High nuclear NADPH oxidase 4 expression levels are correlated with cancer development and poor prognosis in hepatocellular carcinoma

Abstract

NADPH oxidase (NOX) is a key source of reactive oxygen species (ROS). This study aimed to verify NOX2 and NOX4 expression levels in hepatocellular carcinoma (HCC). A total of 134 matched pairs of HCC cells and non-tumour hepatocytes from 134 patients were examined by immunohistochemical staining, and the association of NOX2 and NOX4 expression with clinicopathological parameters was analysed. Western blotting in four HCC cell lines and reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) in 20 pairs of HCC and non-tumour tissue samples were also performed to detect NOX4. Cytoplasmic NOX2 and nuclear NOX4 expression levels were shown by immunohistochemistry to be higher in HCC cells than in non-tumour hepatocytes (p<0.001 each). The western blotting results for NOX4 in four HCC cell lines were consistent with the immunohistochemical results. Increased cytoplasmic expression of NOX2 and NOX4 in HCC cells was significantly correlated with liver cirrhosis (p<0.001 and p<0.031, respectively). However, decreased cytoplasmic expression of NOX2 and NOX4 was significantly correlated with advanced pathological TNM stage (p<0.029 and p<0.007, respectively). Multivariate analysis with clinicopathological parameters showed that high nuclear and low cytoplasmic NOX4 expression levels are correlated with short overall survival (p=0 .021). Our findings imply that cytoplasmic NOX2 and nuclear NOX4 expression is upregulated during HCC development. In particular, NOX4 translocation into the nucleus may affect the development and progression of HCC. NOX2 and NOX4 could be diagnostic markers and have therapeutic implications in HCC.