In vitro development and validation of a non-invasive ¹³C-stable isotope assay for ornithine decarboxylase

Oesophageal cancer is a significant cause of cancer related mortality, with increasing incidence worldwide. Ornithine decarboxylase (ODC) is an enzyme involved in polyamine synthesis and cellular proliferation, and ODC expression and activity has been implicated as a prognostic marker of oesophageal cancer. This study aimed to develop and optimise an in vitro ¹³C-stable isotope assay for ODC activity as a non-invasive marker of oesophageal cancer. Experiments were performed in triplicate (n = 3/group/cell line) using Caco2, HeLa, Flo-1, OE33, TE7 and OE21 cell lines (colorectal, cervical, oesophageal adenocarcinoma and oesophageal squamous carcinoma respectively). Following addition of 2mM ¹³C-ornithine to cells, 10 ml gas samples were collected from the headspace every 20 min for a total of five hours. Gas samples were analysed using isotope ratio mass spectrometry to quantify ¹³CO₂. Assay specificity was determined using the selective ODC inhibitor, N-(4′-Pyridoxil)-Ornithine(BOC)-OMe (POB). All data is expressed as δ ¹³CO₂ from baseline. High ODC activity was detected by ¹³C-ornithine assay in Caco2 (32.00 ±< 1.12 δ ¹³CO₂) in contrast to HeLa cells (5.44 ± 0.14 δ ¹³CO₂) cells. POB inhibited activity in Caco2 cells to 12.87 ± 1.10 δ ¹³CO₂. Differential ODC activity was detected in all oesophageal cancer cells, and 53 h incubation of cell lines with POB reduced activity by 72%, 56%, 64% and 69% in the Flo-1, OE33, OE21 and TE7 cell lines respectively. We have shown that ODC activity can be selectively detected by a non-invasive, stable-isotope ¹³C-ornithine assay. ODC activity was detected in all oesophageal cancer cell lines in vitro. Further studies are indicated to quantify ODC activity in oesophageal cancer patients.