Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: 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 (IC 20 and IC 50 ). Data obtained from treatment period of 24 h are presented in . For IC 50 after 24 h and 72 h see . Doxo, doxorubicin; DDR, DNA damage response; HDAC, histone deacetylase; SD, standard deviation; EHT, Rac1 inhibitor EHT1864.

Article Snippet: Chemicals were obtained from the following providers: Entinostat (MS-275) was obtained from Selleck Chemicals, Doxo from STADA Consumer Health & STADAPHARM GmbH, etoposide, Ehop16, prexasertib (AZD-7762) and dexrazoxane were from MilliporeSigma, cisplatin from Accord Healthcare GmbH, olaparib from APeXBIO Technology LLC, niraparib from MedChemExpress, EHT1864 was purchased from Tocris Bioscience, rabusertib (LY2603618) and ricolinostat (ACY-1215) from MedChemExpress and verapamil (Ver) from Thermo Fisher Scientific, Inc.

Techniques: Variant Assay, Drug Transport Assay, Alamar Blue Assay, Histone Deacetylase Assay, Standard Deviation

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: 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.

Article Snippet: Chemicals were obtained from the following providers: Entinostat (MS-275) was obtained from Selleck Chemicals, Doxo from STADA Consumer Health & STADAPHARM GmbH, etoposide, Ehop16, prexasertib (AZD-7762) and dexrazoxane were from MilliporeSigma, cisplatin from Accord Healthcare GmbH, olaparib from APeXBIO Technology LLC, niraparib from MedChemExpress, EHT1864 was purchased from Tocris Bioscience, rabusertib (LY2603618) and ricolinostat (ACY-1215) from MedChemExpress and verapamil (Ver) from Thermo Fisher Scientific, Inc.

Techniques: Alamar Blue Assay, Fluorescence, Flow Cytometry, Incubation, Control, Standard Deviation

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: 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 μ M; EST, 1 μ M; Ver, 50 μ M), 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 μ M; EST, 1 μ M; Ver, 50 μ M). 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 μ M) and pharmacological inhibitors (EHT, 5 μ M; EST, 1 μ M; Ver, 50 μ M). 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.

Article Snippet: Chemicals were obtained from the following providers: Entinostat (MS-275) was obtained from Selleck Chemicals, Doxo from STADA Consumer Health & STADAPHARM GmbH, etoposide, Ehop16, prexasertib (AZD-7762) and dexrazoxane were from MilliporeSigma, cisplatin from Accord Healthcare GmbH, olaparib from APeXBIO Technology LLC, niraparib from MedChemExpress, EHT1864 was purchased from Tocris Bioscience, rabusertib (LY2603618) and ricolinostat (ACY-1215) from MedChemExpress and verapamil (Ver) from Thermo Fisher Scientific, Inc.

Techniques: Staining, Labeling, Control, Microscopy, Standard Deviation

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: 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.

Article Snippet: Chemicals were obtained from the following providers: Entinostat (MS-275) was obtained from Selleck Chemicals, Doxo from STADA Consumer Health & STADAPHARM GmbH, etoposide, Ehop16, prexasertib (AZD-7762) and dexrazoxane were from MilliporeSigma, cisplatin from Accord Healthcare GmbH, olaparib from APeXBIO Technology LLC, niraparib from MedChemExpress, EHT1864 was purchased from Tocris Bioscience, rabusertib (LY2603618) and ricolinostat (ACY-1215) from MedChemExpress and verapamil (Ver) from Thermo Fisher Scientific, Inc.

Techniques: Alamar Blue Assay

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: 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.

Article Snippet: Chemicals were obtained from the following providers: Entinostat (MS-275) was obtained from Selleck Chemicals, Doxo from STADA Consumer Health & STADAPHARM GmbH, etoposide, Ehop16, prexasertib (AZD-7762) and dexrazoxane were from MilliporeSigma, cisplatin from Accord Healthcare GmbH, olaparib from APeXBIO Technology LLC, niraparib from MedChemExpress, EHT1864 was purchased from Tocris Bioscience, rabusertib (LY2603618) and ricolinostat (ACY-1215) from MedChemExpress and verapamil (Ver) from Thermo Fisher Scientific, Inc.

Techniques: Inhibition, Activation Assay