ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta Т. 18, No. 5, 2025
P. 14-23 , Bibliography 33 , Engl.
UDC 616-006.6-092:577.2:615.357
doi: https://doi.org/10.15407/biotech18.05.014
RAD51-IN-1 INDUCES DNA DAMAGE AND PROMOTES ROS-MEDIATED APOPTOSIS IN OVARIAN CANCER CELLS
Deniz Özdemir1, Can Ali Ağca 2
1Department of Molecular Biology and Genetics, Science Institute, Bingol University, Türkiye
2 Bingöl University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Türkiye
Aim. RAD51 protein is frequently overexpressed in ovarian cancer and plays a critical role in cancer cell development and survival. This study aims to elucidate, how RAD51-IN-1-induced DNA damage and apoptosis contribute to anti-cancer effects in ovarian cancer cell lines (MDAH-2774 and OVCAR-3).
Methods. This research explores the impact of RAD51-IN-1 on cell viability, colony formation, ROS levels, DNA damage, and apoptosis were assessed in MDAH-2774 and OVCAR cell lines through the application of the CVDK-8 viability kit, colony formation assays, DCFDA staining, Comet assays, and AO/ER double staining methods.
Results. Ovarian cancer cell lines were treated with varying doses of RAD51-IN-1, which resulted in a dose-dependent decline in both cell viability and colony formation, with the IC50 value for RAD51-IN-1 being determined. Furthermore, as shown by DCFDA staining, an increase in intracellular reactive oxygen species (ROS) and DNA damage as measured by the Comet assay was observed following RAD51-IN-1 treatment. RAD51-IN-1 was found to induce apoptosis by acridine orange/ ethidium bromide staining.
Conclusion. This study demonstrates that RAD51-IN-1 effectively induces DNA damage and ROS-dependent apoptosis in ovarian cancer cell lines. Although RAD51-IN-1 requires further in vitro and in vivo evaluation as a treatment in ovarian cancer cells, these findings provide preliminary evidence of its potential efficacy.
Key words: RAD51-IN-1, Ovarian Cancer, DNA Damage, Apoptosis.
© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2025
References
1. Tew, B. Y. Kalfa, J., Yang, Z.., Hurth, K.M., Simo, T., Abnoosian, A., Durant, S.T. Hamerlik, P., Salhia, B., (2023). ATM-Inhibitor AZD1390 Is a Radiosensitizer for Breast Cancer CNS Metastasis., Clin. cancer Res. an Off. J. Am. Assoc. Cancer Res., 29(21), 4492–4503, https://doi.org/10.1158/10780432.CCR-23-0290
2. Huang, R. X,. Zhou, P. K., DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct. Target. Ther. 5(1), https://doi.org/10.1038/s41392-020-0150-x
3. Priya, B., Ravi, S., Kirubakaran, S. (2023). Targeting ATM and ATR for cancer therapeutics: Inhibitors in clinic. Drug Discov. Today, 28(8), 103662, https://doi.org/10.1016/j.drudis.2023.103662.
4. Alizzi, Z., Saravi, S., Khalique, S., McDonald, T,. Karteris, E., Hall, M. (2023). Identification of RAD51 foci in cancer-associated circulating cells of patients with high-grade serous ovarian cancer: association with treatment outcomes. Int. J. Gynecol. cancer Off. J. Int. Gynecol. Cancer Soc., 33(9). 1427–1433, https://doi.org/10.1136/ijgc-2023-004483.
5. Feng, Y., Wang, D., Xiong, L,. Zhen, G., Tan, J. Predictive value of RAD51 on the survival and drug responsiveness of ovarian cancer. Cancer Cell Int.21(1). 249. https://doi.org/10.1186/s12935-021-01953-5
6. Nagathihalli, N. S., Nagaraju, G. (2011). RAD51 as a potential biomarker and therapeutic target for pancreatic cancer. Biochim. Biophys. Acta - Rev. Cancer, 1816(2), 209–218, https://doi.org/10.1016/j.bbcan.2011.07.004.
7 Song, J., Cuı, D., Wang, J., Qin,, J., Wang, S., Wang, J., Zhai, K., Ma, H. Ma, D..., liu, Z. (2021). Overexpression of HMGA1 confers radioresistance by transactivating RAD51 in cholangiocarcinoma. Cell Death Discov. 7(1), p. 322, 2021, https://doi.org/10.1038/s41420-021-00721-8
8 Xu, Y. Chen, K. Cai, Y. Cheng, C. Zhang, Z. Xu, G. (2019). Overexpression of Rad51 predicts poor prognosis and silencing of Rad51 increases chemo-sensitivity to doxorubicin in neuroblastoma. Am. J. Transl. Res., 11(9), 5788.
9. Wang, Z., -Jia.R., Wang, L., Yang, O., HU, Fu, O., Zhang, Li, W., Ren, Y. (2022). The Emerging Roles of Rad51 in Cancer and Its Potential as a Therapeutic Target. Front. Oncol. 12, 935593, https://doi.org/10.3389/fonc.2022.935593
10. Bonilla, B. Hengel, S. R. Grundy, M. K., Bernstein, K. A. (2020) RAD51 gene family structure and function, Annu. Rev. Genet., 54(1), 25–46,. https://doi.org/10.1146/annurev-genet-021920-092410
11. Nair, J. Huang, T.T., Murai, J., Haynes, B., Haynes, P.S., Pommier, Y., Lee, J.M. (2020). Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer, Oncogene, 39(33), 5520–5535, https://doi.org/10.1038/s41388-020-1383-4
12. Sun, C., Cao, W., Qiu, C., Li, C., Dongol, S., Zhang, Z., Dong, R., Song K.,...,Zhang, O. (2020). MiR-509-3 augments the synthetic lethality of PARPi by regulating HR repair in PDX model of HGSOC. J. Hematol. Oncol. 13, 1–18, 2020. https://doi.org/10.1186/s13045-020-0844-0
13. Gu, P. Xue,.L., Zhao, Li, W., Jiang, Z., Liu, A., Liu, T., Liu, L.,..., Tang, R. (2022). Targeting the Homologous Recombination Pathway in Cancer With a Novel Class of RAD51 Inhibitors. Front. Oncol. 12, , https://doi.org/10.3389/fonc.2022.885186.
14. Clin, A. Zhao, J., Kumar, S., Chakraborty, C., Talluri, S., Munshi, N.C., Shammas, M.A. (2020). RAD51 Inhibitor Reverses Etoposide-Induced Genomic Toxicity and Instability in Esophageal Adenocarcinoma Cells. Arch. Clin. Toxicol.2(1),. 3–9. https://doi.org/10.46439/toxicology.2.006.
15. Alagpulinsa, D. A. Ayyadevara, S., Reis, R. J. S. (2014). A small molecule inhibitor of RAD51 reduces homologous recombination and sensitizes multiple myeloma cells to doxorubicin. Front. Oncol., 4, no. OCT, pp. 1–11, https://doi.org/10.3389/fonc.2014.00289.
16. Khan, S., Carpenter, M. A., Kemp, M. G. (2022). Rad51 inhibition sensitizes non-replicating quiescent cells to UVB radiation and transcription stress. bioRxiv. https://doi.org/10.1101/2022.09.20.508657.
17. Demeyer, A., Benhelli, Mokrani, H., Chénais, B., Weigel, P., Fleury, F. (2021). Inhibiting homologous recombination by targeting RAD51 protein, Biochim. Biophys. Acta (BBA)-Reviews Cancer, 1876(2). 188597,.
18. Scott, D. E., Newton, N.C., Marsh, M.T., Coyne, A. G., Fisher G., Moschatti, T., Bayly, a. R., Sharpe, T.D., ..., Abel, C. (2021). A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death. Cell Chem. Biol., 28,(6), 835‒847.e5, https://doi.org/10.1016/j.chembiol.2021.02.006.
19. Shkundina, I. S., Gall, A. A., Dick, A., Cocklin, S,. Mazin, (2021). New RAD51 Inhibitors to Target Homologous Recombination in Human Cells. Genes (Basel)., 12(6), https://doi.org/10.3390/genes12060920.
20. Franken, N.A.P., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. (2006). Clonogenic assay of cells in vitro, Nat. Protoc., 1(5), 2315–2319, , https://doi.org/10.1038/nprot.2006.339.
21. Özdemir, D., Şatana, K., Özdemir, D., Çiftci, M., C. A. Agca, (2021). İfosfamid ve Kurkumin Kombinasyonun Küçük Hücreli Dışı Akciğer Kanseri Hücresinde Apoptotik Biyobelirteçler ve Hücre Göçü Üzerine Etkileri,” Türk Doğa ve Fen Derg., vol. 10, no. 2, pp. 295–302, , https://doi.org/10.46810/tdfd.958756.
22. Shailender, G., Kumari, S., Kiranmayi, P. Malla, R. R. (2019). Effect of MMP-2 gene silencing on radiation-induced DNA damage in human normal dermal fibroblasts and breast cancer cells, Genes Environ., 41(1), . 1–7. https://doi.org/10.1186/s41021-019-0131-x
23. Lu, Y., Liu, Y., Yang, C. Evaluating in vitro DNA damage using comet assay, JoVE (Journal Vis. Exp., 128, e56450, 2017.
24. Özdemir, D., Saruhan, S., Agca, C. A. (2023). KAN0438757: A novel PFKFB3 ınhıbıtor that ınduces programmed cell death and suppresses cell mıgratıon ın non-small cell lung carcınoma cells, Biotechnol. Acta, 16(5), 34–44. https://doi.org/10.21203/rs.3.rs-3067846/v1
25. Ward, A., Dong, L., Harris, J.M.,. Khanna, R.R., -Ejeh, A.F., Fairlie, D.P., Wiegmans, A.P., Liu, L. (2017). Quinazolinone derivatives as inhibitors of homologous recombinase RAD51, Bioorg. Med. Chem. Lett., 27(14), 3096–3100, https://doi.org/10.1016/j.bmcl.2017.05.039.
26. Xu, L., Wu, T., 1 , Lu, S., Hao, Junchao Qin, Jang, J., Zhang, X., Liu, Q.,..., Shao, C. (2020). Mitochondrial superoxide contributes to oxidative stress exacerbated by DNA damage response in RAD51-depleted ovarian cancer cells.,” Redox Biol., 36, 101604, https://doi.org/10.1016/j.redox.2020.101604.
27 Xin, Y., Wang, J., Wu, Y. Li, Q., Dong, M., Liu, C., He, Q., Wang, R.,..., Zhang, W. (2022). Identification of Nanog as a novel inhibitor of Rad51, Cell .eath Dis., 13(2), 193, https://doi.org/10.1038/s41419-022-04644-9.
28. Zhu, J., Zhou, l., Wu, g., Konig, h., Lin, l., Li, G., Qiu, X.L., Chen, C.F.,..., Lee, W.H. (2013). A novel small molecule RAD51 inactivator overcomes imatinib‐resistance in chronic myeloid leukaemia, EMBO Mol. Med., 5(3), 353–365, 2013. https://doi.org/10.1002/emmm.201201760
29. Makino, E., Fröhlich, L.M., Sinnberg, T., Kosnopfel, C., Sauer, B., Garbe, C., Schittek,B. (2020). Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition, Cell Death Dis., 11(7), 581, https://doi.org/10.1038/s41419-020-2702-y.
30. Hoppe, M. M., Jaynes, P., Wardyn, G.D., Upadhyayula, S.S., Tan, T.Z., Lie, S., Lim, D.G.Z., , Pang, B.N.K. (2021). Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer, EMBO Mol. Med., 13(5), . e13366, https://doi.org/10.15252/emmm.202013366.
31. Wu, R., Patel, A., Tokumaru, Y., Asaoka, M., Oshi, M., Yan, L. Ishikawa, T., Takabe, K. (2022). High RAD51 gene expression is associated with aggressive biology and with poor survival in breast cancer., Breast Cancer Res. Treat., 193(1), 49–63, https://doi.org/10.1007/s10549-022-06552-0.
32. Hu, J., Zhang, Z., Zhao L., Li, L., Zuo W., Han, L. (2019). High expression of RAD51 promotes DNA damage repair and survival in KRAS-mutant lung cancer cells., BMB Rep., 52(2), 151–156, https://doi.org/10.5483/BMBRep.2019.52.2.213.
33. Klein, H. L. (2008). The consequences of Rad51 overexpression for normal and tumor cells., DNA Repair (Amst)., 7(5), 686–693, doi: https://doi.org/10.1016/j.dnarep.2007.12.008.