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. The following primary antibodies were used: Copper transporting ATPase (ATP7A), extracellular regulated kinase 2 (ERK2), phosphorylated (p)-histone H3 (Ser10) from Thermo Fisher Scientific Inc., cleaved caspase-7 (Asp198), p-Chk1 (Ser 345), cyclin B1, galactosidase β (E2U2I), GAPDH (14C10), MDR1/ABCB1 (D3H1Q), p-P53 (S15), PARP, TopBP1(D8G4L), topoisomerase IIa (D10G9), 53BP1 and Ki67 were from Cell Signaling Technology Inc., pChk2 (T68) [Y171], copper uptake protein 1 (CTR1/SLC31A1) [EPR7936] and Rad51 from Abcam, γH2AX (Ser 139) clone JBW301, p-KAP-1 (S824) and p-RPA32 (S4/S8) from Bethyl Laboratories Inc., organic cation transporter-2 (OCT2) from Biozol Diagnostics Vertrieb GmbH, p16 (F-12) and p21 (C-19) from Santa Cruz Biotechnology, Inc. As secondary antibodies, horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and mouse anti-rabbit IgG were used (Rockland Immunochemicals Inc.).

Parental A2780 ovarian carcinoma cells (A2780) as well as a doxorubicin resistant variant (A2780ADR) were from the European Collection of Authenticated Cell Cultures and were cultured in RPMI-1640 medium (MilliporeSigma) containing 10% fetal calf serum, 1% glutamine and 1% Pen/Strep at 37°C in a humidified atmosphere containing 5% CO₂. Cells were authenticated by STR profiling during the last three years. All experiments were performed with mycoplasma free cells. Immortalized HL-1 cardiomyocytes were provided by Professor W.C. Claycomb (Louisiana State University, New Orleans, LA, USA) (34) and were grown on gelatin (2 mg/ml)/fibronectin (1 mg/ml) (Sigma Aldrich; Merck KGaA) coated dishes and maintained in Claycomb medium, supplemented with 10% FBS and 100 μM norepinephrine (Sigma Aldrich; Merck KGaA). Mouse embryonic stem cells (ESC; LF2) were isolated from the mouse strain 129J (35) and were from Professor A. Smith (University of Oxford, UK). They were cultivated under feeder-free conditions on 0,1% gelatine-coating using knock-out Dulbecco's Modified Eagle's Medium (KO-DMEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with knock-out serum replacement (15%) (Gibco; Thermo Fisher Scientific, Inc.), penicillin/streptomycin (1%), glutamax (1%), β-mercaptoethanol (5×10⁻⁵ M) (Invitrogen; Thermo Fisher Scientific, Inc.) and leukemia inhibitory factor (LIF; MilliporeSigma) (1,000 U/ml) at 37°C in an atmosphere containing 5% CO₂. b4-hiPSC were generated from human foreskin fibroblasts as previously described (36). Cells were cultured on plates coated with reduced growth factor basement membrane matrix (Gibco; Thermo Fisher Scientific, Inc.) in StemMacs medium (Miltenyi Biotec GmbH) supplemented with 10 mM Y-27632 dihydrochloride (MilliporeSigma).

Cell viability was determined using the Alamar blue assay (37). Viable cells are characterized by an effective mitochondrial metabolization of the non-fluorescent dye resazurin (Sigma-Aldrich; Merck KGaA) to fluorescent resorufin (excitation: 535 nm, emission: 590 nm). Relative viability in the untreated control was set to 100%. If not stated otherwise, data are shown as the mean ± standard deviation (SD) of ≥3 independent experiments, each performed in biological quadruplicates. The combination index (CI) was determined (38) for the calculation of additive (CI >0.8<1.2), synergistic (CI≤0.8) or antagonistic (CI≥1.2) drug interactions in the co-treatment experiments.

Doxo import and export were measured by exploiting the inherent red fluorescence of Doxo as previously described (39). Briefly, following 2 h of pulse-treatment with different Doxo concentrations, the fluorescence of the cells was measured as a surrogate marker of drug uptake by flow cytometry (excitation: 450 nm, emission: 560 nm). After a post-incubation period of up to 6 h in the absence of Doxo, the residual intracellular fluorescence was again measured by flow cytometry. The time dependent decrease in Doxo fluorescence was calculated as a surrogate marker of Doxo export.

For flow cytometry-based analysis of cell cycle distribution, cells were trypsinized and combined with floating cells present in the medium. Cells were pelleted (1,000 × g, 10 min, 4°C) and suspended in PBS. DNase-free RNase A (SERVA Electrophoresis GmbH) was added (2 μg/ml, 1 h at room temperature). DNA was stained with propidium iodide (PI) (Sigma-Aldrich; Merck KGaA) for 20 min in the dark. Cell number was adjusted to 10⁶ cells/ml with PBS and analysis was performed using BD Accuri C6 flow cytometer (BD Biosciences).

Cell death by apoptosis was monitored by flow cytometry-based quantitation of the subG₁ fraction, which represents the apoptotic cell fraction. In addition, cleavage of PARP protein and pro-caspase-7 was monitored by western blotting. To monitor senescence β-GAL expression was analyzed by western blotting.

To monitor proliferation, the incorporation of the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) into S-phase cells as well as the percentage of pH3 (Ser10) positive cells (mitotic index) and Ki-67 positive cells were determined. To this end, cells were seeded on cover slips and cultivated for the indicated time period. EdU-incorporation was analyzed using the EdU-Click 488 Kit (baseclick GmbH), which is based on a pulse-labeling of S-phase cells with 10 μM EdU according to the manufacturer's protocol. To determine the mitotic index, cells were fixed with 4% formaldehyde/PBS followed by incubation PBS −0.3% TritonX-100 (5 min; room temperature). After blockage of unspecific binding (5% BSA in 0.3% Triton X-100/PBS (1 h; room temperature), anti-Ser10 phosphorylated histone H3 antibody (pH3; Thermo Fisher Scientific Inc.; dilution 1:1,000; 16 h; 4°C) and Ki-67 antibody (Cell Signaling Technology Inc.; dilution 1:500; 16 h; 4°C) were added. Incubation with Alexa Fluor^® 488 labeled goat anti-rabbit and Alexa Fluor 555 labeled goat anti-mouse secondary antibody was performed for 120 min at room temperature. pH3- and Ki-67-positive cells were counterstained with DAPI-containing Vectashield (Vector Laboratories, Inc.) and analyzed by Olympus BX43 microscope (40× objective) (Olympus Corporation).

The frequency of nuclear foci formed by S139 phosphorylated H2AX (γH2AX foci) is a commonly used surrogate marker of DSBs (40,41) and was assayed by immunocytochemistry-based method. The appearance of nuclear 53BP1 foci, which is another marker of DSBs (42,43), was also determined by immunocytochemistry. Upon treatment of cells grown on cover slips, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS; Merck KGaA; 15 min; room temperature) followed by permeabilization by incubation in PBS −0.3% TritonX-100 (5 min; room temperature). After blocking [1 h; room temperature; blocking solution: 5% BSA (Merck KGaA) in PBS/0.3% Triton X-100 (Sigma-Aldrich; Merck KGaA)], incubation with primary γH2AX antibody (1:2,000; cat. no. 07-727; MilliporeSigma) and 53BP1 antibody (1:500; cat. no. 4937S; Invitrogen; Thermo Fisher Scientific, Inc.) was performed overnight (4°C), followed by incubation with the secondary fluorescence-labelled antibody [Alexa Fluor 488 goat-anti-rabbit IgG (H+L); cat. no. A11008; Invitrogen; Thermo Fisher Scientific, Inc.] (1:500, 2 h; room temperature, in the dark). Cells were mounted in Vectashield Mounting medium (anti-fading; Vector Laboratories, Inc.) containing the blue fluorescent DNA stain 4',6-Diamidin-2-phenylindol (DAPI; cat. no. H-1200; Biozol Diagnostics Vertrieb GmbH) at room temperature and the number of nuclear γH2AX foci and 53BP1 foci was scored using an Olympus BX43 fluorescence microscope (Olympus Corporation). Only nuclei with distinct foci were evaluated. γH2AX foci pan-stained nuclei, which are indicative of apoptotic cells, were excluded from the analyses. If not stated otherwise, data are shown as the mean ± SD from three independent experiments with each ≥50 nuclei analyzed per experimental condition.

The activation of the DDR was investigated by western blotting using total cell extracts obtained by lysing an equal number of cells in 150 μl RIPA buffer (20 min on ice). RCDC-Protein Assay (cat. no. 500-0120; Bio-Rad Laboratories, Inc.) was used for protein determination. After sonication (6 KHz; 5×2 sec; on ice) (EpiShear Probe sonicator; Active Motif, Inc.) and centrifugation (10,000 × g; 4°C; 10 min), Roti^®-Load buffer (5 min; room temperature) was added to the supernatant and proteins were denatured by heating (5 min, 95°C). Afterwards, 20 μg protein was loaded per lane and proteins were separated by SDS-PAGE (6 or 12.5% gel) and transferred onto a nitrocellulose membrane (Cytiva) via the Protean Mini Cell System. After blocking [5% non-fat milk in TBS/0.1% Tween 20 (Merck KGaA; 2 h; room temperature)], the membrane was incubated with the corresponding primary antibody (1:1,000; overnight; 4°C). The following primary antibodies were used: ATP7A (cat. no. PA5-103110; Invitrogen; Thermo Fisher Scientific; Inc.), Caspase-7 cleaved (Asp198; cat. no. 9491S; Cell Signaling Technology, Inc.), Chk1phospho (Ser345; cat. no. 2341, Cell Signaling Technology, Inc.), Chk2phosphoT68 (Y171; cat. no. ab32148; Abcam), CTR1/SLC31A1 (EPR7936; cat. no. ab129067; Abcam), Cyclin B1 (cat. no. 4138, Cell Signaling Technology, Inc.), ERK2 [PA5-32396, Invitrogen/Thermo Fisher Scientific; Carlsbad, USA], Galactosidase beta (E2U2I) (cat. no. 27198, Cell Signaling Technology, Inc.), GAPDH (14C10; cat. no. 2118S; Cell Signaling Technology, Inc.), H2AX phospho (Ser139, cloneJBW301; cat. no. 05-636, Merck KGaA), MDR1/ABCB1 (D3H1Q; cat. no. 12683; Cell Signaling Technology, Inc.), OCT2 (cat. no. MBS9600162, Biozol Diagnostics Vertrieb GmbH), P16 (F-12; cat. no. sc-1661; Santa Cruz Biotechnology, Inc.), p21 (C-19; cat. no. sc-397; Santa Cruz Biotechnology, Inc.), P53 phospho (S15; cat. no. 9284S; Cell Signaling Technology, Inc.), PARP (cat. no. 9542S, Cell Signaling Technology, Inc.), Rad51 (cat. no. ab63801; Abcam), RPA32 phospho (S4/S8; cat. no. ICH-00422; Bethyl Laboratories Inc.), TopBP1 (D8G4L; cat. no. 14342, Cell Signaling Technology, Inc.), Topoisomerase II alpha (D10G9; cat. no. 12286, Cell Signaling Technology, Inc.). After washing (TBS/0.1% Tween 20), the secondary (peroxidase-conjugated) antibody was added (1:2,000; 2 h; room temperature) [Anti-mouse IgG (H&L; goat) Antibody Peroxidase Conjugated (cat. no. 610-1302, Rockland Immunochemicals, Inc.) or Anti-rabbit IgG (H&L; goat) Antibody Peroxidase Conjugated (cat. no. 611-1302, Rockland Immunochemicals, Inc.] The ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.) was used for visualization of the bound antibodies. Image Lab 6.1.0 build 7 (Bio-Rad Laboratories, Inc.) was used for densitometrical analyses. Changes in protein expression of factors of interest were identified by normalization to the expression of a housekeeping protein (such as ERK2).

Total RNA was purified from up to 5×10⁶ cells per preparation using the RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by reverse transcriptase reaction with High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.). RNA extraction, cDNA synthesis and qPCR were performed according the manufacturer's protocols. For each PCR reaction 40 ng of cDNA and 0.25 μM of the corresponding primers (Eurofins MWG Synthesis GmbH) were used. Quantitative real-time PCR analysis was performed in triplicates using the SensiMix SYBR Hi-ROX Kit (Meridian Bioscience, Inc.) and a CFX96 Real-Time System (Bio-Rad Laboratories, Inc.) with the Bio-Rad CFX Manager 3.1 software. PCR runs (35-40 cycles) were performed as follows: 95°C, 10 min; 95°C, 15 sec; 60°C, 30 sec; 72°C, 40 sec; 72°C, 10 min. Melting curves were recorded to ensure the specificity of the amplification reaction. mRNA levels were normalized by geometric averaging using β-actin and GAPDH as internal control genes as reported (44). Unless stated otherwise, relative mRNA expression of untreated control cells was set to 1.0. Primer sequences used for RT-qPCR analyses are depicted in Table SI.

Student's t-test and the One-way ANOVA with Dunnett's post-hoc test were employed to confirm statistically significant differences between different experimental groups. P<0.05 was considered to indicate a statistically significant difference.

The present study employed parental ovarian cancer cells (A2780) and thereof derived Doxo resistant variants (A2780ADR). Comparative analysis of their viability following Doxo treatment for 72 h revealed IC₅₀ of 0.04 and 0.38 μM for A2780 and A2780ADR, respectively (Fig. 1A). A2780ADR cells revealed a profound cross-resistance to the Topo II inhibitor etoposide (Eto; IC₅₀ A2780: 0.14 μM; IC₅₀ A2780ADR: 0.92 μM; Fig. 1B), while being only weakly cross-resistant to the platinating agent cisplatin (CisPt; IC₅₀ A2780: 0.62 μM; IC₅₀ A2780ADR: 1.91 μM; Fig. 1C). Based on their corresponding IC₅₀, A2780ADR cells are characterized by a ~10, ~7 and ~3-fold higher resistance to Doxo, Eto and CisPt, respectively, as compared with the parental A2780 cells. Analyzing viability following a 24 h treatment period, no major differences were observed as concluded from the calculation of the corresponding IC₅₀ values (Fig. S1).

Altered drug transport, as mediated for instance by p-glycoprotein, is one possible mechanism contributing to acquired drug resistance of cancer cells (45,46). Analyzing the mRNA expression of various drug importers and exporters, an elevated mRNA expression of the importer CTR2 and a reduced mRNA expression of the exporter BCRP was observed in Doxo resistant A2780ADR cells as compared with the wild-type cells (Fig. 1D). Notably, while the mRNA expression of MDR1 was clearly detectable in A2780ADR cells, it was below detection limit in A2780 cells (Fig. 1D). Lack of MDR1 expression in the parental cells and high expression in A2780ADR was confirmed on the protein level (Figs. 1E and S2). To monitor the cells' activity of drug import and export, the present study took advantage of the inherent reddish fluorescence of Doxo and comparatively analyzed alterations in the intracellular fluorescence of A2780 and A2780ADR cells following Doxo treatment. The data show a similar increase in fluorescence in both cell lines after 2 h of Doxo pulse-treatment (Fig. 1F), indicating that both cell lines have comparable Doxo uptake capacity (i.e. import). However, analyzing the remaining intracellular Doxo concentration after a subsequent 6 h post-incubation period in the absence of Doxo, residual fluorescence was markedly lower (~50%) in A2780ADR as compared with parental A2780 cells (Fig. 1F). This finding showed that A2780ADR cells were characterized by an about twice as fast drug export as A2780 parental cells, which is very probably due to their elevated MDR1 expression as concluded from the results of the mRNA and protein expression analyses. Thus, increased drug export probably contributes to the high Doxo resistance of A2780ADR cells. However, having in mind the ~10-fold higher Doxo resistance of A2780ADR cells as compared with A2780 cells, it was hypothesized that mechanisms other than just drug export additionally contributed to their pronounced drug resistant phenotype. Since Topo IIα and IIβ are relevant primary targets for the anticancer efficacy of Doxo, we additionally investigated their mRNA and protein expression. A2780 and A2780ADR cells revealed similar mRNA levels of Topo IIα, Topo IIβ and Topoisomerase IIβ binding protein (TopBP1; Fig. 1G). On the protein level, A2780ADR were characterized by a reduced expression of Topo IIα protein under basal situation and 24-72 h following Doxo treatment (Figs. 1H and S2). Overall, these data indicated that both alterations in drug transport catalyzed by MDR1 and the protein expression of Topo IIα contributed to the strongly enhanced Doxo resistance of A2780ADR cells as well as to their profound cross-resistance to etoposide.

To further analyze the outcome of altered drug export in A2780 vs. A2780ADR cells, cell cycle progression was analyzed after 24-72 h of Doxo and Eto treatment. At early time point of analysis (that is, 24 h), A2780 revealed a more pronounced accumulation of cells in G₂/M phase compared with A2780ADR (Fig. 2A). This effect was seen both upon both Doxo and Eto treatment (Fig. 2A). Notably, SubG₁ fraction was not yet enhanced following Doxo or Eto treatment in A2780 cells at this early time point of analysis. Under basal situation, the percentage of SubG₁ phase cells was slightly higher at the 24 h time point of analysis as compared with 72 h. It was hypothesized that this minor effect reflected stress that resulted from the reseeding procedure. After an extended treatment period of 72 h, parental cells revealed a clear increase in the percentage of Doxo- and Eto-treated cells present in the SubG₁ fraction, which was not observed in the drug resistant A2780ADR variants (Fig. 2B). Taken together, these data showed that parental A2780 cells were hypersensitive to G₂/M blocking activity and apoptosis induction triggered by Topo II poisoning anticancer drugs.

To monitor proliferative activity, the percentage of Ki-67 positive cells was analyzed in both cell variants. In addition, mitotic index was calculated by determining the percentage of pH3 positive cells. The data obtained show a marked decrease in the percentage of Ki-67 positive A2780 parental cells following treatment with Doxo but not Eto (Fig. 2C). By contrast, measuring the mitotic index, a marked decrease in the frequency of pH3 positive cells was found in both parental cells and resistant variants following Doxo or Eto treatment (Fig. 2C). Measuring S-phase activity by analyzing the incorporation of EdU, a very strong reduction in the percentage of EdU positive parental cells upon both Doxo and Eto treatment it was again observed, which was much weaker in the resistant A2780ADR cells compared with the wild-type cells (Fig. 2D). Summarizing, the data show that A2780ADR cells are highly resistant to the antiproliferative activity of Topo II inhibitory compounds.

Inhibition of Topo II isoforms leading to the formation of DSB is considered as a major molecular mechanism underlying the anticancer activity of Doxo (47,48). Measuring the formation of nuclear γH2AX foci as well as 53BP1 foci and co-localized γH2AX/53BP1 foci as surrogate markers of DNA damage (that is, DSB), a markedly lower number of DNA damage-associated foci in the A2780ADR cells as compared with the parental A2780 cells was observed, both after treatment with Doxo and Eto (Fig. 3A). Following high-dose treatment with Doxo, a high percentage of γH2AX pan-stained A2780 cells was detectable, which was not observed in A2780ADR cells (Fig. 3A). These data show that the level of Doxo- and Eto-induced DSB was substantially reduced in the drug resistant variant compared with the parental cells. As DSBs are a potent trigger of mechanisms of the DDR, the present study next investigated the activation status of prototypical markers of the DDR by western blotting. An excessive increase in the DDR-related protein levels of γH2AX, p-Kap1, p-Chk2 and p-p53 in Doxo treated A2780 parental cells only, both after Doxo treatment period of 24 and 72 h. By contrast activation of these DDR-related factors was not found in A2780ADR cells (Fig. 3B). Notably, parental cells were also characterized by a great increase in the protein expression of the senescence marker p21, which was not detectable in the Doxo resistant A2780ADR cells (Fig. 3B). Eto treatment of A2780 cells also caused a distinct increase in γH2AX, p-P53 and p21 protein expression (Fig. S3).

Aiming to overcome the Doxo resistance of A2780ADR cells, the present study investigated their response to selected inhibitors of DDR- and DNA repair-related mechanisms, which are considered as promising targets to improve anticancer therapy and to overcome acquired drug resistance of tumor cells (49-52). To this end, the present study comparatively investigated the outcome of a 72 h treatment period of A2780 parental and A2780ADR cells with Poly (ADP-ribose) polymerase (PARP) inhibitors (olaparib and niraparib), checkpoint kinase 1/2 (Chk1/2) inhibitors (prexasertib and rabusertib) and histone deacetylase (HDAC) inhibitors (entinostat and ricolinostat). In addition, the calcium channel blocker Ver, which is reported to interfere with drug transport (53), the catalytic topo II inhibitor dexrazoxane, which is able to protect the heart from Doxo-induced cardiotoxicity by depleting both topo II isoforms independent of metal chelation (54-56) and Rac1 GTPase inhibitors (EHT1864 and Ehop16), which are reported to interfere with Doxo-induced DNA damage formation and DDR activation (57,58), were included into the study. From the IC₅₀ it was concluded that the data revealed a clear cross-resistance of A2780ADR cells to the poly (ADP-ribose) polymerase inhibitor (PARPi) olaparib (>10-fold) and niraparib (>5-fold), the Chk1/2i prexasertib (~4-fold) and rabusertib (~2.5-fold) and the HDAC class I inhibitor entinostat (~3-fold; Fig. 4). A2780ADR cells were neither appreciably cross-sensitive nor hypersensitive to any of the other pharmacological modulators (such as ricolinostat, Ver, EHT1864, EHOP16 and dexrazoxane) employed. Measuring cell viability 24 h after drug treatment, no major differences were observed (Fig. S4). The IC₅₀ and cross-sensitivities are summarized in Table SII.

Based on the results of these extensive studies, the present study addressed the question whether the pharmacological inhibitors under investigation were able to overcome the acquired Doxo resistance of A2780ADR cells. To this end, entinostat, EHT1864, rabusertib, dexrazoxane and Ver were selected for Doxo co-treatment analyses. Each of the selected inhibitors synergistically increased the cytotoxicity of Doxo in the drug resistant A2780ADR cells as concluded from the calculated CI (Fig. 5A). Based on the CI, the most pronounced synergistic toxicity was observed when Doxo was combined with the transport inhibitor Ver or the Rac1 inhibitor EHT1864 (Fig. 5A). The high efficacy of Ver was associated with increased intracellular steady state levels of Doxo under situation of co-treatment (Fig. 5B). It was not found if EHT or EST were used for Doxo co-treatment (Fig. 5B). These data indicated that synergistic toxicity resulting from combined treatment of A2780ADR cells with Doxo and Ver was at least partially due to Ver-mediated inhibition of drug export mechanisms leading to higher concentration of intracellular Doxo. By contrast, synergism observed upon use of EST and EHT1864 is suggested to be independent of drug transport. Notably, the data were in line with published data showing that Rac1 is involved in chemoresistance (59,60) and class I HDACi are useful of overcome acquired anticancer drug resistance of malignant cells (61-64).

Notably, Ver also synergistically increased the cytotoxicity of Doxo in A2780 parental cells (Fig. S5A), showing that the Ver effect is not restricted to cells with acquired Doxo resistance but also pertains to parental tumor cells. The pharmacological inhibitors entinotat (EST), EHT1864 and rabusertib (Rab) were also effective in combination with Doxo whereas dexrazoxane (Dex) was not (Fig. S5A). In addition, verapamil (Ver) also conferred synergistic toxicity in A2780ADR cells in combination with etoposide (Fig. S5B), showing that the Ver effect is not limited to Doxo but also comprises other anticancer drugs. Other inhibitors also conferred synergistic toxicity in combination with Eto, with EHT being most effective (Fig. S5B). To further investigate whether Ver is also able to overcome acquired resistance to anticancer drugs others than Topo II inhibitors, the present study investigated the influence of Ver on the CisPt sensitivity of CisPt-resistant A2780CisR cells, including EHT and EST for control. Data obtained show that all three modulators promoted CisPt-induced cytotoxicity in A2780CisR cells, with Ver and EST being most effective (Fig. S6).

In order to investigate whether the synergistic cytotoxicity evoked by combined treatment with Ver and Doxo was related to an elevated DNA damage induction, the present study measured the outcome of the co-treatments on the formation of DSB. To this end, the number of nuclear γH2AX and 53BP1 foci, which are indicative of DSB, was analyzed. Data obtained show that Ver most markedly increased the number of both nuclear γH2AX and 53BP1 foci as well as γH2AX/53BP1 co-localized foci compared with Doxo mono-treatment (Fig. 6A). In addition, the percentage of γH2AX pan-stained cells was also markedly enhanced upon co-treatment with Ver and Doxo as compared with the mono-treatments (Fig. 6A). Apparently, Ver was able to markedly stimulate the formation of DSB if used in combination with Doxo, demonstrating that Ver potentiates the genotoxic effects of Doxo in A2780ADR cells. Analyzing proliferation by monitoring the percentage of EdU positive cells, the most substantial antiproliferative effects were also observed upon combining Doxo with Ver (Fig. 6B). In addition, Ver also markedly increased the percentage of PI positive cells if used in combination with Doxo (Fig. 6C), showing that Ver potentiates Doxo-induced toxicity.

Aiming to further elucidate the molecular mechanisms underlying the observed synergistic toxicity and to substantiate the assumed increase in DSB formation under situation of Ver + Doxo co-treatment, the present study investigated the outcome of combined treatments on the activation status of a subset of DDR-related factors that are regulating replicative stress responses and cell death by western blotting. The data obtained showed that Ver especially was able to potentiate Doxo-mediated phosphorylation of the DDR-related factors p53, RPA32, Chk2 and H2AX, especially following 72 h of co-treatment (Fig. 7A). Moreover, if Doxo was combined with Ver, PAPR cleavage and cleavage of pro-caspase 7, which are indicative of the activation of apoptotic pathways, was observed (Fig. 7A). Thus, it was hypothesized that specific inhibition of drug transport by Ver conferred a broad re-sensitization of A2780ADR cells by fostering Doxo-induced formation of DSB and, in consequence, by activating DDR-dependent mechanisms that stimulated pro-apoptotic pathways. In addition, co-treatment of A2780ADR cells with Doxo plus Ver increased the mean mRNA expression of pre-selected factors involved in the regulation of oxidative stress response, DNA repair and cell cycle regulation compared with Doxo or Ver mono-treated control (Fig. 7B).

Irreversible cardiotoxicity is a dose-limiting adverse effect of anthracyclines (65,66). Hence, potentiating the anticancer efficacy of Doxo if combined with pharmacological modifiers brings up the concern of elevated side effects of the co-treatment, especially regarding heart damage. To address this aspect, the present study investigated the effect of combination treatments using immortalized murine HL-1 cardiomyocyte cells as an in vitro model. The results revealed more than additive cytotoxicity if Doxo was combined with Ver (Fig. 8A). By contrast, tendentially antagonistic effects were observed if Doxo was combined with the Rac1 inhibitor EHT1864 (Fig. 8A). This is in line with previous studies showing that pharmacological inhibition of Rac1 is able to protect cardiac cell types from Doxo-induced injury in vitro and in vivo (67-70). Moreover, the present study investigated the cytotoxicity of Doxo in combination with Ver, EHT1864 and EST using stem cell lines of murine (mESC) and human (hiPSC) origin. We found that Ver promotes the Doxo sensitivity of both mESC and hiPSC (Fig. 8B and C). By contrast Rac1 inhibition by EHT1864 again did not evoke synergistic toxicity in combination with Doxo in these non-malignant cells (Fig. 8A-C).