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




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Breast Cancer Research

, 7:R681

First Online: 22 June 2005Received: 09 March 2005Accepted: 26 May 2005

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.

Abbreviations5FdUMP5-fluoro-deoxyuridine-monophosphate

5-FU5-fluorouracil

5FUTP5-fluoro-deoxyuridine-triphosphate

AIFapoptosis-inducing factor

CIcombination index

CMFcyclophasphamide-methotrexate-5-fluorouracil

DMEMDulbecco-s modified Eagle-s medium

DMSOdimethylsulfoxide

Doxdoxorubicin

FCSfetal calf serum

FEC = fluorouracilepirubicin, cyclophosphamide

Paclpaclitaxel

SRBsulforhodamine B

TSthymidylate synthase.

Electronic supplementary materialThe online version of this article doi:10.1186-bcr1274 contains supplementary material, which is available to authorized users.

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Author: Wainer Zoli - Paola Ulivi - Anna Tesei - Francesco Fabbri - Marco Rosetti - Roberta Maltoni - Donata Casadei Giunchi - Luc

Source: https://link.springer.com/







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