Downregulation of NADPH oxidase 1 and 5 inhibits TPA-induced cell invasion via PKC/ROS-mediated MMP-9 activation in MCF-7 cells

Abstract

Abstract NADPH oxidases (NOXs) are a family of membrane proteins involved in intracellular reactive oxygen species (ROS) production through transferring electrons across biological membranes. NOXs are activated by protein kinase C (PKC); however, the mechanism underlying NOX activation via PKC during breast cancer invasion remains unclear. In this study, we demonstrated that NOX1 and NOX5 play crucial roles in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloprotease (MMP)-9 expression and invasion of MCF-7 cells. In addition, we show that TPA-induced MMP-9 expression and cell invasion are mediated through PKC, linking the NOX1 and 5/ROS signaling pathways. The expression and activity of NOXs and MMP‑9 were investigated using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The activity of MMP‑9 was determined through zymography. Cell invasion was investigated using the Matrigel invasion assay. ROS levels were determined using a FACSCalibur flow cytometer. NOX inhibitors significantly attenuated TPA-induced MMP-9 expression and invasion in MCF-7 cells. NOX1 and NOX5 specific small interfering RNAs (siRNA) attenuated the TPA-induced MMP-9 expression and cell invasion. TPA-induced ROS levels were suppressed by the knockdown of NOX1 and NOX5. PKC inhibitors suppressed TPA-induced intracellular ROS levels, MMP-9 expression, and NOX activity in MCF-7 cells. Therefore, NOX1 and NOX5 play crucial roles in TPA-induced MMP-9 expression and invasion of MCF-7 breast cancer cells. In addition, TPA-induced MMP-9 expression and cell invasion are mediated through PKC, linking the NOX1 and 5/ROS signaling pathways. These findings provide new insights into the potential mechanisms underlying their anti-invasive effects in breast cancer.