Endocrine Abstracts (2002) 3 P151

Ghrelin, but not GHRP-6 or hexarelin increases proliferation of colonic epithelium via effects on c-myc mRNA

S Khalaf1, BW Ogunkolade1, PA Kelly1, PD Fairclough2, SA Bustin3 & PJ Jenkins1

1Department of Endocrinology, St. Bartholomew's Hospital, London, UK; 2Department of Gastroenterology, St. Bartholomew's Hospital, London, UK; 3Academic Department of Surgery, London Hospital Medical College, London, UK.

Background: We have previously demonstrated colonic expression of the endogenous GH secretagogue, ghrelin, but not the GHS-R, and that ghrelin exerts proliferative effects on colonic epithelial cells. However, whether these are specific to ghrelin, or which genes are affected in response is uncertain.

Aims: Determine the specificity of the proliferative effects of ghrelin on colonic epithelial cells, and effects on mRNA levels of the cancer associated genes, c-myc and Cox-2.

Methods: Cultured human HT-29 cells were incubated in 96 well plates (initial density 104 cells) for upto 72 hours in serum free media with (10 -12 to 10 -7 M) (i) ghrelin, (ii) des-ghrelin (non-octanoylated), (iii) GHRP-6 or (iv) hexarelin. Media only wells were controls. Cell number was assessed by MTS assay. Biopsies of fresh normal colon were obtained at colonoscopy from 5 patients. All gave consent. The biopsies were cultured in serum free media with and without 10 -9 M ghrelin for 60 and 120 minutes. RNA was extracted and mRNA levels (copy no. per microgram of total RNA) of the genes quantitated by real-time RT-PCR.

Results: After 24 hours, 10 -11 and 10 -10 M ghrelin resulted in a 37 and 32% increase in cell no. compared to media only (p<0.005 and 0.05 respectively). There was no effect on cell no. of des-ghrelin, GHRP-6 or hexarelin at any concentration. Ghrelin resulted in increased mRNA levels of c-myc in all 5 samples: mean % increase in copy number from baseline compared to media only at 60 and 120 minutes of 212% v. 135%; p<0.05 and 233% v. 172%; p<0.03 respectively. There was no effect on Cox-2 mRNA levels.

Conclusions: The proliferative effects of ghrelin on colonic epithelium are specific and do not apply to other GH secretagogues or involve the GHS-R 1a. Increased PCNA mRNA supports the increased proliferation which may be mediated by concomitant changes in expression of c-myc. Whether these novel effects of ghrelin are GH dependent remain to be determined.

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