Imbalance in redox status is associated with tumor aggressiveness and poor outcome in lung adenocarcinoma patients

Abstract

Purpose: The expression levels of human antioxidant genes (HAGs) and oxidative markers were investigated in light of lung adenocarcinoma aggressiveness and patient outcome.

Methods: We assayed in vitro the tumoral invasiveness and multidrug resistance in human lung adenocarcinoma (AdC) cell lines (EKVX and A549). Data were associated with several redox parameters and differential expression levels of HAG network. The clinicopathological significance of these findings was investigated using microarray analysis of tumor tissue and by immunohistochemistry in archival collection of biopsies.

Results: An overall increased activity (expression) of selected HAG components in the most aggressive cell line (EKVX cells) was observed by bootstrap and gene set enrichment analysis (GSEA). In vitro validation of oxidative markers revealed that EKVX cells had high levels of oxidative stress markers. In AdC cohorts, GSEA of microarray datasets showed significantly high levels of HAG components in lung AdC samples in comparison with normal tissue, in advanced stage compared with early stage and in patients with poor outcome. Cox multivariate regression analysis in a cohort of early pathologic (p)-stage of AdC cases showed that patients with moderate levels of 4-hydroxynonenal, a specific and stable end product of lipid peroxidation, had a significantly less survival rate (hazard ratio of 8.87) (P < 0.05).

Conclusions: High levels of oxidative markers are related to tumor aggressiveness and can predict poor outcome of early-stage lung adenocarcinoma patients.

Fig. 1

Differential gene expression of HAG network in human lung AdC cell lines. a STRING representation of HAG network gene interactions. b Two-state landscape analysis of HAG expression between cell lines (GSE5846 dataset). Coordinates (X- and Y-axis) represent normalized values of the input network topology. Color gradient (Z-axis) represents the relative functional state mapped onto graph according to the data input from the adenocarcinomas EKVX-a versus A549-b, where z = a/(a + b). The landscape was generated with ViaComplex^® V1.0

Fig. 2

Cell growth is dependent on H₂O₂ in human lung adenocarcinoma cell lines. a Exogenous addition of catalase (125–1,000 U/mL) for 72 h causes dose-dependent inhibition in cell proliferation of AdC cell lines. b CAT washout after 48 h of incubation allowed cells to return to its original proliferation rate. c Dose–response curve against H₂O₂ lung AdC cell lines. d Sublethal doses (<40 μM) of H₂O₂ and aminotriazole stimulate cell growth on EKVX. Data represent mean ± SEM of at least three independent experiments (n = 3), performed in triplicate *different from respective control group (P < 0.05) (Student’s t test)

Fig. 3

4-Hydroxynonenal levels in human lung adenocarcinoma samples. a Representative immunostaining of 4-HNE in AdC biopsies is shown. IHC images represent negative controls (absence of primary antibody) (grade 0), weak (grade 1), moderate (grade 2) and strong stain (grade 3). Images are at ×400 magnification. b Cox multivariate regression analysis was used to estimate HR for cohort clinical covariates and 4-HNE levels. CI confidence interval, BMI body mass index, FEV1 forced expiratory volume in 1 s, FVC forced vital capacity