Submitted on July 28, 2006
Accepted on April 2, 2007
Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway
Patric Delhanty1*, Peter M van Koetsveld1, Carlotta Gauna1, Bedette van de Zande1, Giovanni Vitale1, Leo J Hofland1, and Aart Jan van der Lely1
1 Internal Medicine, Erasmus Medical Centre, Rotterdam, ZH, Netherlands
* To whom correspondence should be addressed. E-mail: p.delhanty{at}erasmusmc.nl.
Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through GHS-R1a. Consequently, it was of interest to us to determine if acylated-ghrelin and its predominant serum isoform, unacylated-ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated-ghrelin in the adrenocortical carcinoma cell-lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHS-R1a, and CRF-R2. Acylated and unacylated-ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated-ghrelin in SW-13 cells was blocked by [D-Lys3]GHRP6, but a CRF-R2 antagonist had no effect on unacylated-ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated-ghrelin suppress the sub-G0/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase 3 and 7 activity in SW-13 cells confirmed that acylated and unacylated-ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA, secrete ghrelin, and [D-Lys3]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated-ghrelin are potential auto/paracrine factors acting through an anti-apoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated-ghrelin stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated-ghrelin is acylated prior to its action, or that ghrelin, unacylated-ghrelin and [D-Lys3]GHRP-6 bind to a novel receptor in these cells.
