggested to be involved in neoplastic proliferation. To examine whether -mediated generation of intracellular ROS confers anti-apoptotic activity, and thus a growth advantage, the current study first analyzed the mRNA expression of family members by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in five human OS cell lines. RT-PCR analysis revealed that and mRNAs were expressed in all the OS cell lines examined, whereas little or no and mRNAs were detected. By RT-qPCR, mRNA expression levels were demonstrated to be higher than mRNA expression levels. The viability of OS cells decreased in a dose-dependent manner with treatment of diphenylene iodonium (DPI), an inhibitor of flavoprotein-dependent oxidase. DPI treatment was observed to reduce intracellular ROS levels by ~50%, and increase the frequency of apoptosis by 30%. Notably, small interfering RNAs (siRNAs) targeting significantly suppressed ROS generation; ROS depletion by DPI or siRNAs induced apoptosis in OS cells. Together, the results of the present study indicate that -mediated ROS generation promotes cell survival and ROS depletion leads to apoptosis, thus highlighting the -ROS signaling pathway as a potential therapeutic target for OS treatment.