Sensitivity of Human and Murine Colonic Tumour Cell Lines to Gamma-aminobutyric Acid (GABA) In Vitro

P FERNANDES1, N Hyland2, A Houston1. 1Department of Medicine, University College Cork, Cork, Ireland, 2Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland

Introduction: Of the many types of cancer, colorectal cancer ranks as the third most common type and is the third leading cause of cancer-related death worldwide. Gamma-aminobutyric acid (GABA) has recently emerged as having potential tumour suppressive effects, with the strongest evidence in favour of an anti-cancer effect found in colon cancer, suggesting that increasing the levels of GABA in the gut may have anti-cancer properties. However, whether GABA exerts a direct effect on colonic tumours is unclear. Therefore our aim was to investigate whether GABA, in a concentration-dependant manner, alters human and murine tumour cell proliferation in vitro, in addition to examining the effects of GABA on tumour cell migration and invasion.

Methods: SW480 and CT26 cells were cultured under standard conditions and were pretreated with GABA and 24 hrs later cell proliferation was measured by resazurin reduction. For migration cells were seeded in serum-free media in Transwell inserts (8µM pore size). Inserts were placed into 24-well plates containing cell culture media (10% serum), incubated for 16hrs in the presence of GABA and migration assessed using a Millipore Colorimetric Migration assay. For invasion inserts were coated with Matrigel and invasion assessed. Data are expressed as mean +/− SEM and represent a minimum of three separate experiments. Statistical differences were determined using a one-way ANOVA. All tests were performed using GraphPad Prism 5 statistical software.

Results: In the human adenocarcinoma cell line, SW480, GABA in a concentration-dependant (5µM - 100µM) manner reduced tumour cell proliferation (P < 0.001). GABA also reduced tumour cell migration by up to 60% at all concentrations tested. Moreover, at 100 µM, GABA reduced the invasive capacity of SW480 cells by 20%. In the murine CT26 colon carcinoma cell line, GABA significantly reduced tumour cell proliferation in the concentration range 2.5 µM - 10 µM, with proliferation returning to control (untreated) levels at the highest concentration of GABA examined (100 µM). As in human adenocarcinoma cells, we observed a 40-50% reduction in the migration of CT26 cells treated with GABA (1 µM - 100 µM). We have also confirmed the expression of GABA B1a, GABA B1b and GABA B Br2 in CT26 cells.

Conclusions: These data demonstrate that exogenous GABA exerts a direct anti-tumorigenic effect on key parameters of tumorigenesis including proliferation, migration and invasive capacity. Furthermore these data would suggest that GABA is exerting a direct effect through interaction with the tumour cells. Therefore these data would suggest that development of novel mechanisms for delivery of GABA to the gut may be therapeutically useful in the treatment of colon cancers.