Abstract 6269: Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway

Abstract

Abstract Background: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. Peptide Receptor Radionuclide Therapy (PRRT) is a novel therapy approved for treating NETs with a treatment response rate of 20-30%. To improve therapeutic response of PRRT, 3-AP (Triapine), which is a ribonucleotide reductase (RR) inhibitor with antineoplastic and radiation sensitizing properties, can be used in combination with PRRT. The purpose of our study is to examine effects of Triapine treatment on cellular proliferation, apoptosis and activation of DNA repair pathway in NET cells in vitro. Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) To determine effective dose range of Triapine (500 - 2500nM), proliferation was measured at 48, 72, and 96 h with sulforhodamine B (SRB) assay. (ii) To evaluate the effects of radiosensitivity, cultures were treated with increasing doses (250 - 5,000nM), received 2 Gy radiation (XRT) 1h later, exposed to Triapine for 24 hours, rinsed, and fed Triapine-free media; colonies were counted and quantified with SRB assays 14 days later and survival curves were generated. (iii) To further characterize cellular responses, cells were treated with Triapine (250 - 5,000 nM), irradiated 1h later, and harvested 24 h later to examine expression levels of Cyclin D1, cleaved PARP, p-DNA-PK (S2056), p-ATM (S1981) and p-ATR (S428) by Western blot. Results: (i) SRB proliferation assay demonstrated the effective dose range, determined by half maximal inhibitory concentration (IC-50), was 1,3 µM in BON cells and 1.73 µM in QGP-1 cells at 96 h. (ii) Exposure to Triapine before irradiation resulted in a significant increase in the radiosensitivity of each of the tumor cell lines with dose enhancement factors at a surviving fraction of 0.10 of 1.8 and 1.6 for BON and QGP-1, respectively. (iii) Triapine treatment alone activated DNA repair pathway in a dose-dependent fashion as determined by pDNA-PK (S2056) expression. Increasing p-ATM (S1981) expression was observed in a dose-dependent fashion in QGP-1 cells, but not in BON cells. In addition, p-ATR (S428) expression was not significantly different in either cell line. (iv) Triapine treatment alone or in combination with 2 and 4 Gy XRT resulted in dose-dependent decrease in cyclin D1 expression in both cell lines, however, no significant increase in cleaved PARP expression at 24 and 48h. Conclusion: Our results demonstrated rapid and dose dependent decrease in cyclin D1 expression after Triapine treatment, dose-dependent activation of DNA-PK, the key component of the non-homologous end joining pathway and significant increase in the radiosensitivity of each of the tumor cell lines. In conclusion, our findings provide a rationale for inclusion of Triapine as a radiosensitizer in combination with PRRT in treating NETs. Citation Format: Zeta Chow, Aman Chauhan, Lowell Anthony, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6269.