Modulation of VEGF Expression and Oxidative Stress Response by Iodine Deficiency in Irradiated Cancerous and Non-Cancerous Breast Cells

Abstract

Breast cancer remains a major concern and its physiopathology is influenced by iodine deficiency (ID) and radiation exposure. Since radiation and ID can separately induce oxidative stress (OS) and microvascular responses in breast, their combination could additively increase these responses. Therefore, ID was induced in MCF7 and MCF12A breast cell lines by medium change. Cells were then X-irradiated with doses of 0.05, 0.1, or 3 Gy. In MCF12A cells, both ID and radiation (0.1 and 3 Gy) increased OS and vascular endothelial growth factor (VEGF) expression, with an additive effect when the highest dose was combined with ID. However, in MCF7 cells no additive effect was observed. VEGF mRNA up-regulation was reactive oxygen species (ROS)-dependent, involving radiation-induced mitochondrial ROS. Results on total VEGF mRNA hold true for the pro-angiogenic isoform VEGF165 mRNA, but the treatments did not modulate the anti-angiogenic isoform VEGF165b. Radiation-induced antioxidant response was differentially regulated upon ID in both cell lines. Thus, radiation response is modulated according to iodine status and cell type and can lead to additive effects on ROS and VEGF. As these are often involved in cancer initiation and progression, we believe that iodine status should be taken into account in radiation prevention policies.

Keywords: MCF12A; MCF7; ROS; VEGF; breast; iodine deficiency; oxidative stress; radiation.

Figure 1

X-ray doses from 0.1 to 3 Gy increase vascular endothelial growth factor (VEGF) mRNA expression in MCF12A breast cells. MCF12A cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by fresh iodine-containing medium and cells were then irradiated with a dose of 0.05, 0.1, or 3 Gy. Cells were harvested 4 h after medium change. VEGF mRNA expression was determined using RT-qPCR. Data are expressed as means ± SEM. p-values < 0.05 were considered as statistically significant. *p < 0.05, **p < 0.01. Statistical test: one-way ANOVA with Dunnett post-hoc test. N = 3, one representative experiment.

Figure 2

Iodine deficiency (ID) and 3 Gy X-rays additively up-regulate VEGF mRNA in MCF12A but not in MCF7 cells. Cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were then irradiated with a dose of 0.05 (A,D), 0.1 (B,E), or 3 Gy (C,F). Cells were harvested 4 or 6 h after medium change. VEGF mRNA expression in MCF12A (A–C) and MCF7 cells (D–F) was determined using RT-qPCR. Data are expressed as means ± SEM. p-values < 0.05 were considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical test: one-way ANOVA with Tukey post-hoc test. A, B, F: N = 5; C: N = 7; D: N = 3; E: N = 4.

Figure 3

ID and radiation differentially up-regulate VEGF protein in MCF12A and MCF7 breast cells. MCF12A (A,B) and MCF7 (C,D) cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were irradiated with a dose of 0.1 (A, C) or 3 Gy (B, D). Cells were harvested 5 (A, B) or 7 (C, D) hours after medium change. VEGF protein expression was visualized by western blot. N = 3.

Figure 4

ID and irradiation modifies VEGF alternative splicing in breast cells. Cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were then irradiated with a dose of 0.1 or 3 Gy. Cells were harvested 4 (A,C) or 6 h (B,D) after medium change. VEGF 165 (A, B) and VEGF165b (C, D) mRNA expression in MCF12A (A, C) and MCF7 cells (B, D) was determined using RT-qPCR. Data are expressed as means ± SEM. p-values < 0.05 were considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical test: one-way ANOVA with Tukey post-hoc test. A: N = 5; B: N = 4; C–D: N = 3.

Figure 5

ID and X-irradiation increase oxidative stress (OS) in breast cells, with an additive effect at 3 Gy in MCF12A cells. Cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were then irradiated with a dose of 0.1 or 3 Gy. 4-HNE adducts were detected by immunofluorescence after 2 and 4 h in MCF12A (A–F) and MCF7 (G–L) cells respectively. Control (A, G), ID (B, H), 0.1 Gy (C, I), ID + 0.1 gy (D, J), 3 Gy X-rays (E, K) and ID + 3 Gy X-rays (F, L). Representative pictures are shown. Scale bars = 20 µm. N = 3.

Figure 6

ID and radiation differentially increase ROS cellular content in MCF12A cells. Cells were cultured in iodine-containing medium for 7 days. Thereafter, the medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were irradiated with a dose of 3 Gy. Cells were harvested 1 (A (two first columns), B) or 2 h (A (two last columns), and C) after medium change. Cellular reactive oxygen species (ROS) content was visualized with DCF-DA and measured by flow cytometry. In irradiated cells, DCF-DA was added 10 min before exposure to radiation (B) or 25 min after the irradiation (C). DCF-DA was added at corresponding timing in non-irradiated cells. Data are expressed as means ± SEM. p-values < 0.05 were considered as statistically significant. *p < 0.05, ***p < 0.001. Statistical test: one-way ANOVA with Tukey post-hoc test. N = 3, one representative experiment.

Figure 7

ID and radiations-induced VEGF mRNA up-regulation depends on cellular ROS production from different origins. Cells were cultured in iodine-containing medium for 7 days. Medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were then irradiated with a dose of 3 Gy. Part of the cells was treated with DPI (A,B) or mitoTEMPO (C,D). Cells were harvested 4 or 6 h after medium change. VEGF mRNA expression in MCF12A (A, C) and MCF7 cells (B, D) was determined using RT-qPCR. Data are expressed as means ± SEM. p-value < 0.05 were considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical test: one-way ANOVA with Newman–Keuls post-hoc test. A: N = 5; B, D: N = 3; C: N = 6.

Figure 8

ID and radiation additively up-regulate antioxidant enzymes mRNA in breast cells. Cells were cultured in iodine-containing medium for 7 days. Medium was replaced by iodine-containing (control) or iodine deficient (ID) medium and cells were then irradiated with a dose of 0.1 or 3 Gy. Cells were harvested 3 (MCF12A) or 6 (MCF7) hours after medium change. SOD1, SOD2, and catalase mRNA expression in MCF12A (A–F) and MCF7 cells (G–L) was determined using RT-qPCR. Data are expressed as means ± SEM. p-value < 0.05 were considered as statistically significant. *p < 0.05, **p < 0.01. Statistical test: one-way ANOVA with Tukey post-hoc test. A: N = 6; B, G: N = 4; C, D, F, K: N = 5; E: N = 8; H, I, J, L: N = 3.