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 (NOX) family enzymes have previously been suggested to be involved in neoplastic proliferation. To examine whether NOX-mediated generation of intracellular ROS confers anti-apoptotic activity, and thus a growth advantage, the current study first analyzed the mRNA expression of NOX family members by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in five human OS cell lines. RT-PCR analysis revealed that NOX2 and NOX4 mRNAs were expressed in all the OS cell lines examined, whereas little or no NOX1 and NOX3 mRNAs were detected. By RT-qPCR, NOX2 mRNA expression levels were demonstrated to be higher than NOX4 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 NOX2 significantly suppressed ROS generation; ROS depletion by DPI or NOX2 siRNAs induced apoptosis in OS cells. Together, the results of the present study indicate that NOX2-mediated ROS generation promotes cell survival and ROS depletion leads to apoptosis, thus highlighting the NOX2-ROS signaling pathway as a potential therapeutic target for OS treatment.