Mitotane cytotoxicity and metabolism after CYP11B1 modulation in H295R cell line.

<p>(A) Mitotane cytotoxicity was not influenced by CYP11B1 interference (p = ns, comparing IC50 doses of CYP11B1 siRna cells and non-targeting siRna control cells). Three replicate wells for each experiment (n = 2) were used to determine each data point. (B) CYP11B1 modulation did not influence uptake of mitotane and its metabolization (p = ns, comparing drug levels of CYP11B1 siRna cells and non-targeting siRna control cells). Four independent experiments were used to determinate each data point, measured in HPLC-UV.</p

CYP11B1 expression after treatment with mitotane and metyrapone, or both.

<p>Data obtained from two independent experiments with two replicates for each experiment, and expressed as fold changes compared to β-actin expression (2-ΔΔCt). A > 2 fold increase was considered significant.</p

Efficiency of siRna transfection and silencing.

<p>(A) H295R cells transfected with a fluorescent siRna indicator. (B) CYP11B1 gene expression in CYP11B1 siRna cells and non-targeting siRna cells. Data obtained from two independent experiments with two replicates for each experiment. (C) Cortisol levels in CYP11B1 siRna cells and non-targeting siRna control cells. Four independent experiments were used to determine each data point.</p

Effects of mitotane and 20 μM metyrapone on H295R cell viability.

<p>Three technical replicate wells for each experiment (n = 3) were used to determine each data point. P = ns comparing mitotane treatment vs. combined treatment with mitotane and metyrapone.</p

Mitotane metabolites in ACC cells: Uptake, bioavailability and metabolization.

<p>Intracellular and extracellular concentration of mitotane and its metabolites in H295R, measured by means of HPLC–UV after metyrapone treatment. Six independent experiments were used to determinate each data point.</p

Effects of 25 μM mitotane and 20 μM metyrapone on cortisol levels.

<p>Four independent experiments were used to determine each data point. Metyrapone or mitotane treatment vs. untreated cells, P <0.05. Combined treatment with mitotane and metyrapone vs. untreated cells. P = 0.005.</p

Impact of combined CYP2W1 immunohistochemistry and initial ENSAT tumor stage on clinical outcome in patients affected by adrenocortical carcinoma treated with mitotane only (n = 72, including both adjuvant and palliative intention).

<p>A: Overall survival; B: Time to progression (TTP).</p

Relationship between CYP2W1 immunoreactivity and baseline clinical or pathological characteristics of patients with adrenocortical carcinoma (only tumour samples derived from primary surgery, n = 196).

<p>*P value with Chi-Square test among different response to treatments.</p><p>DFS = disease free survival, TTP = time to progression.</p

Relationship between CYP2W1 immunoreactivity and response to therapy with mitotane only, according to treatment intention.

<p>A: Response to therapy in patients treated in an adjuvant setting (n = 36). B: Objective response to therapy in patients treated in a palliative setting (n = 32).</p

CYP2W1 immunohistochemistry.

<p>Examples of CYP2W1 immunostaining in normal tissue (i.e. liver and adrenal gland) and neoplastic tissues (i.e. lung cancer and adrenocortical carcinoma). A: Staining with a polyclonal antibody from Thermo Scientific (<b>Ab #1</b>); B: Staining with a polyclonal antibody provided by the Karolinska Institute <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105855#pone.0105855-Edler1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105855#pone.0105855-Gomez2" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105855#pone.0105855-Stenstedt1" target="_blank">[23]</a> (<b>Ab #2</b>). Magnification 20X and 10X.</p