Endocrine Abstracts

Background: In case of inoperable disease or tumor recurrence, there are only a few medical treatment options for patients with adrenocortical carcinoma (ACC). The oral alkylating drug Temozolomide (TMZ) shows efficacy in a subset of patients with melanoma, glioblastoma or pancreatic neurendocrine tumours. Response to TMZ seems determined by the tumoral expression level of the DNA repair enzyme O(6)-Methylguanine-DNA methyltransferase (MGMT).

Aims: To evaluate the effects of TMZ on human ACC.

Methods: The human ACC cell lines H295, HAC-15 and SW13 were treated with increasing concentrations of TMZ (range: 1–100 μM). Cells were harvested for determination of cell number (after 72 and 144 h of incubation) or for DNA fragmentation (apoptosis; after 72 h). By Colony Forming Assay (CFA) we determined the effect of TMZ on surviving fraction (SF) and colony size (CS). MGMT gene promoter methylation was determined with Cobra technique in the cell lines, 4 normal adrenals (NA) and 16 ACC. Expression of MGMT mRNA was determined by RT-qPCR (mRNA) and by Immunohistochemistry (protein) in the cell lines, 4 NA and 7 ACC.

Results: H295, HAC-15 and SW13 cell lines showed a time- and dose-dependent inhibitory response to TMZ (EC50 17.6±1.0 μM; 17.0±1.1 μM and 41.6±1.1 μM, resp.). In addition, TMZ induced a dose-dependent stimulation of DNA-fragmentation. CFA showed that TMZ decreased SF in H295, HAC15 and SW13 (−85±8%, −82±10% and −57±12%, resp). CS was significantly decreased in H295 and SW13 (−45±7% and −31±10%, resp.). Partial methylation of the MGMT promoter was found in the 3 cell lines, in 1/4 NA and in 3/16 ACC. MGMT mRNA expression was present in all cell lines, NA and in 6/7 ACC. The cell lines expressed MGMT protein homogeneously, with the strongest staining in the SW13 cells. NA and 3/7 ACC expressed MGMT in a heterogeneous manner.

Conclusions: ACC variably express MGMT mRNA and protein. TMZ, at pharmacological concentrations, is effective in ACC cell lines expressing MGMT, suggesting that other factors determine sensitivity to TMZ as well. However, the cell line with the highest MGMT mRNA and protein level showed the lowest sensitivity to TMZ. One of the effects of TMZ on ACC cell growth is the induction of apoptosis.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.