Y. R. Somnay1, S. Lubner2, H. Gill1, B. Matsumura1, H. Chen1 1University Of Wisconsin,Endocrine Surgery Research Laboratories, Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Human Oncology,Madison, WI, USA
Introduction: Carcinoids are slow-growing neuroendocrine tumors that often present insidiously with few options for medical management. Monoagent DNA-alkylating chemotherapies such as Dacarbazine (DTIC) are among a paucity of therapies for these tumors, but are limited by host toxicity and intrinsic chemoresistance through the base excision repair (BER) pathway via the activity of poly (ADP-ribose) polymerase (PARP). Inhibitors of PARP are thought to potentiate DNA-damaging chemotherapies by blocking cancer cells’ ability to repair DNA double strand breaks following base excision repair. Here we show that the PARP inhibitor ABT-888 (veliparib) enhances the cytotoxic effects of DTIC in a pancreatic carcinoid cell line and potentiates DNA damage, cell cycle arrest and apoptosis.
Methods: Human pancreatic carcinoid cells (BON) were incubated in ABT-888 (0-20µM), DTIC (0-400µM) or both, for 96 hours. Cell growth was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. Western analysis showed expression levels of Chromogranin A (CgA) a well characterized marker for carcinoid malignancy, in addition to poly(ADP)-ribose polymerase (PARP), cleaved caspase 3, cyclin family kinases, and phosphorylated and total ataxia and telangiectasia mutated kinase (ATM), H2A histone family member X (H2AX), p53 and retinoblastoma gene (Rb). Flow cytometry using propidium iodide staining was used to assess cell cycle kinetics.
Results: Cell survival after treatment with ABT-888 and DTIC was reduced synergistically as combination indices (CI) fell below 1 on the Chou-Talalay scale, as we've previously reported. Notably, ABT-888 alone was non-toxic at therapeutic doses. ABT-888 administered prior to varying DTIC doses reduced CgA expression beyond the combined effect of either drug alone. Of note, the degree of ATM and H2AX phosphorylation, indicative of double strand DNA breaks, was enhanced by ABT-888 prior to DTIC treatment, suggesting BER pathway attenuation via PARP inhibition. This was corroborated by an increase in phospho-p53, a tumor suppressor activated by DNA damage, alongside reductions in cyclin D and downstream inactive phospho-Rb. ABT-888 treatment prior to DTIC decreased levels of cyclin A expression, and accordingly augmented G2 and G0-G1 phase arrest, indicating an enhanced transition into apoptosis. Finally, ABT-888 potentiated DTIC-induced cleavage of terminal apoptotic markers caspase 3 and PARP relative to the effect exerted by either monotherapy.
Conclusion: ABT-888 synergizes with DTIC to suppress carcinoid growth and phenotype. By targeting PARP activity, ABT-888 may impair BER pathway response to DTIC, inducing a futile cycle of persistent DNA strand breakage and potentiating cell cycle arrest and apoptosis through p53 by enhancing Rb hypophosphorylation. By targeting DNA repair pathways that confer cancer cell resistance, ABT-888 may help treat carcinoids that remain refractory to mainstay therapies.