Antineoplastic effects of decitabine, an inhibitor of DNA promoter methylation, in adrenocortical carcinoma cells

Abstract

Hypotheses: Decitabine recovers expression of silenced genes on chromosome 11q13 and has antineoplastic effects in adrenocortical carcinoma (ACC) cells.

Design: NCI-H295R cells were treated with decitabine (0.1-1.0 microM) over 5 days. Cells were evaluated at 24-hour intervals for the effects of decitabine on ACC cell proliferation, cortisol secretion, and cell invasion. Expression was quantified for 6 genes on 11q13 (DDB1, MRPL48, NDUFS8, PRDX5, SERPING1, and TM7SF2) that were previously shown to be underexpressed in ACC.

Setting: Academic research. Study Specimen Human ACC cell line.

Main outcome measures: Adrenocortical carcinoma cell proliferation, cortisol secretion, and cell invasion were measured using immunometric assays. Quantitative reverse transcription-polymerase chain reaction was used to measure gene expression relative to GAPDH.

Results: Decitabine inhibited ACC cell proliferation by 39% to 47% at 5 days after treatment compared with control specimens (P < .001). The inhibitory effect was cytostatic, time dependent, and dose dependent. Decitabine decreased cortisol secretion by 56% to 58% at 5 days after treatment (P = .02) and inhibited cell invasion by 64% at 24 hours after treatment (P = .03). Of 6 downregulated genes on 11q13, decitabine recovered expression of NDUFS8 (OMIM 602141) (P < .001) and PRDX5 (OMIM 606583) (P = .006).

Conclusions: Decitabine exhibits antitumoral properties in ACC cells at clinically achievable doses and may be an effective adjuvant therapy in patients with advanced disease. Decitabine recovers expression of silenced genes on 11q13, which suggests a possible role of epigenetic gene silencing in adrenocortical carcinogenesis.

Figure 1

Effects of decitabine on cortical secretion in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cell counts were measured using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Compared with control specimens, decitabine caused time-dependent (A) and dose-dependent (B) inhibitory effects on adrenocortical carcinoma cell proliferation, with significant 39% (*P=.02) and 47% (†P< .001) decreases in cell counts observed at 5 days after treatment. Serum (10%) is Nu-Serum (BD Biosciences, San Jose, California).

Figure 2

Effects of decitabine on cortisol concentration in NCI-H295R cells (ATCC, Rockville, Maryland). Cells were treated with decitabine (in dimethyl sulfoxide [DMSO] vehicle) at 0.1μM and 1.0μM doses for 5 days, and cortisol concentrations in cell media were measured using an immunometric assay kit (Parameter; R&D Systems, Minneapolis, Minnesota). Media taken from decitabine-treated cells had decreased cortisol concentration over time (A), with significant decreases at 5 days after treatment of 58% (*P=.001) and 56% (†P=.02) for the 0.1μM and 1.0μM doses, respectively (B). The inhibitory effect of decitabine on cortisol concentration per cell (C) was significant only with the lower 0.1μM dose (‡P=.03).

Figure 3

Effects of decitabine on NCI-H295R cell (ATCC, Rockville, Maryland) invasion. Cells were starved of serum for 48 hours and then placed in a modified Boyden chamber for 24 hours. Cells that invaded through the extracellular matrix barrier were counted using an assay kit (CyQUANT; Invitrogen, Carlsbad, California). Decitabine had a dose-dependent inhibitory effect on NCI-H295R cell invasion, with a 64% decrease in cell invasion using the 1.0μM dose (*P=.03). DMSO indicates dimethyl sulfoxide.