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


CONCLUSIONS
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.
Zoli
Wainer
W
Division of Oncology and Diagnostics, Morgagni Pierantoni Hospital, Forlì, Italy. w.zoli@ausl.fo.it
Ulivi
Paola
P
Tesei
Anna
A
Fabbri
Francesco
F
Rosetti
Marco
M
Maltoni
Roberta
R
Giunchi
Donata Casadei
DC
Ricotti
Luca
L
Brigliadori
Giovanni
G
Vannini
Ivan
I
Amadori
Dino
D
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D017239:Paclitaxel / Q000494:pharmacology*; D019014:fas Receptor / Q000235:genetics
D019014:fas Receptor; D004317:Doxorubicin; D017239:Paclitaxel; D005472:Fluorouracil