Endocrine Abstracts

Background

Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of adrenocorticotropic hormone (ACTH) from an anterior pituitary corticotroph adenoma. Clinical manifestations include diabetes, hypertension, osteoporosis, and depression. If untreated, CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke or raised vulnerability to infection. Currently, transsphenoidal surgery is considered the first-line treatment but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest.

Aims

To investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH-encoding POMC mRNA thereby reducing secretion of the hormone. To transfect mouse AtT20 cells (cells that secrete high levels of ACTH) with ASOs against POMC at varying doses to determine which is the most effective at reducing ACTH secretion.

Methods

An AtT20 cell line that secretes high levels of ACTH was used as the in vitro model system. ASOs were designed to specifically target exon 3 of the POMC gene. Transfection of AtT20 cells was carried out using Lipofectamine, and secreted ACTH levels were determined by immunoassay. Statistical analysis was done using ANOVA; P values < 0.05 considered significant.

Results

ASOs that targeted POMC exon 3 (ASO-2 and ASO-3) were transfected into AtT20 cells at 10 and 100 nM. Control ASOs were ASO-1 (matched to POMC sense strand) and ASO-4 (a scrambled version of ASO-3). Experiments included untreated AtT20 cells and AtT20 cells treated with transfection reagent or ASOs alone. The results of six experiments indicated that ACTH secretion from AtT20 cells was reduced after transfection with ASO-2 and ASO-3 at 100 nM (ANOVA, P = < 0.05) and 10 nM (ANOVA, P < 0.05) when compared with untreated AtT20 cells. ASO-1 and ASO-4 had no effect on ACTH secretion by AtT20 cells (ANOVA, P > 0.05).

Conclusions

ASOs against POMC can reduce ACTH secretion from AtT20 cells and may be useful as a novel therapy for CD.