Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

Abstract

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti-acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long-term exposure to concentrations of aluminium-in the form of aluminium chloride (AlCl₃ )-in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl₃ form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl₃ . Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well-established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens.

Keywords: aluminium; antiperspirants; breast cancer; carcinogen; metastasis; mouse models of cancer.

Figure 1

(a) Phase contrast view of NMuMG cells incubated for 4 months in the presence of AlCl₃ 100 μM (bottom) or the same volume (1/1,000) of solvent (H₂O) alone as a control (top). AlCl₃ or H₂O were renewed twice a week with fresh culture medium. Bar = 100 μm. (b) Approximately 80% confluent NMuMG cells cultured for 6 months in the presence of AlCl₃ 100 μM, or of the same dilution—1/1,000—of solvent (H₂O) alone as a control were analysed for the expression of E‐cadherin, or β‐actin as a loading control, by Western Blotting. One representative gel (out of 6) is shown. The bands presented were from the same gel. The graph shows the mean values for E‐cadherin expression, obtained in densitometric analysis, ± SEM, from six experiments, normalized with respect to β‐actin levels. Control (H₂O) was set to 1. p H₂O versus AlCl₃ 100 μM = 0.00038 (two‐tailed t‐test). (c) Same experiment as in (b), except that (i) cells were approximately 50% confluent; (ii) analysis was for N‐cadherin; (iii) the number of experiments was 4; (iv) p H₂O versus AlCl₃ 100 μM = 0.0023 (two‐tailed t‐test). [Color figure can be viewed at wileyonlinelibrary.com.]

Figure 2

AlCl₃ transforms NMuMG cells in vitro. (a) NMuMG cells cultured for 4 months in the presence of AlCl₃ 100 μM or the equivalent volume of H₂O as control were resuspended in agarose gels at the density of 2 × 10⁴ cells/ml and grown for 14 days in the presence of complete culture medium. AlCl₃ was not added to the soft agar assay. Bar = 100 μm. (b) The growth in agarose gels was quantified by measuring the diameter of the structures (single cells or multicellular colonies) formed after 14 days. At least 50 randomly selected structures (single cells or multicellular colonies) from two independent experiments/condition were measured. p AlCl₃ versus H₂O = 6.71E‐04 (two‐tailed t‐test). (c) The graph shows the growth in agarose of NMuMG cells from a different source (ATCC), cultured for 7 months in the presence of AlCl₃ 10 μM, AlCl₃ 100 μM or the equivalent volume of H₂O as control, as indicated. The soft agar assay was as described in a, b. p AlCl₃ 10 μM versus H₂O = 0.02; p AlCl₃ 100 μM versus H₂O = 2.79E‐05 (two‐tailed t‐test). [Color figure can be viewed at wileyonlinelibrary.com.]

Figure 3

AlCl₃ promotes tumorigenesis and lung metastasis in NMuMG cells injected subcutaneously into NSG mice. Five million NMuMG cells cultured for 6 months in the presence of AlCl₃ 100 μM or the equivalent volume of H₂O as control were resuspended in 200 μl of Matrigel and injected subcutaneously into the flank of 6‐week‐old NSG female mice. Five mice/condition were used. The mice were sacrificed 6 weeks after injection. The graphs show (a) the volume of each tumor (calculated according to the formula 4/3π × a × b × c, with a, b, c corresponding each to ½ of the three axis of the tumor, approximated to an ellipsoid, respectively) or (b) the weight of each mouse at the time of sacrifice. Tumor incidence was 100% (all the mice formed tumors). Statistics were as follows. For tumor volume, H₂O versus AlCl₃ 100 μM: p = 0.01, two‐tailed t‐test. For mouse weight, H₂O versus AlCl₃ 100 μM: p = 0.01, two‐tailed t‐test. (c) Macroscopic apical view (upper panel) or ventral view (lower panel) of the lungs of a NSG female mouse injected with AlCl₃‐treated NMuMG cells or H₂O‐treated NMuMG cells, as indicated, at the time of sacrifice.

Figure 4

HE stained sections of the lungs of a NSG female mouse injected with (b) NMuMG cells cultured in the presence of AlCl₃ 100 μM for 6 months or (a) NMuMG cells incubated for 6 months in the presence of a 1/1,000 dilution of H₂O. The mice were from the same experiment described in Figure 3. In (b), the inset shows necrotic cells. Bar in a and b = 50 μm.

Figure 5

AlCl₃ promotes tumorigenesis and brain metastasis in NMuMG cells injected subcutaneously into NSG mice. (a) HE stained sections of the cerebellum of a NSG female mouse injected with NMuMG cells cultured in the presence of AlCl₃ 100 μM for 6 months. The mouse was from the same experiment described in Figure 3. (b) Cytokeratin 7 or (c) cytokeratin 19 immunohistochemistry on adjacent sections. Bar in a, b, c = 50 μm.

Figure 6

AlCl₃ promotes tumorigenesis and metastasis in NMuMG cells injected subcutaneously into NOD SCID and nude mice. (a) Five million NMuMG cells cultured for 8 months in the presence of AlCl₃ 100 μM or the equivalent volume of H₂O as control were resuspended in 200 μl of Matrigel and injected subcutaneously into the flank of 6‐week‐old NOD SCID female mice. Five mice/condition were used. Cell growth at the site of injection was measured with a caliper. The graph shows the volume ± SEM of such growth (calculated according to the formula V (mm³) = d ² (mm²) × D (mm)/2, where d and D are the smallest and largest tumor diameters, respectively) at the indicated number of days after injection. Tumor incidence was 100% (all the mice injected with AlCl₃‐treated cells formed tumors). p values (two‐tailed t‐test) comparing the two experimental groups were as follows: 7 days: p = 0.37; 13 days: p = 0.02; 24 days: p = 0.001; 32, 39 and 45 days: p < 0.001. (b) Five million NMuMG cells cultured for 8 months in the presence of AlCl₃ 100 μM or the equivalent volume of H₂O as control were resuspended in 200 μl of Matrigel and injected subcutaneously into the flank of 6‐week‐old Swiss nude female mice. Five mice were used for AlCl₃‐treated cells, and four mice for control cells (the fifth mouse planned to be injected with control cells died during anesthesia at the time of injection). Cell growth at the site of injection was measured with a caliper. The graph shows the volume ± SEM of such growth (calculated according to the formula V (mm³) = d ² (mm²) × D (mm)/2, where d and D are the smallest and largest tumor diameters, respectively) at the indicated number of days after injection. Tumor incidence was 100% (all the mice injected with AlCl₃‐treated cells formed tumors). p values comparing the two experimental groups were as follows: 7 days: p = 0.11; 14 days: p = 0.0049; 21 days: p = 0.0017; 28 and 35 days: p < 0.003 (two‐tailed t‐test). (c) HE stained section (left) or cytokeratin 19 immunohistochemistry on an adjacent section (right) showing a lung metastasis in a Swiss nude mouse injected with AlCl₃‐treated NMuMG cells. Bar = 50 μm.