Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

Abstract

Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H₂O₂), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H₂O₂ determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

Keywords: Bcl-2; E-cadherin; GPX1; MnSOD; Sirt3; hydrogen peroxide.

Figure 1. GPX enzyme activity and MnSOD levels are expressed in MCF-7 transfectants

A. Lysates from MCF-7 transfected with the empty vector (C), MCF-7 GPX1, MCF-7 MnSOD^Val and MCF-7 MnSOD^Ala cell lines were used to determine GPX enzyme activity using a spectrophotometric assay that determines the GPX dependent consumption of NADPH. B. Lysates from MCF-7, GPX1, MnSOD^Val, and MnSOD^Ala cells were analyzed for MnSOD protein levels by western blot using anti-MnSOD antibodies. β-Actin was used as an endogenous protein loading control. Protein levels were quantified using fluorescence detection and normalized to β-Actin. Error bars indicate the standard deviation (n=3) (* = p<0.05).

Figure 2. The effects of the expression of GPX1 and MnSOD on the levels of E-Cadherin, p-Akt, Bcl-2 and Sirt3

Lysates from MCF-7 cells transfected with the empty vector (C), GPX1, MnSOD allele-specific constructs were analyzed for selected protein levels by western blot using protein-specific antibodies. β-Actin was used as an endogenous protein loading control. Protein levels were quantified using fluorescence detection and normalized to β-Actin. Error bars indicate the standard deviation (n=3) (* = p<0.05). Proteins analyzed were A) E-Cadherin, B) p-Akt, C) Bcl-2 and D) Sit3.

Figure 3. H₂O₂ levels in cells expressing GPX1^A7L and MnSOD variants

H₂O₂ levels were assessed in the transfectants using the Amplex Red indicator. Fluorescence was quantified and normalized to protein content as determined using the BCA protein assay. Error bars indicate the standard error (n=4) (* = p<0.05).

Figure 4. Catalase levels in MCF-7 GPX1 and MnSOD transfectants

Lysates from MCF-7 transfected with the empty vector (C), GPX1, MnSOD transfected MCF-7 cells were analyzed for catalase levels by western blot using anti-catalase antibodies. β-Actin was used as an endogenous protein loading control. Protein levels were quantified using fluorescence detection and normalized to β-Actin. Error bars indicate the standard deviation (n=3) (* = p<0.05).