Ovarian cancer cells SKOV3, ES-2, OVCA420, and HeyA8 were purchased from the American Type Cell Culture (ATCC). SKOV3 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. ES-2, OVCA420, and HeyA8 cells were cultured in DMEM/High Glucose medium containing 10% FBS and 1% penicillin/streptomycin. These cell lines were cultured at 37°C with 5% CO₂ and 95% humidity. Olaparib (OP) was purchased from MedChemExpress and 100 mM DMSO stock solution was prepared. Disulfiram (DSF) was purchased from Target Molecule, Niraparib (NP) was provided by Zai Lab Co., Ltd., (Shanghai) and a 25 mM stock solution in DMSO was prepared for each drug.

We seeded SKOV3 and ES-2 cells on 96-well plates at a density of 5×10³ cells per well. Cells were treated for 72 h with DMSO, olaparib only (50 μM per well), screening compounds (10 μM per well), and the combination (50 μM Olaparib/10 μM compound per well)., and cell viability was measured using the sulforhodamine B (SRB) assay to determine the relative cell proliferation (15). The SRB test results were analyzed using GraphPad Prism 8 software to calculate the half-inhibition rate of cell proliferation (IC₅₀) of the compounds (16), and the results are expressed as the means of triplicate measurements.

According to the IC₅₀value of each drug in the cell lines, the final concentration gradient was set as 0.5 IC₅₀, 0.75 IC₅₀, 1.0 IC₅₀, and 1.25 IC₅₀. The CI and fraction affected (FA) values were calculated using Calcusyn software, which was based on the Chou-Talalay theorem (17). FA refers to the fraction of cell viability affected. Survivability plots and CI value scatter plots were made in GraphPad Prism 8.

SKOV3 and ES-2 cells were seeded in 6-well cell culture plates in triplicate at a concentration of 5×10³ cells per well in 2 mL medium supplemented with 10% FBS and incubated overnight. The media was removed and fresh media containing drugs was added, and the same volume of DMSO was added as a control. The cells were incubated at 37 °C for one week until the colonies were visible to the naked eye. The cells were then fixed with 4% paraformaldehyde for 25 to 30 min, washed with PBS, and stained with 2% crystal violet solution for 15 to 20 min. Finally, the cells were washed with water and air-dried. The number of cell colonies in the wells was counted and the clone formation rate was calculate as: clone formation rate (%)/clone formation rate (control)(%) (18).

SKOV3 cells were seeded in medium containing PARP inhibitors and Disulfiram, and the same volume of DMSO was added as a control. After 48 h of treatment, the supernatants and digested cell suspension were collected. Whole cells in the binding buffer suspension were stained with 1 µL RNA enzyme (Sigma, USA), 2 μL annexin V–FITC (BD, USA), and 2 μL propidium iodide (PI) (Sigma, USA) for 15 min at room temperature in the dark. Unstained cells and single-stained cells were prepared as controls. These samples were detected using flow cytometry, and the stained cells were analyzed using a FACS Calibur (BD). Data were analyzed with FlowJo software (v10).

SKOV3 and ES-2 cells were seeded in 10 cm dishes. Cells were collected after 48 h of drug treatment. Proteins were extracted using RIPA lysis buffer (Sigma) and protein concentrations were determined using the BCA assay. SDS-PAGE (Shanghai Sangon Biological Engineering and Technological Service Company, China) was done according to instruction on the Cell Signaling Technology webstie (19). The following antibodies were used: rabbit anti-PARP antibody (9532s), rabbit anti-γH2AX antibody (9718s), and rabbit anti-GAPDH (ab9485, Abcam). Membranes were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences), and data were quantified using the Image Studio Lite software.

Glass coverslips were placed into 24-well plates and 8×10³ cells were seeded per well. The cells were treated with Disulfiram and Olaparib at different concentrations and incubated 37°C with 5% CO₂ and 95% humidity for 48 h. Fixed cells were permeabilized with 0.2% Triton (Sangon, China) in 1×PBS for 30 min. Cells were incubated in 1% BSA (Sangon) in 0.2% Triton/PBS for 30 min. Cells were then incubated with primary rabbit anti-γH2AX antibody (1:400) at 4°C overnight. Cells were then washed with 0.2% triton/PBS three times for 3 min per wash and incubated with a secondary anti-rabbit 800 antibody for 1 h in the dark. Cell nuclei were counterstained with DAPI (D9542, Sigma) for 5 min, and washed with 0.2% triton/PBS three times for 5 min per wash. Images were taken using an Olympus inverted fluorescence microscope.

SKOV3, ES-2, HeyA8, and OVCA420 cells were treated for 8 h with 15 μM Disulfiram, and total RNA was isolated using the TRIzol reagent (Invitrogen). RNA was extracted and reverse transcribed into cDNA with the Prime Script RT Reagent Kit (Takara). The cDNA was then used as the template for the RT-qPCR reaction that was performed using SYBR-Green (Takara) on QuantStudio^®3 Real-Time PCR System (Applied Biosystems). GAPDH was used as an internal control. The reaction parameters were as follows: 5 min at 25°C, 30 min at 42°C, 5 min at 85°C, and then held at 16°C. The PCR profile was 95°C for 2 min, followed by 40 cycles of 95°C for 10s and 60°C for 30s. Data were analyzed using GraphPad Prism (version 8; GraphPad Software), and relative gene expression was calculated using the 2-ΔΔCT method.

The ES-2 xenograft tumor models were developed by injecting 1×10⁷ cells into female nude mice (6–8 weeks old). The mice were grouped randomly when the volume of the tumor nodules reached 100 mm³ and were then treated with the indicated compounds or vehicle via intraperitoneal injection for 18 days. Body weight and tumor dimension were measured. Tumor volume was calculated using the following equation: tumor volume = length × width (2) × 0.52. After the study, the mice were euthanized, and tumors and major organs were collected.

Tissue ections were cut from formalin-fixed paraffin-embedded xenografts. For IHC staining, samples were stained using the VECTASTAIN ABC kit (Vector). Anti–Ki67 (1:250; Catalogue #ab15580, Abcam) was used as the primary antibody. Hematoxylin and eosin (HE) staining was performed following standard protocols.

The results are expressed as the mean ± SD. All experiments were performed at least three times, except for the animal experiments. Statistical significance of the difference between two groups was determined by Student’s t-test. Two-way ANOVA was used to analyze animal data. The statistical analyses were performed using GraphPad Prism 7.0. The significant differences in the means were determined at the level of *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

To identify drugs that might enhance the effect of PARPis, we screened 170 drug molecules retrieved from the FDA/CFDA compound library using SKOV3 and ES-2 cells. The experiments were performed with the screening compounds alone and the combination with Olaparib. DMSO and Olaparib alone were used as controls. Cell viability of the cells treated with the screening compounds vs the drug combination with Olaparib was plotted ( Figures 1A, B ). In both cases, each dot represents one compound. Dots located below the orange dashed line (slope of 1) indicates that the cell viability ratio of +Olaparib to -Olaparib is below one in the presence of these compounds; the dots located above the dashed line indicated that the cell viability ratio of +Olaparib to -Olaparib is above one. Ninety compounds decreased SKOV3 cell viability, and 151 compounds decreased ES-2 cell viability, indicating that the combinatorial effects of Olaparib were greater in the ES-2 cells ( Figure 1B ). The average survival rate ratios following combined treatment with PARPis vs the compound alone were 0.55 (37.61 vs 68.22%) and 0.22 (4.58 vs 21.28%) for the SKOV3 and ES-2 cells, respectively. Disulfiram (red dot) had the biggest effect on decreasing ES-2 cell viability among the 170 compounds ( Figure 1B ). The chemical structure of Disulfiram is shown in Figure 1C .

To further confirm the effect of Disulfiram in combination with PARPis, we examined the effect of Disulfiram with PARPis (Olaparib or Niraparib) on cell viability in two additional cell lines, OVCA420 and HEYA8. We first determined the IC₅₀ values of Olaparib and Niraparib and in combination with Disulfiram, resepectively. The IC₅₀ of Olaparib was higher compared to the IC₅₀ of Niraparib, which was relatively low ( Table S1 ), consistent with the literature (20). Interestingly, Disulfiram showed relatively low IC₅₀ values with in all four cell lines.

We used the CI (combination index) to evaluate whether the effect Disulfiram was additional or synergistic (17). Based on the different IC₅₀ values of Olaparib, Niraparib, and Disulfiram ( Figure S1 ), we set the concentration of Olaparib or Niraparib with Disulfiram at 50, 75, 100, or 125% of its IC₅₀. Figure 2 shows cell growth at the different concentrations of Olaparib or Niraparib in combination with Disulfram. The green trace of each figure represents the cell growth in the presence of Olaparib (left column) or Niraparib (right column) alone from 0 IC₅₀ to 1.25 IC₅₀; the purple trace of each figure represents the cell growth in the presence of Disulfiram alone from 0 to 60 µM; and the red trace of each figure represents the cell growth in the presence of Disulfram/Olaparib (left column) and Disulfram/Niraparib (right column) at different concentrations. In all figures, red traces decreased more compared to the corresponding green and purple traces with increasing drug concentrations, indicating that cell growth was more inhibited by the combination of Disulfiram compared to the PARPis alone. The mean CI values of Disulfiram combined with Olaparib or Niraparib in the SKOV3, ES-2, HeyA8, and OVCA420 cells are denoted at the bottom of each figure. The CI values were all below one, indicating that Disulfiram works synergistically with Olaparib or Niraparib to inhibit ovarian cancer cell growth, especially in SKOV3 cells. Moreover, combinational matrix (DSF+OP and DSF+NP) showed effect of Disulfiram in combination with and Olaparib or Niraparib on ovarian cancer cell growth. ( Figures S2A–D ).

We further examined the effect of Disulfiram in combination with PARPis on colony formation. Compared with Disulfiram or PARPis alone, the rate of colony formation in the combination group was significantly reduced ( Figure 3 ). The average colony formation rate for the combination of Olaparib (5 μM) and Disulfiram (0.194 μM) in SKOV3 cells was reduced to 6.04%, and dropped to 8.33% with Olaparib (4 μM) and Disulfiram (0.194 μM) in ES-2 cells ( Figures 3A–D ). These results are consistent with the findings of cell growth inhibition ( Figure 2 ).

To understand the synergist effect of Disulfiram and PARPis, we measured cell apoptosis of SKOV3 cells after treatment with Disulfiram and Olaparib alone and in combination. After 48 h of drug treatment the total apoptosis rate of Disulfiram (30 µM) with Olaparib (200 µM) was 23.89%, which was approximately 4-fold higher than the apopotosis rate of Olaparib (6.90%) and approximately 10-fold higher than Disulfiram (2.59%) alone. When the concnetration of Disulfiram and Olaparib was doubled, the total apoptosis rate of Disulfiram with Olaparib was 30.3%, approximately 3-fold higher than the apopotosis rate of Olaparib (9.87%) and approximately 3.5-fold higher than Disulfiram (8.52%) alone ( Figure 3E ) and analyzed apopotosis rate ( Figure S3B ). In OVCA420, the proportion of apoptotic cells after drug addition was also detected ( Figure S3A ). Compared with the single drug group, the combination drug induced the generation of apoptotic cells ( Figure S3C ). These results indicate that the combination of Disulfiram with Olaparib increases apoptosis of SKOV3 and OVCA420 cells, which is consistent with the findings for cell growth.

As shown in Figure 3 , the combination of Disulfiram with PARPis increased cell apoptosis. Therefore, we next measured the level of cleaved PARP, which is considered a markers of apoptosis (21), in SKOV3 and ES-2 cells in the presence of Disulfiram and PARPis. We also measured the level of γH2AX (phosphorylation of H2AX, which is one of the most conserved histone H2AX variants), a widely recognized marker of DNA double-strand cleavage (22). Figures 4A, B show that Dislfiram in compbination with PARPis increased H2AX protein expression and PARP cleavage compared to PARPis or Disulfiram alone, indicating that the combined treatment increased DNA double-strand cleavage in SKOV3 and ES-2 cells. Apparently, the densitometry analysis showed that the formation of H2AX increased after drug addition or combined drug group in SKOV3 ( Figure 4C ) and ES-2 ( Figure 4D )

Immunofluorescence assays showed that the percentage of the cells with H2AX foci >10 was 91.72% in the combination group, which was much higher than the Disulfiram (21.67%) or Olaparib (79.05%) alone groups ( Figures 4E–G ). These results confirmed that Disulfiram in combination with Olaparib enhances DNA double-strand damage ( Figure 3 ). It is worth noting that the level of DNA damage induced by Disulfiram alone was not significantly different from the Control group (18.46%).

HRD cells are more sensitive to PARP inhibitors due to the synthetic lethalilty and because the HRR pathway is not limited to the most common BRCA1/2 mutations. Deletions or mutations in other genes can be directly or indirectly involved in the HRR pathway, which could affect cancer cells sensitivity to PARP inhibitors. We used real-time PCR to determine the transcripton levels of BRCA1, BRCA2, RAD51, RAD52, ATR, ATM, PALB2. Figure 5 shows the expression level of these genes in SKOV3, ES-2, HeyA8, and OVCA420 cells that were treated with 15 μM Disulfiram for 8 h. Compared to the control, Disulfiram significantly inhibited the expression of all-tested genes in the four cell lines, except for RAD51 in HeyA8 and OZVCA420 cells. These results suggest that the effect of Disulfiram alone on ovarian cancer cells might involve the HRR pathway. However, we did not observe much difference in gene expression in response to the combination of Disulfiram with PARPis vs Disulfiram alone (data not shown).

Based on the cellular level data, we established an ovarian cancer xenograft model in vivo by subcutaneously injecting ES-2 cells into nude mice. Figure 6 shows that Disulfiram combined with Niraparib suppressed ES-2-derived xenograft tumor growth in vivo. Female nude mice bearing ES-2-derived tumors were randomized into four treatment groups: DMSO, Disulfiram, Niraparib, and Niraparib+Disulfiram. Figures 6A, B show the change in tumor volume after DMSO (blue trace), Disulfiram (green trace), Niraparib (red trace), and Niraparib+Disulfiram (purple trace) treatment for 18 days. Compared to the DMSO control, both Disulfiram and Niraparib inhibited tumor growth approximately 2-fold after 18 days of the injection, the combination of Disulfiram and Niraparib dramatically inhibited tumor growth compared to the other groups. The change in tumor weight exhibited a similar trend ( Figure 6C ), but the body weight of the mice after the injection of different compounds was very similar across the groups, indicating that Disulfiram, Niraparib, and Niraparib+Disulfiram have negligible toxicity ( Figure 6D ). We did not observe any abnormal behavior or side effects in any of the groups during treatment. This result was confirmed by HE staining of the heart, kidney, lung, liver, and spleen ( Figure 6E ). In immunohistochemistry experiment (23), compared to the control, the proliferation marker Ki67 was dramatically decreased in the Disulfiram+Niraparib group, which confirmed the anticancer effect shown in vivo ( Figure 6F ). These in vivo data demonstrate that Disulfiram in combination with Niraparib is an effective anti-cancer treatment strategy with minimal to no toxicity.