Increasing dietary selenium elevates reducing capacity and ERK activation associated with accelerated progression of select mesothelioma tumors

Abstract

To study the effect of the micronutrient selenium on malignant mesothelioma (MM) progression, we cultured four different MM cell lines in media containing increasing amounts of sodium selenite (30, 50, and 80 nmol/L). Increasing selenium levels increased density-dependent proliferation and mobility for CRH5 and EKKH5 but not AB12 and AK7. Comparing these cell lines revealed that extracellular regulated kinase (ERK) phosphorylation was sensitive to a selenium increase in CRH5 and EKKH5 but not AB12 and AK7 cells. Stable expression of a dominant-negative mutant ERK eliminated the effects of increasing selenium. Because ERK is redox sensitive, we compared the MM cell lines in terms of glutathione levels and the capacity to reduce exogenous hydrogen peroxide. Increasing selenium levels led to higher glutathione and reducing capacity in CRH5 and EKKH5 but not AB12 and AK7. The reducing agent N-acetylcysteine eliminated the effects of selenium on ERK activation, proliferation, and mobility. Mice fed diets containing increasing levels of selenium (0.08, 0.25, and 1.0 ppm) showed increased tumor progression for CRH5 but not AB12, MM cells, and in vivo N-acetylcysteine treatment eliminated these effects. These data suggest that the effects of dietary selenium on MM tumor progression depend on the arising cancer cells' redox metabolism, and the tumors able to convert increased selenium into a stronger reducing capacity actually benefit from increased selenium intake.

Figure 1

Increasing selenium levels increases the proliferation of select MM cell lines. Cellular GPx activity increases with increasing selenium levels [low (30 nmol/L), medium (50 nmol/L), and high (80 nmol/L)] in all MM cell lines (AK7, AB12, EKKH5, and CRH5). A: A total of 10³ cells/well were plated and allowed to grow to confluency with media changes daily. B: Proliferation of AK7 and AB12 cells was not affected by selenium levels, whereas CRH5 and EKKH5 cells showed an increase in proliferation with increasing levels of selenium during the late stages of growth. Data represent means ± SEM. N = 3. Means of each selenium group were compared at each time point. Each experiment was repeated at least twice. ^∗P < 0.05. MFI, mean fluorescence intensity.

Figure 2

Increasing selenium levels affect the mobility of CRH5 and EKKH5 MM cell lines similar to proliferation. Four different MM cell lines were cultured in media containing 30, 50, and 80 nmol/L selenium for 1 week, cells were replated, and allowed to grow to confluency. Each experiment was repeated twice. Images from the scratch assays captured at 0 and 6 hours show mobility. A: Representative images at 6 hours are shown. B: Quantification of the scratch volume was calculated by ImageJ software. Data represent means ± SEM. ^∗P < 0.05.

Figure 3

The effects of selenium on the proliferation and mobility of MM cell lines are mediated by ERK activation. A: Western blot analysis on cell lysates from 1-week-old cells show an increase in pERK levels with increasing selenium [low (30 nmol/L), medium (50 nmol/L), and high (80 nmol/L)] in CRH5 and EKKH5, but not AK7 and AB12, cell lines. CRH5 also showed a slight increase in pAKT with increasing selenium. Total ERK and AKT levels were equivalent, as was β-actin (loading control). Levels of GPx1 confirmed equivalent use of bioavailable selenium for each MM cell line. Data are representative of two independent experiments. C-fos mRNA levels (B and E), mean fluorescence intensity (MFI) (C and F), and scratch area (D and G) in CRH5 and EKKH5 cells are shown. The cells were stably transfected with a plasmid encoding a dominant-negative form of ERK (TAYE-ERK). Levels of c-fos mRNA were compared with housekeeping β-actin mRNA for each sample using real-time PCR. The TAYE-ERK CRH5 and TAYE-ERK EKKH5 cells were cultured in media containing 30, 50, and 80 nmol/L sodium selenite for 1 week and the mobility and proliferation were evaluated. Each experiment was repeated twice. Data represent means ± SEM. ^∗P < 0.05.

Figure 4

Reducing capacity with increasing selenium is a crucial mechanism by which proliferation and mobility are affected in selenium-sensitive cells. Cellular GSH (A), ROS levels (B), and Western blot analysis (C) in MM cell lines cultured in media containing low (30 nmol/L), medium (50 nmol/L), and high (80 nmol/L) selenium for 1 week. For ROS levels the cells were challenged with 250 mmol/L H₂O₂. For Western blot analysis 0.4 mmol/L NAC was added as a reducing agent for 14 hours before protein extraction and Western blot analysis. No differences were found in pERK or pAKT for any of the cell lines, and levels of GPx1 confirmed equivalent use of bioavailable selenium for each MM cell line. Data are representative of two independent experiments. All four MM cell lines cultured in increasing selenium with NAC added as described earlier showed no differences in proliferation (D) or mobility (E). Each experiment was repeated twice. Data represent means ± SEM. ^∗P < 0.05.

Figure 5

Increasing dietary selenium in vivo promotes redox-dependent tumor progression of CRH5, but not AB12, MM cells. Male Balb/c mice were fed defined diets containing low (0.08 ppm), medium (0.25 ppm), and high (1.0 ppm) selenium as sodium selenite for 4 weeks, and then 10⁶ AB12 or CRH5 MM cells were injected s.c. and tumor volume measurements were performed weekly. CRH5 also was injected into mice maintained on drinking water containing 0.4 mmol/L NAC for the remainder of the experiment. Mice were sacrificed when tumor volumes threatened the health of the mice. Each experiment was repeated twice. Data represent means ± SEM. Means of each selenium group were compared at each time point. N = 5 per group. ^∗P < 0.05.