Combined PARP and Dual Topoisomerase Inhibition Potentiates Genome Instability and Cell Death in Ovarian Cancer

Abstract

Although ovarian cancer is a rare disease, it constitutes the fifth leading cause of cancer death among women. It is of major importance to develop new therapeutic strategies to improve survival. Combining P8-D6, a novel dual topoisomerase inhibitor with exceptional anti-tumoral properties in ovarian cancer and compounds in preclinical research, and olaparib, a PARP inhibitor targeting DNA damage repair, is a promising approach. P8-D6 induces DNA damage that can be repaired by base excision repair or homologous recombination in which PARP plays a major role. This study analyzed benefits of combining P8-D6 and olaparib treatment in 2D and 3D cultures with ovarian cancer cells. Measurement of viability, cytotoxicity and caspase activity were used to assess therapy efficacy and to calculate the combination index (CI). Further DNA damage was quantified using the biomarkers RAD51 and γH2A.X. The combinational treatment led to an increased caspase activity and reduced viability. CI values partially show synergisms in combinations at 100 nM and 500 nM P8-D6. More DNA damage accumulated, and spheroids lost their membrane integrity due to the combinational treatment. While maintaining the same therapy efficacy as single-drug therapy, doses of P8-D6 and olaparib can be reduced in combinational treatments. Synergisms can be seen in some tested combinations. In summary, the combination therapy indicates benefits and acts synergistic at 100 nM and 500 nM P8-D6.

Keywords: PARP inhibitor; dual topoisomerase inhibitor; ovarian cancer.

Figure 1

Mode of action of dual topoisomerase inhibition and PARP inhibition. The schematic representation shows the proposed targets of PARPi and topoisomerase inhibitors. PARP is involved in different DNA repair pathways, including BER and HR. Here, PARP contributes to the detection of lesions and the initialization of repair. Combining PARPi with P8-D6 causes an increase in genomic instability. P8-D6 stabilizes the topoisomerase I and II-DNA covalent complex, thereby leading to an increase of DNA damage in the form of SSBs and DSBs. By SSB repair, if DNA repair functions via TDP1 and PARP, the cell survives. Inhibition of PARP and/or TDP1 results in the accumulation of SSBs and DSBs, which can be repaired by HR. Moreover, the inhibition of Topo II generates DSB. If there is an HR deficiency, cells cannot be repaired via the HR pathway and go into induced apoptosis.

Figure 2

Anti-tumor responses in combined treatment of P8-D6 and olaparib in OC 2D culture. OvCar8 and A2780 cells were treated in a 2D monolayer cell culture with different concentrations of olaparib and P8-D6 in single and combination therapy. Subsequently, the viability and caspase activity were determined. (A) Treatment schedule. Camera icon indicates imaging timepoints. (B,D) The IC₅₀ value of each olaparib concentration was calculated by using the viability data. (C,E) The apoptosis is represented as relative caspase activity. Data are means + SEM one-way ANOVA, * (p < 0.05), ** (p < 0.01), **** (p < 0.0001).

Figure 3

Combination index (CI) analysis of olaparib in combination with P8-D6 in OC 2D culture. (A) Combination index (CI) for drug combinations by olaparib and P8-D6 in OvCar8 cells. CI values computed according to CompuSyn software with viability data. Combinations were considered synergistic when CIs were below 1.0. The fraction affected (fa)-value represents the fraction of cell viability affected by therapy. (B) The monotherapy column defines the concentrations that are needed in monotherapy to affect a certain fraction of cells by therapy. DRI values represent the order of magnitude (fold) of dose reduction in combination setting compared with each drug alone.

Figure 4

Anti-tumor responses in combined treatment of P8-D6 and olaparib in 3D culture. OvCar8 spheroids were maintained in ULA plates for 72 h and subsequently treated with olaparib and P8-D6 in single or combination treatment as illustrated in the schedule. (A) Treatment schedule. Camera visualizes the measurement time points by microscope. Subsequently, the viability and caspase activity were measured. (B) The IC₅₀ value of each olaparib concentration was calculated using the viability data. (C) The apoptosis is represented as relative caspase activity. Data are means + SEM one-way ANOVA, * (p < 0.05), **** (p < 0.0001).

Figure 5

Combination index (CI) analysis of olaparib in combination with P8-D6 in OC spheroids. (A) Combination index (CI) for drug combinations by olaparib and P8-D6 in OvCar8 spheroids. CI values computed accordingly by CompuSyn software with viability values. Combinations were considered synergistic when CIs were below 1.0. The fraction affected (fa)-value represents the fraction of cell viability affected by therapy. (B) The monotherapy column defines the concentrations that are needed in monotherapy to affect a certain fraction of cells by therapy. DRI values represent the order of magnitude (fold) of dose reduction in combination setting compared with each drug alone.

Figure 6

Toxicity of PARPi with dual topoisomerase inhibitor in 3D tumor spheroids of OvCar8. OvCar8 spheroids were maintained in ULA plates for 72 h and subsequently treated with olaparib and P8-D6 in single or combination treatment as described in the schedule in Figure 4A. (A) After treatment the cell toxicity was measured by fluorescence microscopy using CellTox™ Green (timepoint 144 h after seeding); scale bars, 500 µm. (B) The fluorescence signals after treatment were quantified (relative fluorescence units RFU) and shown in the heat map. (C) Spheroids were stained after treatment with PI (red), calcein-AM (green), Hoechst 33342 (blue) and imaged by fluorescence microscopy; scale bars, 500 µm. (D) Scanning electron microscopy images of spheroids, which were treated with P8-D6, olaparib, combination or PBS were taken; scale bars, 500 × 100 µm, 5000 × 10 µm.

Figure 7

Olaparib potentiates γH2A.X and RAD51 recruitment for strand break induced by P8-D6 in OvCar8. (A) Immunofluorescence images of OvCar8 2D monolayer: DAPI-stained nuclei (blue), γH2A.X (green) and RAD51 (red) after treatment with olaparib and/or P8-D6; scale bars, 50 µm. (B) The intensity of the γH2A.X and RAD51 signal in relation to DAPI in OvCar8 2D monolayer after treatment. (C) Confocal fluorescence z-stack images of OvCar8 spheroids were obtained with immunostaining against γH2A.X, RAD51 and Hoechst 33342. Spheroids were treated with olaparib and/or P8-D6, permeabilized with glycerol, followed by antibody staining; scale bars, 100 µm. (D) γH2A.X and RAD51 fluorescence intensities of different treatments were compared between different treatments.

Figure 8

Protein expressions modulated by combinational treatment of PARP and P8-D6. Western blot analysis was performed for several cellular proteins associated with DNA damage repair systems using total OvCar8 cell lysate. HSP90 and β-Actin were used as loading control. O2.5—olaparib 2.5 µM, O10—olaparib 10 µM, C2.5—combination of olaparib 2.5 µM and P8-D6 0.1 µM, C10—combination of olaparib 10 µM and P8-D6 0.1 µM.