Oral presentations

Abstract

Introduction: We have previously utilized proteomic and immuno-histochemical data to validate that high levels of acid ceramidase (AC) expression confers poorer neoadjuvant chemoradiotherapy response in rectal cancer. Biological (siRNA) AC knockdown improved radiosensitivity in-vitro. We aimed to assess the effect of AC plasmid over-expression and the radiosensitizing potential of carmofur, a 5-FU derivative, for locally advanced rectal cancer. Method: Cells were transfected with 2 μg AC plasmid, siRNA and lipofectamine control. Optimal carmofur dosing using standard ELISA activity assays with DMSO control was established in multiple colorectal cancer cell lines to achieve siRNA comparable AC knockdown. Western blotting confirmed altered expression of AC. Standard clonogenic assays assessed cell survival following increasing irradiation. Result: Plasmid overexpression increased radio-resistance across cell lines. ELISA revealed reduced expression of AC to 10% with 8 μm concentration of carmofur in LIM-1215, 16% with 4 μm HCT-116, 24% 8 μm HT-29. Carmofur clonogenic assays demonstrated reduced colony formation efficiency (colonies/number of cells plated–CFE) and improved radiosensitivity across cell lines. HCT 116 showed 0.438(CFE) control v 0.183(CFE) carmofur at 1Gy, 0.140(CFE) control v 0.076(CFE) at 2Gy (p value= 0.000563). Conclusion: Plasmid over-expression of AC increases radio-resistance compared to control and siRNA inhibition in-vitro using these cell lines. Initial work demonstrates that pharmacological inhibition of AC with carmofur produces comparative radiosensitizing effects in-vitro to siRNA with these cell lines. This work further solidifies acid ceramidase as a potential therapeutic biomarker, however further work is needed to recapitulate these findings in 3D-models and ultimately in-vivo to establish a translatable clinical role in this setting. Take-home message: Our work aims to enable optimisation of neoadjuvant radiotherapy for patients with locally advanced rectal cancer by personalising treatment according to tumour protein consistency.