Manganese superoxide dismutase promotes interaction of actin, S100A4 and Talin, and enhances rat gastric tumor cell invasion

Abstract

It has been demonstrated that cancer cells are under high levels of oxidative stress and express high levels of Manganese superoxide dismutase (MnSOD) to protect themselves and support the anabolic metabolism needed for growth and cell motility. The aim of this study was to identify proteins that may have a correlation with invasion and redox regulation by mitochondrial reactive oxygen species (ROS). MnSOD scavenges superoxide anions generated from mitochondria and is an important regulator of cellular redox status. Oxidative posttranslational modification of cysteine residues is a key mechanism that regulates protein structure and function. We hypothesized that MnSOD regulates intracellular reduced thiol status and promotes cancer invasion. A proteomic thiol-labeling approach with 5-iodoacetamidofluorescein was used to identify changes in intracellular reduced thiol-containing proteins. Our results demonstrate that overexpression of MnSOD maintained the major structural protein, actin, in a reduced state, and enhanced the invasion ability in gastric mucosal cancer cells, RGK1. We also found that the expression of Talin and S100A4 were increased in MnSOD-overexpressed RGK1 cells. Moreover, Talin bound not only with actin but also with S100A4, suggesting that the interaction of these proteins may, in part, contribute to the invasive ability of rat gastric cancer.

Keywords: MnSOD; ROS; actin binding protein; cancer invasion; reduced form of actin.

Fig. 1

Invasion assay. (A) Matrigel Matrix coating on the BD Biocoat Matrigel Invasion Chamber was utilized for invasion assay. After incubation at 37°C in a 5% CO₂ humidified atmosphere for 22 h, the cells invaded through Matrigel were observed. (B) Quantitative assessment of the invasion index. The invasion index is expressed as the ratio of the % of invasion of MnSOD overexpressed cells over the % of invasion of vector alone cells. The invasion index in MnSOD overexpressed cells in RGK1 was significantly increased compared with control cells. Bar: mean ± SE; Student t test. *p<0.05.

Fig. 2

The detection of intracellular reduced thiols by 5-IAF labeling. (A) The cells were stained with 5-IAF in order to detect reduced intracellular thiols, and images were taken by laser microscope. The 5-IAF fluorescence intensity in MnSOD overexpressed cells increased compared with control cells in both RGM1 (rat normal gastric mucosal cell lines) and RGK1 [the N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced cancer cells of the original RGM-1]. (B) Quantitative analysis of fluorescence intensity. Significant increase in fluorescence of intracellular reduced thiols in MnSOD overexpressed cells is shown. Bar: mean ± SE; Student t test. *p<0.05. (C) Separation of cellular protein via 1D Gel after 5-IAF labeling. The 45 kDa band of MnSOD overexpressed cells and RGK1 was increased in both RGM and RGK1 compared with control cells. Interestingly, MnSOD overexpressed cancer cells were dramatically increased, but normal cells were not much different than the control and MnSOD overexpressed cells. (D) CBB stain represents the protein loading control.

Fig. 3

The detection of intracellular reduced thiols by 5-IAF labeling after hydrogen peroxide treatment. (A) After 0.5 mM hydrogen peroxide treatment for 20 min, the 5-IAF fluorescence intensity of the 45 kDa band in MnSOD overexpressed cells increased compared with control cells in RGM1, but a slight change occurred in RGK1 cells. (B) Quantitative assessment of the intensity of each band normalized CBB.

Fig. 4

Expression of EMT markers. (A) Expression of EMT markers were detected by Western blot analysis. Total cell lysates (20 µg/lane) of each sample were subjected to SDS PAGE in 7.5–15% gels for resolution of Snail1, Twist1, E-cadherin, N-cadherin. β-actin was used to standardize loading. (B) Quantitative assessment of the intensity of each band. There were no significant difference in the expression of EMT markers between the control and MnSOD overexpressed cells.

Fig. 5

Expression of actin binding proteins and linkage of actin binding proteins. (A) Expression of actin binding proteins was detected by Western blot analysis. Total cell lysates (20 µg/lane) of each sample were subjected to SDS-PAGE in 7.5–15% gels for resolution of Talin, Vinculin, α-actinin, S100A4. β-actin was used to standardize loading. (B) Quantitative assessment of the intensity of each band. Levels of Talin and S100A4 in MnSOD overexpressed cells were higher than those in control cells, but the expression of Vinculin and α-actinin did not change in the control or MnSOD overexpressed cells. Bar: mean ± SE; Student t test. *p<0.05, **p<0.01. (C) Linkage of actin binding proteins was detected by immunoprecipitation. Linkages of S100A4 and Talin, S100A4 and actin, Talin and actin are shown.