Cadmium modifies the cell cycle and apoptotic profiles of human breast cancer cells treated with 5-fluorouracil

Abstract

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.

Figure 1

FACScan analysis via Annexin V-FITC/PI staining was used to observe the induction of apoptosis in MCF-7 cells at high doses. (A) Representative images of the flow cytometry analysis. Cells in the lower right quadrant indicate the percentage of Annexin-positive, early apoptotic cells. Cells in the lower left quadrant indicate the percentage of Annexin-negative/PI-negative, viable cells. Cells in the upper right quadrant indicate the percentage of Annexin-positive/PI-positive, late apoptotic cells. Cells in the upper left quadrant indicate the percentage of PI-positive, necrotic cells; (B) Graphic representation of apoptotic levels (early plus late apoptosis) after treatment with Cd and/or 5-FU for 24 and 48 h. Data are expressed as mean ± SEM of three independent experiments. Q, Quadrant.

Figure 1

FACScan analysis via Annexin V-FITC/PI staining was used to observe the induction of apoptosis in MCF-7 cells at high doses. (A) Representative images of the flow cytometry analysis. Cells in the lower right quadrant indicate the percentage of Annexin-positive, early apoptotic cells. Cells in the lower left quadrant indicate the percentage of Annexin-negative/PI-negative, viable cells. Cells in the upper right quadrant indicate the percentage of Annexin-positive/PI-positive, late apoptotic cells. Cells in the upper left quadrant indicate the percentage of PI-positive, necrotic cells; (B) Graphic representation of apoptotic levels (early plus late apoptosis) after treatment with Cd and/or 5-FU for 24 and 48 h. Data are expressed as mean ± SEM of three independent experiments. Q, Quadrant.

Figure 2

Determination by qRT-PCR of gene expression after treatment with Cd and/or 5-FU for 6, 24, or 48 h. The fold-increase in expression was quantified after normalizing expression levels for those in control MCF-7 cells. Gene expression of bcl-2 (A); p53 (B); bax (C); caspase 8 (D); caspase 9 (E); c-myc (F); cyclin D1 (G); cyclin A1 (H).

Figure 3

Modification of cell cycle, proliferation and apoptotic proteins analysed by western blot on the MCF-7 cell line after treatment with 5-FU and/or Cd for 6, 24 or 48 h. Representative images of three experiments.