Addition of 5-fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines

Abstract

Introduction: The aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5-fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells.

Methods: The study was performed on MCF-7 and BRC-230 cell lines. The cytotoxic activity was evaluated by sulphorhodamine B assay and the type of drug interaction was assessed by the median effect principle. Cell cycle perturbation and apoptosis were evaluated by flow cytometry, and apoptosis-related marker (p53, bcl-2, bax, p21), caspase and thymidylate synthase (TS) expression were assessed by western blot.

Results: 5-FU, used as a single agent, exerted a low cytotoxic activity in both cell lines. The Dox-->Pacl sequence produced a synergistic cytocidal effect and enhanced the efficacy of subsequent exposure to 5-FU in both cell lines. Specifically, the Dox-->Pacl sequence blocked cells in the G2-M phase, and the addition of 5-FU forced the cells to progress through the cell cycle or killed them. Furthermore, Dox-->Pacl pretreatment produced a significant reduction in basal TS expression in both cell lines, probably favoring the increase in 5-FU activity. The sequence Dox-->Pacl-->48-h washout-->5-FU produced a synergistic and highly schedule-dependent interaction (combination index < 1), resulting in an induction of apoptosis in both experimental models regardless of hormonal, p53, bcl-2 or bax status. Apoptosis in MCF-7 cells was induced through caspase-9 activation and anti-apoptosis-inducing factor hyperexpression. In the BRC-230 cell line, the apoptotic process was triggered only by a caspase-dependent mechanism. In particular, at the end of the three-drug treatment, caspase-8 activation triggered downstream executioner caspase-3 and, to a lesser degree, caspase-7.

Conclusion: In our experimental models, characterized by different biomolecular profiles representing the different biology of human breast cancers, the schedule Dox-->Pacl-->48-h washout-->5-FU was highly active and schedule-dependent and has recently been used to plan a phase I/II clinical protocol.

Figure 1

Effect of different drug treatments on the survival of MCF-7 and BRC-230 breast cancer cell lines. (a) 5-FU, (b) Dox→Pacl sequence and (c) Dox→Pacl→5-FU treatment. D1, D2, D3 and D4 represent the doses of the three drugs used in the sequence (see Materials and methods). Each data point is the average of at least three independent experiments performed in octuplet. The standard deviation never exceeded 5%.

Figure 2

BRC-230 cells after Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout→5-FU (1 μg/ml) treatment. Apoptotic nuclei stained with DAPI show intense fluorescence corresponding to chromatin condensation (arrow heads) and fragmentation (arrows).

Figure 3

Inhibition of apoptosis. Results from TUNEL assay showing inhibition induced by Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout→5-FU (1 μg/ml) treatment in (a) BRC-230 cells in the presence of caspase-3 (casp-3) inhibitor and (b) MCF-7cells in the presence of caspase-9 (casp-9) inhibitor.

Figure 4

Protein levels of bcl-2, bax, p53 and p21 following different drug exposures. Protein (50 μg) was loaded for the controls and treated samples: (a) untreated cells; (b) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml); (c) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout; (d) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout→5-FU (1 μg/ml).

Figure 5

Western blot analysis of caspases and apoptosis-inducing factor (AIF) proteins following different treatments. Protein (50 μg) was loaded for the controls and treated samples: (a) untreated cells; (b) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml); (c) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout; (d) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout→5-FU (1 μg/ml).

Figure 6

Western blot analysis of thymidylate synthase and ternary complex. (a) Untreated cells; (b) 5-FU (1 μg/ml); (c) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml); (d) Dox (0.1 μg/ml)→Pacl (0.1 μg/ml)→48-h washout→5-FU (1 μg/ml). 50 μg of protein were loaded for the controls and treated samples.