Sodium channel γENaC mediates IL-17 synergized high salt induced inflammatory stress in breast cancer cells

Abstract

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. In this report, using three breast cancer-related cell lines, we determined the molecular basis of the potential synergistic inflammatory effect of sodium chloride (NaCl) with interleukin-17 (IL-17). Combined treatment of high NaCl (0.15M) with sub-effective IL-17 (0.1 nM) induced enhanced growth in breast cancer cells along with activation of reactive nitrogen and oxygen (RNS/ROS) species known to promote cancer. Similar effect was not observed with equi-molar mannitol. This enhanced of ROS/RNS activity correlates with upregulation of γENaC an inflammatory sodium channel. The similar culture conditions have also induced expression of pro-inflammatory cytokines such as IL-6, TNFα etc. Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.

Keywords: Cancer; Cytokine; Epithelial sodium channel (ENaC); Inflammation; Interleukin-17.

Figure 1

Induction of nitric oxide pathway by high sodium chloride in breast cancer cells. (A) Cell viability analysis of MDA-MB-231 in the presence of varying NaCl concentration (0.1 -0.3 mM). It is important to note that 0.1 M NaCl is the basal sodium chloride concentration in the RPMI1640 culture media and therefore 0.1 M NaCl refers to basal media treatment control group. (B) Cell viability analysis of MDA-MB-231 in the presence of varying interleukin (IL-)17 concentration (0.1 - 500 nM) in basal (0.1 M NaCl) media. (C) Lack of cell death with NaCl (0.15 M) and IL-17 (0.1 nM) combination. (D) Cell viability was similar among various breast cancer (MDA-MB-231, MCF-7) and normal breast (MCF10A) cell lines with NaCl (0.3 mM) and IL-17 (0.1 nM) combination; (E) iNOS, inducible nitric oxide synethsase (E), nitric oxide (F) and RNS, reactive nitrogen species (G) expression in all three cell lines mentioned above following treatment with NaCl (0.15 M) and/or IL-17 (0.1 nM in basal 0.1 M NaCl media). Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).

Figure 2

High sodium chloride induced reactive oxygen species in breast cancer cells. (A) Western blot analysis of anti-oxidant enzyme (SOCS, SOD1, SOD2) expression following treatment with NaCl (0.15 M) and/or IL-17 (0.1 nM in basal 0.1 M NaCl media) in three cell lines; (B) ROS, reactive oxygen species expression in all three cell lines mentioned above following treatment with NaCl (0.15 M) and/or IL-17 (0.1 nM). (C-E) mRNA analysis for SOCS, SOD1, SOD2 expression in various breast cancer cell lines (MDA-MB-231, MCF-7 and MCF-10a) following co-treatment with high salt (0.15 M NaCl) and 0.1 nM IL-17. Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).

Figure 3

γENaC mediates high sodium chloride induced inflammatory stress. (A) Western blot analysis of epithelial sodium channel (ENaC) isoforms (-α, -β, -γ) following treatment with NaCl (0.15 M) and/or IL-17 (0.1 nM in basal 0.1 M NaCl media) in three cell lines; (B) mRNA analysis for γENaC expression in various breast cancer cell lines (MDA-MB-231, MCF-7 and MCF-10a) following co-treatment with high salt (0.15 M M NaCl) and 0.1 nM IL-17. (C) Efficient knock-down of γENaC by specific siRNA, while scramble siRNA does not decrease expression of γENaC. (D-F) Inhibition of nitric oxide (C), RNS (D) and ROS (E) release following siRNA knock-down of γENaC. Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).

Figure 4

Lipid rafts play critical role in γENaC mediated inflammatory stress. (A) Western blot analysis of γENaC following depletion of lipid rafts in three cell lines; (B) (B-D) Inhibition of nitric oxide (B), RNS (C) and ROS (D) release following βMCD mediated removal of lipid rafts. Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).

Figure 5

ERK-1/2 protein kinase upregulation in γENaC induced inflammatory stress. (A) Western blot (phospho-blot) analysis of signaling factors (ERK-1/2, p-ERK-1/2, p38, c-Jun) following treatment with NaCl (0.15 M M) and/or IL-17 (0.1 nM in basal 0.1 M NaCl media) in three cell lines; (B) Western blot (phospho-blot) analysis of protein kinase, (p-)ERK-1/2, following siRNA knock-down of γENaC and/or lipid rafts in three cell lines; (C) Efficient knock-down of ERK-1/2 by specific siRNA, while scramble siRNA does not decrease expression of ERK-1/2. (D-F) Inhibition of nitric oxide (C), RNS (D) and ROS (E) release following siRNA knock-down of ERK-1/2. Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).

Figure 6

Upregulation of inflammatory cytokines and chemokine following sodium chloride induced cancer cell stress. ELISA based analysis of cytokines, IL-6 (A), TNFα (B); chemokines, CCL5 (C), CXCL-12 (D), MIP-1δ (E); and angiogenic growth factor VEGF (F) following treatment with NaCl (0.15 M mM) and IL-17 (0.1 nM) in three cell lines. Basal condition refers to 0.1 M NaCl which is the basal concentration of NaCl in regular RPMI1640 media. Data represented mean values ± SEM from five independent experiments. Student-t-test performed for statistical analysis (significance p<0.05).