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. 2023 Sep 28;18(9):e0291981.
doi: 10.1371/journal.pone.0291981. eCollection 2023.

Synergistic combination of doxorubicin with hydralazine, and disulfiram against MCF-7 breast cancer cell line

Zainab Lafi  1 , Walhan Alshaer  2 , Lobna Gharaibeh  1 , Dana A Alqudah  2 , Baidaa AlQuaissi  2 , Banan Bashaireh  2 , Abed Alqader Ibrahim  3
Affiliations

Synergistic combination of doxorubicin with hydralazine, and disulfiram against MCF-7 breast cancer cell line

Zainab Lafi et al. PLoS One. .

Abstract

Disulfiram and hydralazine have recently been reported to have anti-cancer action, and repositioned to be used as adjuvant in cancer therapy. Chemotherapy combined with other medications, such as those that affect the immune system or epigenetic cell profile, can overcome resistance with fewer adverse effects compared to chemotherapy alone. In the present study, a combination of doxorubicin (DOX) with hydrazine (Hyd) and disulfiram (Dis), as a triple treatment, was evaluated against wild-type and DOX-resistant MCF-7 breast cancer cell line. Both wild-type MCF-7 cell line (MCF-7_WT) and DOX-resistant MCF-7 cell line (MCF-7_DoxR) were treated with different combination ratios of DOX, Dis, and Hyd followed by measuring the cell viability using the MTT assay. Synergism was determined using a combination index, isobologram analysis, and dose-reducing index. The anti-proliferation activity and mechanism of the triple combination were investigated by apoptosis analysis. The results showed a reduction in the IC50 values of DOX in MCF-7_WT cells (from 0.24 μM to 0.012 μM) and MCF-7_DoxR cells (from 1.13 μM to 0.44 μM) when treated with Dis (0.03μM), and Hyd (20μM) combination. Moreover, The triple combination DOX/Hyd/Dis induced significant apoptosis in both MCF-7_WT and MCF-7_DoxR cells compared to DOX alone. The triple combination of DOX, Dis, and Hyd showed a synergistic drugs combination to decrease the DOX dose needed to kill both MCF-7_WT and MCF-7_DoxR cancer cells and enhanced chemosensitivity to DOX.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
The chemical structures of disulfiram (A), hydralazine (B), and doxorubicin (C).
Fig 2
Fig 2. STRING analysis of protein–protein interactions.
(A) Interaction between proteins involved in drug resistance STAT3, NOTCH, DNMT, (B) Illustration of combination of HDACi and DNMTi pathway, (C) combination of HDACi and DNMTi and the effect of cell apoptosis proteins, (D) interactions of DNMTi and proteins ivoved in cell cycle, (E) Effect of ALDH enzymes on P53 proteins, (F) Interactions between ALDH and glutathione (GSH), (G) Interactions between ALDH, ABCC1 and p53 proteins, (H) Signalling pathway showed interaction of DNMT, ALDH, P53, TOP2A, ABCB1. STAT3, NOCH1. (thickness of edges indicate confidence).
Fig 3
Fig 3. Diagnostic graphics produced for synergistic affect quantification.
(A) Isobologram for Dis/Hyd combination at different concentration points, (B) The Fa-DRI plot (Chou-Martin plot) for the non-constant ratios of Dis/Hyd combination, (C) The fraction affected (Fa) versus combination index (CI) plot after treatment with Dis/Hyd combination, that most of CI values are <1 in the range 0.4–0.95, (D) Table of IC50 of Dis, Hyd and Dis/Hyd combination.
Fig 4
Fig 4
A) Induced apoptosis of MCF-7_WT cells treated with Dis and Hyd alone and combined B) Induced apoptosis of MCF-7_DoxR cells treated with Dis and Hyd alone and combined.
Fig 5
Fig 5. Diagnostic graphics produced for synergistic effect quantification of DOX/Dis/Hyd combination against MCF-7_WT cells.
(A) Dose -Fa curve for DOX alone and for DOX with (DOX/Dis/Hyd combination) at different concentration points (B) Table of IC50 of DOX, Dis, Hyd alone and combination (C) The fraction affected (Fa) versus combination index (CI) plot after treatment with DOX/Dis/Hyd combination, that most of CI values are <1 for the range of 0.5–0.95(D) The Fa-DRI plot for the non-constant ratios of DOX/Dis/Hyd combination.
Fig 6
Fig 6. Diagnostic graphics produced for synergistic effect quantification of DOX/Dis/Hyd combination against MCF-7_DoxR cells.
(A) Dose -Fa curve for DOX alone and for DOX with (DOX/Dis/Hyd combination) at different concentration points (B) Table of IC50 of DOX, Dis, Hyd alone and combination (C) The fraction affected (Fa) versus combination index (CI) plot after treatment with DOX/Dis/Hyd combination, that most of CI values are < 1 for the range of 0.25–0.8 (D) The Fa-DRI plot for the non-constant ratios of DOX/Dis/Hyd combination.
Fig 7
Fig 7
IC50 values after treatment with DOX/Dis/Hid. The MCF-7_WT and MCF-7_DoxR cells were treated with DOX, Dis, Hyd and DOX/Dis/Hyd to assess the cytotoxicity levels (A) The dose-response curve for cells treated with Hyd and; (B) The dose-response curve for cells treated with Dis; (C)The dose-response curve for cells treated with DOX; (D) The dose-response curve for cells treated with DOX/Dis/Hyd in combination, the curve showing DOX IC50 with and without combination with Dis/Hyd (0.03/20 μM). All cytotoxicity values represent the average ± SD of three independent experiments.
Fig 8
Fig 8. Apoptosis Induced by DOX/Dis/Hyd single treatment and combinations.
A) and B) MCF-7_WT cells, C) and D) MCF-7_DoxR cells.
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