Effects of iron deficiency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer

Abstract

Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre- and postmenopausal women. In this study, we have tested a hypothesis that, in addition to estrogen, both iron deficiency due to menstruation and iron accumulation as a result of menstrual stop play important roles in menopause-related BC outcomes. We first tested this hypothesis in cell culture models mimicking the high-estrogen and low-iron premenopausal condition or the low-estrogen and high-iron postmenopausal condition. Subsequently, we examined this hypothesis in mice that were fed iron-deficient and iron-overloaded diets. We show that estrogen only slightly up-regulates vascular endothelial growth factor (VEGF), an angiogenic factor known to be important in BC recurrence. It is, rather, iron deficiency that significantly promotes VEGF by stabilizing hypoxia-inducible factor-1α. Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron-deficiency-mediated proangiogenic environment could contribute to the high recurrence of BC in young patients, and iron-accumulation-associated pro-oxidant conditions could lead to the high incidence of BC in older women.

Conflict of interest statement

Competing Interests Statement: None

Copyright © 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1. Effects of high E2 and low Fe versus low E2 and high Fe on VEGF formation, in vitro angiogenesis, and HIF-1α stabilization

(A) MCF-7 cells grown under pre- (high E2 and low Fe) or postmenopausal conditions (low E2 and high Fe) were exposed to 1% O2 for 6 h, followed by overnight culture under normoxia (hypoxia + culture). (B) MCF-7 grown under the two conditions were exposed to normoxia or hypoxia (1% O2) for 6 h and then lysed for HIF-1α blotting. A representative gel from three independent experiments was displayed. Bar graph below shows quantitation by densitometry after normalizing to the housekeeping gene and then to the control under normoxia. Ni was used as a positive control for HIF-1α induction and β-tubulin as a loading control of proteins. Results are reported as the mean ± SD. *: Significantly different among the groups compared by Student’s t test (n=6).

Figure 2. Effects of E2, ferritin, and transferrin on VEGF formation

MCF-7 cells were grown in α-MEM media in the presence of various concentrations of (A) E2, (B) ferritin, (C) apo-transferrin (no iron), and (D) the same amounts of transferrin (5 μg/ml) but with different iron saturations. After 24 h treatment, cell culture media were collected for VEGF measurements. E2: 17β-estradiol; Ftn: ferritin, Tf sat: transferrin saturation. Significantly different among the groups compared (n=4).

Figure 3. Effects of high E2 and low Fe versus low E2 and high Fe onlipid peroxidation, oxidant formation, and MAPK phosphorylation

(A) MCF-7 cells were grown under pre- and postmenopausal conditions. After overnight culture, cells were collected or further exposed to 10 μM H2O2 for 4 h before lipid peroxidation measurements. (B) MCF-7 cells grown under the two conditions were treated with or without 10 μM H2O2 for various times. Cells were lysed and probed for phosphorylated ERK, p38, and JNK (shown) and non-phosphorylated ERK, p38, and JNK (data not shown). A representative gel from three independent experiments was displayed. β-tubulin was used to show an equal loading of proteins. Ratios indicate quantitation by densitometry after normalizing to the β-tubulin and then to the untreated control. a.u.: Arbitrary units. Significantly different among the groups compared (n=6).

Figure 4. Body iron status in mice fed four different levels of iron diets

(A) Serum iron and transferrin saturation rate in mice fed 3.5 ppm iron diet (iron deficient), 35 ppm and 350 ppm iron diets (normal low and normal high iron levels), and 3500 ppm iron diet (iron overload). (B) mRNA levels of EPO by qRT-PCR in kidneys of mice fed different levels of iron diets. (C) mRNA levels of hepcidin by qRT-PCR in livers of mice fed different levels of iron diets. Significantly different among groups compared (n=3) per group.

Figure 5. Effects of iron deficiency and overload on liver HIF-1α stabilization and VEGF formation

(A) Protein expressions of HIF-1α and ferritin heavy chain in livers of mice fed four different levels of iron diets. Bar graphs on the right represent average level of HIF-1α and heavy-chain (H)-ferritin from three mice per group. The band intensities were first quantitated by densitometry, normalized to the loading control, and then the control group fed 350 ppm iron diet. (B) Levels of liver VEGF in the mice fed four different levels of iron diets (n=3).

Figure 6. Effects of iron deficiency on mammary mRNA levels of HIF-1α and VEGF and iron overload on liver lipid peroxidation

(A) Levels of mRNAs of HIF-1α (A) and VEGF (B) by qRT-PCR in mammary fat pads of mice fed different levels of iron diets. (C) Levels of lipid peroxidation from the liver samples of the same mice and data were expressed as a.u. per mg protein (n=3). Significant different among the groups compared (n=3).