Involvement of superoxide and nitric oxide in BRAFV600E inhibitor PLX4032-induced growth inhibition of melanoma cells

The BRAF(V600E) inhibitor PLX4032 (Vemurafenib) is an FDA-approved new drug for the treatment of metastatic melanomas, which specifically inhibits the RAS/MEK/ERK signaling pathway to control cell proliferation and adhesion. However, no study has been carried out to investigate the role of intracellular oxidative balance in PLX4032-induced tumor growth inhibition. Herein, for the first time, superoxide (O-2(center dot-)) and nitric oxide (NO) generated from PLX4032-challenged melanoma cells were monitored using electrochemical sensors and conventional fluorescein staining techniques. Impacts of superoxide dismutase (SOD) and NG-monomethyl-L-arginine monoacetate (L-NMMA), a nitric oxide synthase inhibitor, were also examined to demonstrate the specificity of ROS/NO generation and its biological consequences. PLX4032 specifically triggers production of O-2(center dot-) and NO from BRAF(V600E) mutant A375 cells. SOD and L-NMMA could abolish the PLX4032-induced increase in intracellular O-2(center dot-) and NO production, thereby rescuing cell growth in BRAF mutant A375 cells (A375BRAF(V600E)). In addition, PLX4032 treatment could decrease the mitochondrial membrane potential in A375BRAF(V600E) cells. The results suggest that PLX4032 can selectively cause ROS production and depolarization of mitochondrial membranes, potentially initiating apoptosis and growth inhibition of PLX4032-sensitive cells. This work not only proposes a new mechanism for PLX4032-induced melanoma cell inhibition, but also highlights potential applications of electrochemical biosensors in cell biology and drug screening.