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Article Open Access

Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity

  • Authors:
    • Elvira Mukinovic
    • Sina Federmann
    • Larissa Messling
    • Marlena Sekeres
    • Julia Mann
    • Lena Abbey
    • Matthias U. Kassack
    • Gerhard Fritz
  • View Affiliations / Copyright

    Affiliations: Institute of Toxicology, Medical Faculty and University Hospital, Heinrich Heine University Duesseldorf, D‑40225 Duesseldorf, Germany, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Duesseldorf, D‑40225 Duesseldorf, Germany
    Copyright: © Mukinovic et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 48
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    Published online on: February 24, 2026
       https://doi.org/10.3892/ijo.2026.5861
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Abstract

The efficacy of anticancer therapeutics is limited by acquired drug resistance of tumor cells. The present study aimed to characterize and overcome resistance mechanisms to the anthracycline derivative doxorubicin (Doxo). To this end, comparative analyses of Doxo‑induced stress responses of parental A2780 ovarian carcinoma cells and Doxo‑resistant A2780ADR variants were performed. A2780ADR cells revealed cross‑resistance to multiple compounds, including anticancer drugs [cisplatin (CisPt) and etoposide (Eto)] and DNA repair/DNA damage response (DDR) inhibitors (olaparib, niraparib, entinostat, prexasertib and rabusertib). A2780ADR cells formed markedly fewer DNA double‑strand breaks (DSB) following Doxo exposure compared with parental A2780 cells, resulting in a mitigated DDR, reduced proliferation inhibition and attenuated apoptosis. Potential resistance mechanisms identified to contribute to Doxo resistance of A2780ADR cells include increased Doxo efflux due to increased multi‑drug resistance gene 1 (MDR1) expression and reduced topoisomerase IIα protein expression. Substantial resensitization of A2780ADR cells to Doxo was achieved by both the RAC1 GTPase inhibitor EHT1864, the histone deacetylase inhibitor entinostat (EST) and, most effectively, the calcium channel blocker verapamil (Ver). Notably, Ver‑mediated sensitization also pertains to Eto and CisPt. The synergistic effect of Ver in combination with Doxo, which is reflected by low combination index (CI<0.8), probably involves inhibition of MDR1‑mediated drug export, increased intracellular steady state levels of Doxo and elevated DSB formation, eventually promoting pro‑toxic mechanisms of the DDR. However, combination treatment with Doxo and Ver also increased the cytotoxic response of non‑malignant murine cardiomyocytes, murine embryonic stem cells and human induced pluripotent stem cells. Taken together, the present study suggested inhibition of MDR1‑mediated Doxo efflux by Ver a useful approach to overcome acquired drug resistance of A2780ADR cells by stimulating DDR‑related cytotoxicity, yet at the price of a potentially increased risk of normal tissue toxicity.

View Figures

Figure 1

Comparative analysis of the response
of A2780 and A2780ADR cells to treatment with anticancer drugs
(Doxo, Eto and CisPt) and effect of mechanisms of drug transport.
Logarithmically growing parental A2780 and A2780ADR variant cells
were treated with the anticancer drugs (A) Doxo, (B) Eto and (C)
CisPt at the indicated concentrations. At 72 h after drug addition,
viability was monitored by use of the AlamarBlue assay as described
in methods. Data shown are the mean ± SD from three independent
experiments each performed in biological quadruplicates (n=3; n=4).
Dashed lines indicate inhibitory concentrations (IC20
and IC50). For viability data (IC50) after 24
and 72 h of treatment also see Fig.
S1 and Table SII. (D)
Comparative analysis of the mRNA expression of selected
transporters in A2780 and A2780ADR. Data shown are the mean ± SD
from triplicate determinations. mRNA expression of transporters was
normalized to GAPDH mRNA levels and set to 1.0 in the parental
A2780 cells. The dashed lines indicate changes in mRNA levels of
≥2.0 and ≤0.5, which are considered as biologically relevant. (E)
Comparative analysis of the protein expression of representative
drug transporters under basal situation and after 24 h treatment
with Doxo (0.1 and 1.0 μM). Data shown are from a
representative western blotting using ERK2 protein levels as
loading control. Data obtained after 72 h are presented in Fig. S2 (left panel). (F) Intracellular
Doxo fluorescence was measured by flow cytometry-based methods
after 2 h Doxo pulse-treatment (0.25 and 1.0 μM) and was
taken as indicative of drug import. To measure drug export, Doxo
pulse-treated cells were post-incubated for 6 h in the absence of
the drug before fluorescence was monitored. Data shown in the left
panel are representative results obtained from flow cytometry
analyses. C, control; I, import, E, export. The histogram in the
right panel depicts quantitative data obtained from n=3 independent
experiments each performed in biological triplicates (n=3).
***P≤0.001. (G) Analysis of basal mRNA expression of
topoisomerase II isoforms TOP2A, TOP2B and TopBP1. Data shown are
the mean ± SD from triplicate determinations. Relative mRNA level
in A2780 cells was set to 1.0. The dashed lines indicate changes in
mRNA levels of ≥2.0 and ≤0.5, which are considered as biologically
relevant. (H) Comparative analysis of the protein expression of
TOP2A and TopBP1 under basal situation and after 24 h treatment
with Doxo (0.1 μM, 1.0 μM). Data shown are from a
representative western blotting using ERK2 as protein loading
control. Data obtained after 72 h Dox treatment are presented in
Fig. S2 (right panel). Doxo,
doxorubicin; Eto, etoposide; CisPt, cisplatin; SD, standard
deviation; Nd, not detectable TOP2A, topoisomerase IIα; TOP2B,
topoisomerase IIβ; TOPBP1, topoisomerase binding protein 1; MDR1,
multi-drug resistance gene 1; ATP7A, copper transporting ATPase;
OCT2, organic cation transporter-2; CTR1, copper uptake protein 1;
Topo IIa, topoisomerase IIα; ERK2, extracellular regulated kinase
2.

Figure 2

Cell cycle progression and
proliferation following treatment of A2780 and A2780ADR cells with
Topo II inhibitors. Logarithmically growing cells were treated with
the indicated concentrations of Doxo or etoposide Eto for (A) 24 or
(B) 72 h. Afterwards, cell cycle distribution was analyzed by flow
cytometry and the percentage of cells present in different phases
of the cell cycle (SubG1-, G1-, S- and
G2/M-phase) was quantified. Data shown in the histogram
(left panel) are the mean ± SD from n=3 independent experiments
each performed in biological triplicates. The table on the right
panel summarizes the mean values and indicates statistical
differences between the individual groups. *P≤0.05;
**P≤0.01 (A2780 vs. A2780ADR); #P≤0.05;
##P≤0.01 (Con vs. treated group). (C) Logarithmically
growing parental A2780 and Doxo resistant A2780ADR cells were
treated with the indicated concentrations of Doxo (0.1 and 1.0
μM) or Eto (1.0 and 10 μM). At 24 h later the
percentage of Ki-67 positive or pH3 positive cells was determined
as described in methods. Total magnification, ×400. Quantitative
data shown in the histogram are the mean ± SD from n=3 independent
experiments, each performed with n=5 biological replicates.
*P≤0.05; **P≤0.01; ****P≤0.0001
(A2780 vs. A2780ADR). Con vs. treatment: #P≤0.05;
##P≤0.01; ###P≤0.001. (D) Logarithmically
growing parental A2780 and Doxo resistant A2780ADR cells were
treated with the indicated concentrations of Doxo (0.1 and 1.0
μM) or Eto (1.0 and 10 μM). At 24 h later cells were
pulse-labeled with EdU for 2 h as described in Methods and the
percentage of EdU positive cells was determined microscopically
(total magnification, ×400). Quantitative data shown in the
histogram are the mean ± SD from five biological replicates. A2780
as compared with A2780ADR: **P≤0.05;
****P≤0.0001. Con vs. treatment: ##P≤0.01;
###P≤0.001. Doxo, doxorubicin; Eto, etoposide; SD,
standard deviation.

Figure 3

Effect of Topo II inhibitors on DNA
damage formation and activation of DDR-related mechanisms in A2780
and A2780ADR cells. (A) At 24 h after treatment of logarithmically
growing cells with the indicated concentrations of Doxo or Eto, the
number of nuclear γH2AX-foci, 53BP1-foci, γH2AX/53BP1 co-localized
foci and γH2AX pan-stained cells was analyzed. The upper part of
the figure shows representative images (total magnification,
×1,000). Quantitative data depicted in the histogram are the mean ±
SD from n=3 independent experiments with each five images being
analyzed per experimental condition. *P≤0.05;
**P≤0.01; ***P≤0.001 (A2780 vs. A2780ADR);
#P≤0.05; ##P≤0.01; ###P≤0.001
(treated vs. untreated control). Control experiments performed by
use of 1st or 2nd antibody only or no antibody at all did not
interfere with the signal of main interest (that is, nuclear foci;
data not shown). (B) Logarithmically growing cells were treated
with the indicated concentrations of Doxo for 24 or 72 h.
Afterwards, the protein expression of DDR-related factors was
analyzed by western blotting using EKR2 protein expression as
loading control. Doxo, doxorubicin; Eto, etoposide; p-,
phosphorylated; Chk1/2, checkpoint kinase 1/2; ERK2, extracellular
regulated kinase; γH2AX, Ser139 phosphorylated histone H2AX; 2;
Kap1, KRAB-associated protein 1; PARP, poly (ADP-ribose)
polymerase; p21, cyclin-dependent kinase inhibitor 1; p53, tumor
suppressor p53.

Figure 4

Analysis of cross-sensitivity of
parental A2780 and Doxo-resistant A2780ADR cells to selected
inhibitors of DDR- and DNA repair-related mechanisms.
Logarithmically growing parental A2780 and A2780ADR variant cells
were treated with selected pharmacological inhibitors of DNA repair
(olaparib and niraparib), DDR (prexasertib and rabusertib), HDAC
(ricolinistat and entinostat), Rac1 GTPase (EHT1864 and Ehop16),
drug transport (verapamil) and Topo II (dexrazoxane) at the
indicated concentrations. At 72 h after drug addition, viability
was monitored by use of the AlamarBlue assay as described in
methods. Data shown are the mean ± SD from three independent
experiments each performed in biological quadruplicates (n=3; n=4).
Dashed lines indicate inhibitory concentrations (IC20
and IC50). Data obtained from treatment period of 24 h
are presented in Fig. S4. For
IC50 after 24 h and 72 h see Table SII. Doxo, doxorubicin; DDR, DNA
damage response; HDAC, histone deacetylase; SD, standard deviation;
EHT, Rac1 inhibitor EHT1864.

Figure 5

Combined treatment of A2780ADR with
Doxo and selected inhibitors causes synergistic toxicity. (A)
Logarithmically growing Doxo resistant A2780ADR cells were
co-treated with Doxo and selected pharmacological inhibitors at the
indicated concentrations. At 72 h after drug addition, viability
was monitored by use of the AlamarBlue assay and CI was calculated
as described in methods. Data shown are the mean ± SD from three
independent experiments each performed in biological quadruplicates
(n=3; n=4). (B) Intracellular Doxo fluorescence was measured by
flow cytometry-based method after co-treatment with Doxo and
selected pharmacological inhibitors as described in methods. To
measure drug export, Doxo pulse-treated cells were post-incubated
for 6 h in the absence of the drug before fluorescence was
monitored. Data shown in the left panel are representative results
obtained from flow cytometry analyses. C, control; I, import, E,
export. The histogram in the right panel depicts quantitative data
obtained from n=3 independent experiments each performed in
biological triplicates (n=3). Statistical significance:
*P≤0.05; **P≤0.001. Doxo, doxorubicin; CI,
combination index; SD, standard deviation; EST, entinostat; EHT,
Rac1 inhibitor EHT1864; Dex, dexrazoxane; Rab, rabusertib; Ver,
verapamil.

Figure 6

Influence of combined treatment of
A2780ADR with Doxo and selected inhibitors on DNA damage formation,
proliferation and cell death. (A) At 24 h after treatment of
logarithmically growing cells with the indicated concentrations of
Doxo and inhibitors (EHT, 5 μM; EST, 1 μM; Ver, 50
μM), the number of nuclear γH2AX-foci, 53BP1-foci,
γH2AX/53BP1 co-localized foci and γH2AX pan-stained cells was
analyzed as described in methods. The upper part of the figure
shows representative images (total magnification, ×1,000).
Quantitative data depicted in the histogram are the mean ± SD from
n=5 microscopical images analyzed per experimental condition.
*P≤0.05, **P≤0.01, ***P≤0.001
mono-treatment vs. co-treatment; #P≤0.05,
##P≤0.01, ###P≤0.001 untreated vs. treated
group). (B) Logarithmically growing Doxo resistant A2780ADR cells
were co-treated with the indicated concentrations of Doxo and
selected pharmacological inhibitors (EHT, 5 μM; EST, 1
μM; Ver, 50 μM). At 24 h later, cells were
pulse-labeled with EdU to monitor proliferation as described in
methods and the percentage of EdU positive cells was determined
microscopically (total magnification, ×400). Quantitative data
shown in the histogram are the mean ± SD from five replicates.
**P≤0.05; ##P≤0.01; ###P≤0.001
(vs. untreated control). (C) PI staining of mono- and co-treated
A2780ADR cells 72 h after treatment with Doxo (0.1 μM) and
pharmacological inhibitors (EHT, 5 μM; EST, 1 μM;
Ver, 50 μM). Left panel: representative images; right panel:
percentage of PI positive cells (mean ± SD from n=5 microscopical
images analyzed per experimental condition) (40× microscope
objective). *P≤0.05; **P≤0.01. mono-treatment
vs. co-treatment; #P≤0.05; ##P≤0.01, Con vs
treated group. Doxo, doxorubicin; EST, entinostat; EHT, Rac1
inhibitor EHT1864; Ver, verapamil; SD, standard deviation; PI,
propidium iodide.

Figure 7

Influence of combined treatment of
A2780ADR cells with Doxo and selected inhibitors on mechanism of
the DDR and mRNA expression of selected susceptibility-related
genes. (A) Logarithmically growing A2780ADR cells were co-treated
with the indicated concentrations of Doxo and selected
pharmacological inhibitors (concentrations see Fig. 5) for 24 or 72 h. Afterwards, the
protein expression of DDR-related factors was analyzed by western
blotting. For loading control, blots were reprobed with ERK2
antibody. (B) Reverse transcription-quantitative PCR of the mRNA
expression of selected factors known to contribute to different
mechanisms of drug sensitivity. Data shown are mean ± SD from
triplicate determinations as described in methods. Relative mRNA
level in untreated A2780ADR cells was set to 1.0. Doxo,
doxorubicin; DDR, DNA damage response; p-, phosphorylated; nd, not
detectable; Bax, Bcl-2 associated protein X; Bcl-2, B-cell
lymphoma; BBC3, Bcl-2 binding component 2; BRCA1, 2, breast cancer
associated gene 1,2; Cl casp-7, cleaved caspase 7; Chk, checkpoint
kinase; CXCL8, chemokine ligand 8 (interleukin 8); p21, CDK
inhibitor 1; p16, CDK inhibitor 2; CDKN1A/2A, cyclin dependent
kinae inhibitor 1A/2A; CCNB1, Cyclin B1; b-Gal, beta-galactosidase;
FASL, FAS ligand; FASR, FAS receptor; GADD, growth arrest and DNA
damage inducible GPX1, glutathione peroxidase 1; GSTM1, glutathione
S-transferase 1; HMOX1, heme oxygenase 1; γH2AX, Ser139
phosphorylated histone H2AX; p53, tumor suppressor p53; PARP, poly
(ADP-ribose) polymerase; PCNA-proliferating cell nuclear antigen;
PGC1A, PPARG coactivator 1; PPARGC1A, peroxisome
proliferator-activated receptor gamma coactivator 1-alpha; RAD51,
radiation damage gene 51; RPAreplication protein A; SOD1,
superoxide dismutase 1; Ver, verapamil.

Figure 8

Effects of co-treatment of Doxo with
selected pharmacological inhibitors on non-malignant cells.
Logarithmically growing (A) non-malignant murine HL-1 cardiomyocyte
cells, (B) mESC and (C) hiPSC were treated with the indicated
concentrations of Doxo and selected pharmacological inhibitors for
72 h. Afterwards, cell viability was analyzed by the use of the
AlamarBlue assay as described in methods. Data shown are the mean ±
SD from n=1-3 independent experiments each performed in biological
quadruplicates. *P≤0.05; **P≤0.001 (mono- vs.
co-treated group). Dashed lines indicate 50% viability. Doxo,
doxorubicin; mESC, murine embryonic stem cells; hiPSC, human
induced pluripotent stem cells; EHT, Rac1 inhibitor EHT1864; EST,
entinostat; Ver, verapamil.

Figure 9

Hypothetical model of Ver-mediated
resensitization of Doxo-resistant tumor cells. It was hypothesized
that Ver increased the anticancer efficacy of Doxo in a synergistic
manner in anticancer drug resistant ovarian A2780ADR cells. This is
probably due to inhibition of MDR1-mediated drug export, leading to
higher intracellular steady-state concentrations of Doxo. In
consequence, Doxo-mediated Topo II poisoning is promoted,
eventually causing increased DNA damage (that is, DSB) formation
and activation of DSB-related pro-toxic signaling mechanism which
impair cell proliferation and stimulate cell death- and
senescence-related pathways. Apart from verapamil, inhibition of
Rac1 GTPase-regulated signaling by EHT1864 and inhibition of HDAC
class I by entinostat are also useful to overcome acquired Doxo
resistance of A2780ADR cells, yet with the exact molecular
mechanisms involved being unclear. Ver, verapamil; Doxo,
doxorubicin; DSB, DNA double-strand breaks.
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Copy and paste a formatted citation
Spandidos Publications style
Mukinovic E, Federmann S, Messling L, Sekeres M, Mann J, Abbey L, Kassack MU and Fritz G: <p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>. Int J Oncol 68: 48, 2026.
APA
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L. ... Fritz, G. (2026). <p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>. International Journal of Oncology, 68, 48. https://doi.org/10.3892/ijo.2026.5861
MLA
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L., Kassack, M. U., Fritz, G."<p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>". International Journal of Oncology 68.4 (2026): 48.
Chicago
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L., Kassack, M. U., Fritz, G."<p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>". International Journal of Oncology 68, no. 4 (2026): 48. https://doi.org/10.3892/ijo.2026.5861
Copy and paste a formatted citation
x
Spandidos Publications style
Mukinovic E, Federmann S, Messling L, Sekeres M, Mann J, Abbey L, Kassack MU and Fritz G: <p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>. Int J Oncol 68: 48, 2026.
APA
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L. ... Fritz, G. (2026). <p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>. International Journal of Oncology, 68, 48. https://doi.org/10.3892/ijo.2026.5861
MLA
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L., Kassack, M. U., Fritz, G."<p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>". International Journal of Oncology 68.4 (2026): 48.
Chicago
Mukinovic, E., Federmann, S., Messling, L., Sekeres, M., Mann, J., Abbey, L., Kassack, M. U., Fritz, G."<p>Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil‑mediated promotion of DNA damage‑driven cytotoxicity</p>". International Journal of Oncology 68, no. 4 (2026): 48. https://doi.org/10.3892/ijo.2026.5861
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