THE EFFECT OF NOVEL GHRELIN RECEPTOR AGONISTS ON HEK-293 CELL GROWTH

Robert Tolhurst1, Sylvia Els2, Annette Beck-Sickinger2, Birgitte Holst3, Thue Schwartz3, Helen Cox1. 1Wolfson Centre for Age-Related Diseases, King's College London, SE1 1UL, London, UK, 2Institute of Biochemistry, Leipzig University, 04103, Leipzig, Germany, 3Department of Neuroscience and Pharmacology, Copenhagen University, 2200, Copenhagen, Denmark

Ghrelin receptor 1a (GHSR1a) mediates the effects of ghrelin, a major hunger-inducing peptide that is produced mainly in the stomach. GHSR1a has high constitutive activity, being able to increase IP3 levels and activate both serum and CREB response-element reporter assays in cells in the absence of ghrelin. Ghrelin treatment increases activation of these signaling pathways and novel agonists, such as wFw-Isn-NH2 (a partial agonist) and KwFwLL (an inverse agonist) have been used to further study GHSR1a signaling, with an aim to develop anti-obesity therapies without side effects (Sivertsen et al. 2011).

Ghrelin can increase the growth of different types of cancer cell lines, via mitogen activated protein kinase (MAPK) activation (Chopin et al. 2011). Therefore, we aimed to use two different cell lines, 1) HEK-293 (which have endogenous levels of GHSR1a and ghrelin) and 2) HEK-GHSR1a (HEK-293 cells stably transfected with GHSR1a) in a sulphorhodamine B growth assay (Vichai & Kirtikara, 2006) to study the effects of novel GHSR1a agonists and an antagonist on growth.

HEK-GHSR1a cells showed significantly increased growth when treated with serum (ANOVA, P < 0.001), compared to HEK-293 cells (but not when cells were grown in the absence of serum). We found that ghrelin increased growth in a concentration-dependent manner in HEK-293 and HEK-GHSR1a cells (EC50 0.25 (0.04 – 1.58) and 0.03 (0.004 – 25.84) nM respectively) and this was MAPK mediated as the MAPK inhibitor, PD98059, inhibited 1 nM ghrelin-induced growth significantly (P < 0.01) at 1µM (and with IC50 values of 0.34 and 0.46 µM, respectively).

Surprisingly, we found that the inverse agonist KwFwLL increased growth in both cell lines (EC50 0.1 (0.02 – 0.59) nM and 7.76 (0.99 – 60.66) pM respectively), with similar efficacies to that of ghrelin. However, the partial agonist wFw-Isn-NH2 only increased growth significantly in HEK-293 cells (EC50 <0.01 (0.002 – 0.02) pM) and exhibited parital agonism (at 10-14 - 10-12 M) in stably transfected cells. Furthermore, a novel GHSR1a antagonist per se also increased growth in HEK-293 cells (EC50 6.4 (1.2 – 34.0) pM) and in HEK-GHSR1a cells (EC50 1.73 (0.6 – 5.02) pM). Taken together this data demonstrates a very different ligand pharmacology in terms of growth response, from their original classification as determined by IP3 accumulation.

Supported by: EU 7th Framework Programme, Grant Agreement: no.223057 (GIPIO).

Chopin L, Walpole C, Seim I, Cunningham P, Murray R, Whiteside E et al. (2011). Ghrelin and Cancer. Mol Cell Endocrinol 340: 65-69.

Sivertsen B, Lang M, Frimurer TM, Holliday ND, Bach A, Els S, et al. (2011). Unique interaction pattern for a functionally biased ghrelin receptor agonist. J Biol Chem 286: 20845–20860.

Vichai V & Kirtikara K (2006). Sulphorhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols 179: 1112-1116.