Click to download
| 207P London, UK Pharmacology 2016 |
RGL1 as a novel target for the anti-cancer effects of celecoxib
Introduction: Cyclo-oxygenase (COX)-2 inhibitors such as celecoxib are widely used anti-inflammatory drugs which also have the potential to prevent and treat cancer especially colon cancer. However, the use of these drugs in cancer has been limited by concerns over the cardiovascular toxicity they produce. Looking for safer ways to target this pathway, we have recently identified ral guanine nucleotide dissociation stimulator-like 1 (RGL1), as a novel target of COX-2 which is down-regulated in tissue from COX-2 knockout mice. RGL1 is key a component of the highly oncogenic Ras/Ral pathway suggesting that Rgl1 down-regulation could play a role in the anti-cancer effects of COX-2 inhibitors. Here we have examined whether pharmacological COX-2 inhibition by celecoxib can regulate Rgl1 expression in human colon cancer cells and whether this is associated with its anti-cancer effects.
Methods: Caco2 human colon cancer cells cultured in DMEM media were treated with celecoxib (3-100μM) for 24-96hrs. RGL1 gene expression was measured by qPCR using TaqMan expression assays and normalised to 18S and GAPDH housekeeping genes. Cell viability was determined using alamarBlue metabolic activity assay and confirmed by annexin-V staining for apoptosis. Data are given as mean±SEM for n independent experiments and considered statistically (*) where p<0.05 by one-way ANOVA or Student’s t-test.
Results: In Caco2 colon cancer cells, pharmacological COX-2 inhibition with celecoxib (100μM) produced RGL1 down-regulation (Figure 1a; n=4). The maximum effect was seen within 24 hrs and at the lowest concentration tested (3μM; n=4) with no further down-regulation seen up to 100μM (Figure 1b). Celecoxib also showed anti-cancer effects in these cells where it reduced viability (Figure 1c,d) and increase apoptosis (vehicle: 3.2±2.8cells/field; celecoxib 100μM: 34.5±12.2cells/field; p=0.002). However, the time course and concentrations required for these effects were different to those required for RGL1 down-regulation, with a maximum effect of celecoxib on cell viability apparent at 96 hrs (Figure 1c; n=10) and requiring higher concentrations of celecoxib (no effect at 3uM; -logIC50: 4.4±0.2; Figure 1d; n=10).
Figure 1: Effect of celecoxib on Rgl1 expression and cell viability in Caco2 colon cancer cells.
Conclusions: These data demonstrate that pharmacological COX-2 inhibition can produce down-regulation of RGL1 expression in human colon cancer cells, validating our previous observations in gene knockout mice. However, differences in the time-course and concentration of celecoxib required for RGL1 down-regulation versus its anti-cancer effects may suggest that the mechanism of cancer cell killing is not solely dependent on changes in RGL1 expression.