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

Breast Cancer Res. 2005;7(5):R681-9. doi: 10.1186/bcr1274. Epub 2005 Jun 22.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Combined Chemotherapy Protocols / administration & dosage
  • Apoptosis / drug effects*
  • Breast Neoplasms / pathology*
  • Cell Cycle / drug effects*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Doxorubicin / pharmacology*
  • Female
  • Fluorouracil / pharmacology*
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Kinetics
  • Paclitaxel / pharmacology*
  • fas Receptor / genetics


  • fas Receptor
  • Doxorubicin
  • Paclitaxel
  • Fluorouracil