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Article Open Access

Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells

  • Authors:
    • Ali Haider Alhammer
    • Fadhel M. Lafta
    • Shahad Ali Mudhafar
  • View Affiliations / Copyright

    Affiliations: Department of Medical and Molecular Biotechnology, Biotechnology Research Center, Al‑Nahrain University, Baghdad 10072, Iraq, Biological and Medical Applications Branch, Institute of Laser for Postgraduate Studies, University of Baghdad, Baghdad 10071, Iraq
    Copyright: © Alhammer et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].
  • Article Number: 86
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    Published online on: August 21, 2026
       https://doi.org/10.3892/wasj.2026.501
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Abstract

Triple‑negative breast cancer (TNBC) is an aggressive subtype of cancer with limited treatment options. Despite the use of both doxorubicin (Doxo) and 5‑fluorouracil (5‑FU) as part of the FAC regimen, their interaction in TNBC has not yet been studied in detail, at least to the best of our knowledge. As reducing toxicity while preserving therapeutic efficacy remains a key therapeutic goal, the present in vitro study investigated whether the combination of Doxo and 5‑FU exerts synergistic effects on MDA‑MB231 cells and enables dose reduction. MTT assays were used to measure the viability of the TNBC cell line, MDA‑MB‑231 and non‑transformed rat embryo fibroblasts (REFs) following exposure to Doxo and 5‑FU, alone or in combination, for 72 h. Synergistic effects were evaluated using the combination index (CI) calculated using CalcuSyn software. Cell morphology was assessed by inverted microscopy, and apoptosis was assessed using acridine orange/ethidium bromide dual staining followed by fluorescence microscopy. The dose‑response data revealed anti‑proliferative effects in MDA‑MB‑231 cells (GI50=8.57 and 34.38 µM for Doxo and 5‑FU, respectively) and REF cells (GI50=1.3 and 48.66 µM, respectively). Combination treatment reduced the concentrations of Doxo and 5‑FU required to achieve the GI50 by 27.9 and 85.5%, respectively, compared with monotherapy. This dose‑sparing effect was accompanied by a modest increase in apoptosis and marked morphological alterations. To the best of our knowledge, the present study is the first to quantitatively characterize the synergistic interaction between Doxo and 5‑FU in TNBC cells, providing evidence of clinically relevant dose reduction beyond the historical empirical use of the FAC regimen.
View Figures

Figure 1

Cytotoxic effects of Doxo and 5-FU as
single agents on MDA-MB-231 cells and REFs. MTT assays were used to
assess cell viability after the (A and B) MDA-MB-231 cells, and (C
and D) REFs were exposed to increasing concentrations of (A and C)
Doxo or (B and D) 5-FU for 72 h. Data points are displayed as the
mean percentage survival ± SEM. (A and B) GI50 values
for MDA-MB-231 cells were determined from three independent
biological replicates; (C and D) GI50 values for REF
cells were determined from a single biological replicate with
technical triplicate measurements. Doxo, doxorubicin; 5-FU,
5-fluorouracil; REFs, rat embryo fibroblasts.

Figure 2

Synergistic cytotoxic effects of Doxo
and 5-FU in MDA-MB-231 cells. (A) Dose-response curves of
MDA-MB-231 cells incubated for 72 h with fractions of the
GI50 of 5-FU alone, Doxo alone, or a combination of
both, prior to the assessment of cell viability by MTT assay. (B)
Mean CI values computed using CalcuSyn software at the ED50, ED75
and ED90 (one-way ANOVA, P=0.0020; Tukey's multiple comparisons
test): n.s, not significant (ED50 vs. ED75); **P<0.01
(ED50 vs. ED90); *P<0.05 (ED75 vs. ED90). (C)
Isobologram illustrating the Doxo/5-FU drug interaction points at
different effective doses in relation to their respective
additivity lines. Points falling below the line indicate synergism,
whereas those approaching or above the line reflect additive or
antagonistic effects. (D) Fold reduction in the doses of Doxo and
5-FU required to achieve the GI50 with combination
therapy compared with monotherapy. *P<0.05 and
****P<0.0001 vs. a theoretical value of 1 (one-sample
t-test performed separately for Doxo and 5-FU). Data points on the
(A) survival curves and (B and D) bars were generated based on the
average of two biological replicates with triplicate measurements,
expressed as the mean ± SEM. Doxo, doxorubicin; 5-FU,
5-fluorouracil; conc, concentration.

Figure 3

Effects of the combination of 5-FU
and Doxo on the morphology and induction of apoptosis in MDA-MB-231
cells. MDA-MB231 cells were treated with control (untreated), 5-FU
(10 µM), Doxo (10 µM), or both drugs for 72 h prior to the
examination of (A) cell morphology using an inverted microscope at
x200 magnification (scale bar, 50 µm), and (B and C) apoptosis
using acridine orange/ethidium bromide staining and fluorescence
microscopy. (B) Representative images of MDA-MB-231 cells at x400
magnification obtained via fluorescence microscopy (scale bar, 25
µm). (C) Bar chart indicates the percentage of cells in various
apoptotic phases per treatment. V, viable (green color); EA, early
apoptotic (yellow color); and LA, late apoptotic (orange/red
color). Doxo, doxorubicin; 5-FU, 5-fluorouracil.
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Copy and paste a formatted citation
Spandidos Publications style
Alhammer AH, Lafta FM and Mudhafar SA: Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells. World Acad Sci J 8: 86, 2026.
APA
Alhammer, A.H., Lafta, F.M., & Mudhafar, S.A. (2026). Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells. World Academy of Sciences Journal, 8, 86. https://doi.org/10.3892/wasj.2026.501
MLA
Alhammer, A. H., Lafta, F. M., Mudhafar, S. A."Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells". World Academy of Sciences Journal 8.5 (2026): 86.
Chicago
Alhammer, A. H., Lafta, F. M., Mudhafar, S. A."Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells". World Academy of Sciences Journal 8, no. 5 (2026): 86. https://doi.org/10.3892/wasj.2026.501
Copy and paste a formatted citation
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Spandidos Publications style
Alhammer AH, Lafta FM and Mudhafar SA: Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells. World Acad Sci J 8: 86, 2026.
APA
Alhammer, A.H., Lafta, F.M., & Mudhafar, S.A. (2026). Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells. World Academy of Sciences Journal, 8, 86. https://doi.org/10.3892/wasj.2026.501
MLA
Alhammer, A. H., Lafta, F. M., Mudhafar, S. A."Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells". World Academy of Sciences Journal 8.5 (2026): 86.
Chicago
Alhammer, A. H., Lafta, F. M., Mudhafar, S. A."Dose‑reducing synergistic effects of doxorubicin and 5‑fluorouracil on triple‑negative breast cancer cells". World Academy of Sciences Journal 8, no. 5 (2026): 86. https://doi.org/10.3892/wasj.2026.501
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