Abstract
Context NCI-H295 cells are the most widely used model for adrenal
steroidogenesis and adrenocortical carcinoma and have been used for decades in
laboratories worldwide. However, reported steroidogenic properties differ
considerably.
Objective To evaluate heterogeneity of steroidogenesis among NCI-H295
cell strains, clarify the influence of culture media and test response to
inhibitors of steroidogenesis by using liquid chromatography tandem mass
spectrometry (LC-MS/MS).
Methods NCI-H295 cells were obtained from two cell banks and cultivated
in different media. An LC-MS/MS-based panel analysis of thirteen
steroids was adapted for cell culture supernatant. Cells were treated with
metyrapone, abiraterone and mitotane.
Results Mineralocorticoid synthesis was strongly affected by passaging as
reflected by reduction of aldosterone secretion from 0.158±0.006 to
0.017±0.001 µg/106 cells
(p<0.05). Relevant differences were also found for cells from two
vendors in terms of aldosterone secretion (0.180±0.001 vs.
0.09±0.002 µg/106 cells,
p<0.05). Selection of medium strongly impacted on cortisol secretion
with>4-fold difference (40.6±5.5 vs.
182.1±23 µg/106 cells) and
reflected differential activation of the glucocorticoid pathway. Exposure to
abiraterone, metyrapone and mitotane resulted in characteristic steroidogenic
profiles consistent with known mechanism of drug action with considerable
differences in metabolites upstream of the blocked enzyme.
Conclusion We demonstrate that steroid hormone secretion in NCI-H295
cells is strongly affected by the individual strain, passage and growing
conditions. These factors should be taken into account in the evaluation of
experiments analyzing steroid parameters directly or as surrogate parameters of
cell viability.
Key words
Steroid hormones - glucocorticoids - androgens - adrenocortical carcinoma - cell culture
