Abstract
IntroductionThe 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.MethodsThe 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.Results5-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.ConclusionIn 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.
Highlights
The aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells
In the light of these preclinical and clinical observations, the present study aimed to investigate the cytotoxic effect produced by the combination of Dox, Pacl and 5-FU in human breast cancer cell lines and to define, at the preclinical level, the most effective treatment scheme
Cell lines The study was performed on two human breast cancer cell lines: a commercial line (MCF-7; 40-h doubling time, obtained from the American Type Culture Collection (Rockville, MD), and a cell line established in our laboratory (BRC-230; 30-h doubling time) [25]
Summary
The aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells. Adjuvant chemotherapy has been shown to provide disease-free and overall survival benefits for patients with node-positive breast cancer in large meta-analyses conducted by the Early Breast Cancer Trialists' Collaborative Group [2]. A study by Weiss et al [3], showed that the overwhelming majority (80%) of node-positive patients relapse and die within 26 years of diagnosis despite the use of cyclophasphamide-methotrexate-5fluorouracil (CMF)-based adjuvant chemotherapy. The development of drugs and strategies to improve relapse-free and overall survival remains a high priority.
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