Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^??) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^?? and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^?? and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.