Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress

Abstract

Background: Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro.

Principal findings: Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300-600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes.

Significance: The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.

Figure 1. Cytotoxicity after 24 h exposure to H₂O₂.

(A) Cellular viability measure as LDH retention (n = 4 experiments, in triplicate). (B) Cellular metabolism measured as the reduction of MTT (n = 12 experiments, in triplicate). (•) Cos-7 cells, (○) Cos-7/pcDNA3 cells (▪), Cos-7/pFNR cells and (▴) Cos-7/pFld cells. ^a different from Cos-7 (p<0.05), ^b different from Cos-7/pcDNA3 (p<0.05), ^c different from Cos-7/pFNR (p<0.05).

Figure 2. Oxidative damage to lipids after 24 h exposure to H₂O₂.

(A) MDA levels. (B) Peroxide levels (FOX assay). (•) Cos-7 cells, (○) Cos-7/pcDNA3 cells, (▪) Cos-7/pFNR cells and (▴) Cos-7/pFld cells. ^a different from Cos-7 (p<0.05), ^b different from Cos-7/pcDNA3 (p<0.05), ^c different from Cos-7/pFNR (p<0.05), ^d different from Cos-7/pFld (p<0.05). n = 4 experiments, in triplicate.

Figure 3. Cytotoxicity after 48 h exposure to MV.

(A) Cellular metabolism (MTT reduction). (B) Lipid peroxidation levels (FOX assay). Black bars: Cos-7 cells, dark grey bars: Cos-7/pcDNA3 cells, light grey bars: Cos-7/pFNR cells and white bars: Cos-7/pFld cells. ^a different from Cos-7 (p<0.05), ^b different from Cos-7/pcDNA3 (p<0.05), ^c different from Cos-7/pFNR (p<0.05), ^d different from Cos-7/pFld (p<0.05). n = 4 experiments, in triplicate.