Abstract NTOC-086: PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS
Authors: Crosley Powel , Agopsowicz Kate , Pihlajoki Marjut , Heikinheimo Markku , Färkkilä Anniina , Hitt Mary 
Published: June 2017

BACKGROUND: Granulosa cell tumour (GCT) constitutes ~5% of ovarian neoplasms. Surgery remains the primary treatment modality due to a generally poor response to chemotherapy in GCT patients.

Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from the Caspase-3 (CASP3) zymogen allowing CASP3 to automature and execute apoptosis. PAC1 has demonstrated safety and efficacy in vivo against several cancers and is currently in Phase I trials for advanced malignancies.

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptosis ligand that can bind membrane-bound death receptors. This binding triggers the extrinsic apoptotic pathway resulting in activation of CASP3 to execute its proteolytic role and programmed cell death. TRAIL has been found to induce apoptosis selectively in cancer cells in vitro and in vivo, and has been well tolerated in human patients.

We hypothesise that combining PAC1 activation of CASP3 with induction of apoptotic signaling by exogenous TRAIL will significantly heighten biologic effect, reduction of disease, and that these effects will be obtained at doses lower than those required by either agent alone.

Here we report on in vitro experiments in support of this hypothesis, suggesting that combining PAC1 with TRAIL may be effective therapy for treatment of GCT.

METHODS: The GCT cell line KGN was treated in vitro with a 6-log range of PAC1 concentration for 48 hours to establish a dose-response curve (using a real-time cell analyzer, RTCA). In parallel, KGN cells were treated with a 6-log range of TRAIL concentration to establish its dose-response curve. Calculated EC50 values were then used for both PAC1 (20 μM) and TRAIL (10 ng/mL) to evaluate the biologic response of simultaneous treatment with PAC1 and TRAIL, and TRAIL delayed 24 hours after PAC1 treatment (using resazurin viability assay and RTCA). Separately, cells from fresh primary and recurrent tumour samples were cultured in vitro for 5 days, then treated with PAC1 (20 μM), TRAIL (10 ng/mL), or the combination, and finally assayed for viability and caspase 3/7 activity 48 and 72 hours later.

RESULTS: Dose-response assays indicate treatment with PAC1 strongly reduces viability of KGN cells compared to untreated control (p&lt;0.05) in a dose-dependent manner and treatment with PAC1 alone significantly reduced viability compared to untreated control (p&lt;0.05). Similar assays with TRAIL only reduced viability of KGN cells at the highest concentration tested (1 µg/mL). The assays also suggest a ~24 hour delay in PAC1 reduction of GCT viability while TRAIL appears to display a time-limited response. Combination treatment was assessed using calculated EC50 concentrations for both PAC1 (20 µM) and TRAIL (10 ng/mL). Assays for both KGN and patient-derived primary GCT cells tested each drug alone, both drugs applied concurrently and TRAIL applied 24 hours after PAC1. Combination of PAC1 with TRAIL was dramatically more cytotoxic than TRAIL or PAC1 treatment alone (p&lt;0.05)

CONCLUSION: Combining CASP3 activator PAC1 with apoptosis-inducing agents may be an effective strategy for treatment of GCT and warrants preclinical assessment.

Citation Format: Powel Crosley; Kate Agopsowicz; Marjut Pihlajoki; Markku Heikinheimo; Anniina Färkkilä; Mary Hitt. PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-086.