Endocrine Abstracts

Background: Previously we have demonstrated expression of GH, IGF-1 and ghrelin, but not GHRH, in normal and malignant breast tissue. In vitro studies have suggested an inhibitory effect of ghrelin on proliferation of breast cancer cell lines. However the use of cell lines and dose of ghrelin may not represent physiological conditions.

Objectives: (1) To establish a viable method for culturing fresh breast tissue explants, (2) to use this to investigate the effects of exogenous ghrelin on local expression of GH, IGF-I, PCNA and c-myc (two tumour associated genes).

Methods: 6 paired samples of normal and malignant breast tissue were obtained at surgery. 10-20mg explants were individually cultured in serum-free media for up to 96 hours. Tissue viability and cell death were assessed every 24 hours by colorimetric MTS and LDH assays respectively. With conditions optimised, exogenous ghrelin (10^-9M) was added to 5 subsequent paired breast samples and incubated for 4 and 24 hours. Total RNA was extracted and mRNA expression quantitated by RT-PCR (Taqman) assay.

Results: Over 96 hours, 5 of 6 paired normal and malignant samples showed a mean increase in MTS absorbance of 53%, and LDH release remained stable in all samples. Ghrelin had no effect on GH mRNA levels but down-regulated IGF-1 mRNA levels from a median of 1.4x10⁶ copies/micrograms total RNA to zero expression after 24 hours in 4 of 5 paired samples. Its effects on PCNA and c-myc mRNA expression showed interpatient variability, but were consistent within the same individuals.

Conclusion: We have established a novel physiologically relevant system for culture of breast tissue explants for up to 96 hours. Using this system ghrelin exerts direct transcriptional effects on breast tissue, the most consistent of which is marked down-regulation of IGF-I. This may explain the previous reports of its inhibitory effects on cell proliferation.