Cancer Res Treat.  2021 Jul;53(3):819-828. 10.4143/crt.2020.1013.

Enhanced Efficacy of Combined Therapy with Checkpoint Kinase 1 Inhibitor and Rucaparib via Regulation of Rad51 Expression in BRCA Wild-Type Epithelial Ovarian Cancer Cells

Affiliations
  • 1Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
  • 2Department of Obstetrics and Gynecology, Hallym University Dongtan Sacred Heart Hospital, Hwaseong, Korea
  • 3Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Korea

Abstract

Purpose
This study aimed to evaluate anticancer effects of combination treatment with poly(ADP-ribose) polymerase (PARP) and checkpoint kinase 1 (Chk1) inhibitors in BRCA wild-type ovarian cancer. PARP inhibitors can function as DNA-damaging agents in BRCA wild-type cancer, even if clinical activity is limited. Most epithelial ovarian cancers are characterized by a TP53 mutation causing dysfunction at the G1/S checkpoint, which makes tumor cells highly dependent on Chk1-mediated G/M phase cell-cycle arrest for DNA repair.
Materials and Methods
We investigated the anticancer effects of combination treatment with prexasertib (LY2606368), a selective ATP competitive small molecule inhibitor of Chk1 and Chk2, and rucaparib, a PARP inhibitor, in BRCA wild-type ovarian cancer cell lines (OVCAR3 and SKOV3).
Results
We found that combined treatment significantly decreased cell viability in all cell lines and induced greater DNA damage and apoptosis than in the control and/or using monotherapies. Moreover, we found that prexasertib significantly inhibited homologous recombination–mediated DNA repair and thus showed a marked anticancer effect in combination treatment with rucaparib. The anticancer mechanism of prexasertib and rucaparib was considered to be caused by an impaired G2/M checkpoint due to prexasertib treatment, which forced mitotic catastrophe in the presence of rucaparib.
Conclusion
Our results suggest a novel effective therapeutic strategy for BRCA wild-type epithelial ovarian cancer using a combination of Chk1 and PARP inhibitors.

Keyword

Chk1 inhibitor; PARP inhibitor; Rad51; Ovarian neoplasms

Figure

  • Fig. 1 Prexasertib or rucaparib reduce cell viability and proliferation and induce apoptotic cell death in BRCA wild-type ovarian cancer cell lines. The cells were treated with either prexasertib (0–100 μM) or rucaparib (0–100 μM) for 72 hours after cells were seeded. The cell viability (A) and proliferation (B) of prexasertib and rucaparib was determined by PrestoBlue or Cell Titer-Glo assay in SKOV-3 and OVCAR-3 cells. (C) The activity of caspase-3 was measured by luciferase assay using Caspase-Glo 3/7 reagent. Cell viability, proliferation, and caspase-3 activity were calculated relative to 0.01% dimethylsulfoxide-treated control cells. (D) Representative images of immunoblotting data for protein levels of checkpoint kinase 1 (Chk1), poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved PARP proteins after prexasertib or rucaparib treatment. Alpha-tubulin was used as a loading control. Blue and red colors indicate prexasertib and rucaparib, respectively. Values are expressed as the mean±standard deviation. *p < 0.05 compared to 0 μM, **p < 0.01 compared to 0 μM, ***p < 0.001 compared to 0 μM, #Control group (CTL).

  • Fig. 2 Combination treatment promotes suppression of cell viability and induction of apoptotic cell death in ovarian cancer cells. SKOV-3 and OVCAR-3 cells were treated with either prexasertib (0–100 μM) or rucaparib (0, 10, and 50 μM) for 72 hours after cells were seeded. (A) Cell viability was by PrestoBlue in SKOV-3 and OVCAR-3 cells. (B) Apoptotic cell analysis was measured using an Annexin V assay by fluorescence activatdetermineded cell sorting. (C) Caspase-3 activity was measured by luciferase assay using Caspase-Glo 3/7 reagent. (D) Representative images of immunoblotting data for levels of checkpoint kinase 1 (Chk1), poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved PARP proteins in combination treatment conditions. Alpha-tubulin was used as a loading control. *p < 0.05 compared to 0 μM, **p < 0.01 compared to 0 μM, ***p < 0.001 compared to 0 μM, #Control group. Values are expressed as the mean±standard deviation.

  • Fig. 3 Combination treatment increases mitotic entry of sub G1 phase cells and activates the DNA damage signaling pathway. (A) Cell-cycle analysis was determine using FxCycle propidium iodide (PI)/RNase staining solution by fluorescence activated cell sorting in both SKOV-3 and OVCAR-3 cell lines. (B) Representative images of immunoblotting data for levels of phospho-Ser345 checkpoint kinase 1 (Chk1), phospho-Ser296 Chk1, Rad51, γH2AX, and phosphor-Ser10 Histone H3 proteins in combination treatment conditions. Alpha-tubulin was used as a loading control. Values are expressed as the mean±standard deviation. **p < 0.01 compared to 0 μM, ***p < 0.001 compared to 0 μM, #Control group.

  • Fig. 4 Regulation of Rad51 expression affects anticancer effect by poly(ADP-ribose) polymerase (PARP) inhibition. (A, B) Knockdown of Rad51 expression was performed by Lipofectamine in ovarian cancer cells. After inhibition of Rad51, SKOV-3, and OVCAR-3 cells were treated with 50 μM rucaparib for 48 hours. Cell viability and caspase-3 activity were measured by PrestoBlue and Cell Titer-Glo assays, respectively. (C) Representative images of immunoblotting data for protein levels of Rad51, checkpoint kinase 1 (Chk1), PARP, γH2AX, cleaved caspase-3, and cleaved PARP under specific conditions. Alpha-tubulin was used as a loading control. Values are expressed as the mean±standard deviation. **p < 0.01 compared to 0 μM, ***p < 0.001 compared to 0 μM, #Control group.


Reference

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