Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane

in Endocrine-Related Cancer
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  • 1 Endocrinology, Research Center, Diagnostica Molecolare Avanzata, II Faculty of Medicine, S Andrea Hospital, ‘Sapienza’ University of Rome, 00189 Rome, Italy

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p′-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, α-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-β isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

Abstract

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p′-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, α-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-β isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

Introduction

Mitotane is an adrenocorticolytic drug used for primary treatment and the recurrence of disease in patients affected by adrenocortical carcinoma (ACC; Hahner & Fassnacht 2005). Mitotane acts selectively on the adrenal cortex leading to cell destruction and the impairment of steroidogenesis (Fang 1979, Martz & Straw 1980). At higher concentrations, mitotane produces a dose-related cellular toxic effect with damage on the fasciculata/reticularis areas causing rupture of mitochondrial membranes, but with a minimal effect in the glomerulosa area (Schteingart et al. 1993). It is usually well tolerated in the plasmatic narrow range between 14 and 20 mg/l. Unfortunately, in some cases, its use is limited by a strong toxicity and a relative higher percentage of treated patients show side effects, particularly gastrointestinal and neurological ones (Cai et al. 1997).

Recently, there has been a strong interest in applying proteomics to foster a better understanding of disease processes, mechanisms of action, and new pharmacological drug targets (Hanash 2003). Analyzing the protein expression by comparing the two-dimensional gel electrophoresis patterns of proteomes under different conditions enabled to identify the proteins whose levels significantly vary after treatment with specific compounds. In this study, we have described the effects of mitotane on growth, steroidogenesis, and proteomic profile on H295R cells, a model of ACC able to produce all the adrenocortical steroids (Rainey et al. 1994). Mitotane-induced different expression of proteins involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis. This work represents the first proteomic study performed on an ACC cell line and the effects induced by the main drug used for the treatment of this neoplasia.

Materials and methods

Cell culture and treatments

H295R adrenocortical cells were supplied from the ATCC (Rockville, MD, USA). Cells were cultured in DMEM/HAM'S F-12 and medium supplemented with penicillin/streptomycin 50 U/ml. 24 h post seeding, the cells were treated with mitotane at 10−5 M final concentration. This dose has been used to evaluate the mitotane effect on cell growth and cell cycle at different times (24, 48, 72, 96 and 120 h). Cell viability was evaluated by using trypan blue dye exclusion test.

Cell cycle analysis

The cell cycle was studied by using propidium iodide (PI) staining. Treated and untreated cells were harvested, washed in cold PBS, fixed in 70% ethanol, and stained with a solution containing 50 μg/ml PI (Sigma Chemical) and 75 U/ml RNase (Sigma Chemical) in PBS. Samples were then measured at a different time after mitotane treatment by using a FACScan cytofluorimeter (Becton Dickinson, Sunnyvale, CA, USA).

Steroid determination

Hormone levels were determined in the cell supernatant. Progesterone, testosterone and cortisol were measured by ECLIA (Roche). Aldosterone was measured by immunoenzymatic assay (DiaMetra, Milan, Italy).

Protein extracts

Whole cell pellets were lysed in 0.1% SDS/2.3% DTE. Proteins were then precipitated by adding 20% (v/v) cold acetone and incubating at −20 °C. Protein pellets were dissolved in 8 M urea/4% CHAPS solution. A mitochondria-enriched fraction was obtained using the Mitochondria/Cytosol fractionation Kit (MBL International Corporation, Woburn, MA, USA).

Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was performed as described by Gorg et al. (1988). 60 μg proteins were isoelectrofocused on 18 cm Immobiline DryStrip (IPG strip, Amersham Biosciences) with a 3–10 non-linear (NL) pH gradient. The second dimension electrophoresis was run on 9–16% linear gradient polyacrylamide gels. Gels were silver stained as described by Shevchenko et al. (1996).

Analysis of two-dimensional gels

Gels were scanned on a Bio-Rad GS-800 calibrated imaging densitometer (Bio-Rad) and spot analysis was performed using the Bio-Rad PDQuest software. For each sample, four gel replicates derived from two independent experiments were run. Spot volume was normalized to the total density in valid spots.

Protein identification by MALDI-ToF MS

Protein spots of interest were manually cut out of the gel and destained with 7.5 mM potassium ferricyanide/25 mM sodium thiosulfate solution. After washing in H2O, spots were washed 20 min in 200 mM NH4HCO3, dehydrated with 100% acetonitrile and in gel digested with 0.5 ng/μl trypsin (Trypsin Gold, mass spectrometry grade, Promega). The generated peptides were filtered through micro ZipTip C18 pipette tips (Milllipore, Bedford, MA, USA) and the mass spectra were obtained using a Voyager-DE MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). Peptide mass fingerprinting database searching was performed using MASCOT (Matrix Science) in the NCBInr/Swiss-Prot databases, setting the parameters to allow one missed cleavage for peptide and a mass tolerance of 0.5 kDa.

Western blotting

Cellular lysates were sonicated on ice, clarified by centrifugation and stored at −80 °C. 70 μg of the fractions were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane and incubated with prohibitin (PHB; N-20 Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (AC-15 Sigma), HSP71 and β-tubulin (Upstate Biotechnology), and adrenodoxin reductase (C-15 Santa Cruz Biotechnology Inc.) antibodies. Immunoblots were developed using ECL Kit (Amersham); its quantification was performed by densitometric analysis.

Results

Cell proliferation and treatment

Schteingart et al. (1993) reported that mitotane at 10−5 M concentration inhibits cortisol secretion in H295R cell line, with minimal effects on cell viability. Therefore, we have used this dose to evaluate the mitotane effect on cell growth and cell cycle at different times on these cells. As shown in Fig. 1, the drug induced a moderate cell growth inhibition of about 15% at 72 h after treatment with minimal effect on cell viability. This effect was partially lost during the following hours, decreasing after 120 h. To evaluate whether mitotane could affect cell cycle, FACscan was performed. It demonstrated a reversible delay in G2-phase after mitotane treatment (data not shown).

Figure 1
Figure 1

Effects of mitotane treatment on cell proliferation.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Impairment of steroid hormones secretion in mitotane-treated H295R cells

Since H295R cells are able to secrete several steroid hormones, the measurements of progesterone, testosterone, cortisol, and aldosterone levels were determined in both control and mitotane-treated cells after 24, 48, and 72 h. As shown in Fig. 2 at 24 h the progesterone secretion was inhibited by 26%, reaching 68% at 72 h (s.d. 0.04 ng/ml), testosterone and aldosterone inhibition was 8% and 24% respectively at 24 h compared with control cells. At 72 h, the inhibition percentage increased to 55% for testosterone (s.d. 0.018 ng/ml) and to 49% for aldosterone (s.d. 15 pg/ml). The cortisol level reached 70% of inhibition at 72 h (s.d. 0.12 mcg/dl), in agreement with the mitotane role in cortisol inhibition (Hahner & Fassnacht 2005). These results demonstrated that mitotane exerts significant inhibition on several hormones, probably acting upstream of steroidogenic cascade, as proved by progesterone-reduced level.

Figure 2
Figure 2

Inhibition percentage of steroid hormones synthesis in mitotane-treated H295R cells assayed by the determination of hormone concentration in cell culture medium after 24 h (white bars), 48 h (grey bars) and 72 h (black bars) following drug addition. Androgens, glucocorticoids, and mineralocorticoids levels were respectively inhibited.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Proteomic analysis of H295R cells

Protein separation was obtained by two-dimensional gel electrophoresis of H295R total protein that generated a two-dimensional map in which about 350 spot features are detectable (Fig. 3). The protein spots identified by peptide mass fingerprinting are listed in Table 1.

Figure 3
Figure 3

Proteomic map obtained by the two-dimensional gel electrophoresis of the total protein extract of H295R cells. Spots marked by arrows correspond to the identified proteins listed in Table 1.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Table 1

List of identified proteins in two-dimensional proteomic maps of H295R cells

Spot numberProtein nameSwiss-Prot ANaTheoretical Mr/pIScoreb
1CalreticulinP2779746 466/4.29130
2, 3, 460 kDa heat shock proteinP1080957 962/5.2466
5c, 6cD-3-phosphoglycerate dehydrogenaseO4317556 519.31/6.3186
7Retinal dehydrogenase 1P0035254 730/6.3068
8c, 9cα-enolaseP0673347 037/6.9971
1040S ribosomal protein SAP0886532 722/4.79105
11, 12, 13Actin, cytoplasmic 1P6070941 736/5.29132
14l-lactate dehydrogenase B chainP0719536 507/5.7278
15, 16, 17Fructose-bisphosphate aldolase AP0407539 288/8.3987
18, 19, 20Glyceraldehyde-3-phosphate dehydrogenaseP0440635 922/8.58116
21cProhibitinP3523229 804/5.5787
22c, 23c, 24c, 25cTriosephosphate isomeraseP6017426 823/6.51104
26Rho-GDP-dissociation inhibitor 1P5256523 207/5.0367
27cHeat shock protein β-1P0479222 782/5.98127
28cPeroxiredoxin 6P3004124 903/6.02106
29cPeroxiredoxin 2P3211921 891/5.6693
30Phosphatidylethanolamine binding proteinP3008620 925/7.4382
31Cofilin-1P2352818 371/8.2693
32, 33, 34, 35, 36Peptidyl-prolyl cis-trans isomerase AP6293718 109/99
37cHistidine triad nucleotide binding protein 1P4977313 670/6.4660
38c, 39c, 40cProfilin-1P0773714 923/8.47125
dTubulin β-2 chainP0743749 670/4.78102
dHeat shock cognate 71 kDa proteinP1114270 898/5.3778
dPeroxiredoxin-1 Q0683022 110/8.2782
dHeterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/hnRNP B1)P2262637 429/8.9794
dNucleoside diphosphate kinase BP2239217 298/8.597
dCathepsin D heavy chainP0733926 628/5.5672
dHeat shock 70 kDa protein 1AQ5SP1770 038/5.4810
dAdrenodoxin reductaseP2257053 836/8.7290

Protein scores >60 are significant (P<0.05).

Entry name and accession number according to Swiss-Prot (http://expasy.org).

Protein score represents the probability that the observed match is a random event.

Proteins showing differential expression in proteomic profiles of H295R total extracts following mitotane treatment.

Proteins showing differential expression in proteomic profiles of H295R extracts enriched in mitochondria following mitotane treatment.

Since molecular targets of mitotane and kinetics of cellular response induction to the drug are still largely undefined, a comparative study of proteomic profiles of total cells extracts from mitotane-treated and untreated H295R cells was performed at different times: 15 min, 1 h, 5 h, and 24 h in order to detect early events induced by mitotane. Since the block in steroid synthesis, induced by mitotane, occurs between 24 and 48 h, the sample enriched in mitochondrial proteins was examined at these times in order to detect potential variation of proteins involved in steroidogenesis.

Computer-assisted differential analysis of maps derived from total cell lysates of untreated cells shows that several proteins underwent a time-dependent modulation. It suggests that mitotane affects temporal profile expression of many proteins belonging to different functional classes.

Protein expression changes following mitotane treatment in total cellular extracts

In proteomic two-dimensional maps, derived from crude cell extracts of H295R cells, we identified 18 protein spots, whose expression levels were equal or higher than 1.5-fold, following mitotane treatment in at least one of the examined time point. Fourteen out of the 18 protein spots, corresponding to 9 proteins, were unambiguously identified (Table 2). Some of these proteins appeared in the two-dimensional map as multiple isoforms with changed PIs, thus reflecting different degrees of post-translational modifications. Triose phosphate isomerase protein presented two major isoforms (spots 22 and 25) whose expression appeared constant, and two additional isoforms (spots 23 and 24) were detectable in treated cells already after 1 h, but their level at 24 h was 2.0-fold lower than that of control cells. α-enolase isoforms (spots 8 and 9) showed a 2.0-fold decrease for the most acid isoform (spot 8) after 1 h up to 1.5-fold decrease for the least acid one (spot 9) after 5 h in treated samples. D-3-phosphoglycerate-dehydrogenase (D-3-PGDH) has been identified as two spots. The most acid isoform (spot 5) showed a progressive 2.9-fold increase after 1 h, followed by a significant 3.4- and 1.9-fold reduction after 5 and 24 h respectively, compared with control cells. The least acidic isoform (spot 6) showed a −2.0-fold change after 5 h in mitotane-treated cells, without significant differences in the previous and in the following times. Peroxiredoxin VI (Prx VI) (spot 28) showed an increase of 1.5- and of 1.6-fold 1 and 5 h after drug treatment and the same positive regulation was observed for Prx II (spot 29) at 15 min (3.6-fold), 1 h (7.4-fold), and 5 h (2.2-fold). Heat shock protein-β1 (HSP27; spot 27) underwent an over-expression (+2.2-fold change) at 15 min with a progressive reduction to 24 h in treated samples. Histidine triad nucleotide binding protein (Hint; spot 37) was markedly increased with +8-fold change after 15 min and +3-fold change at 5 h following drug addition. Finally, profilin-1 (spots 38, 39, and 40) showed different temporal expressions. The spot 38, the most acid one, was already detectable at 15 min and was well expressed with 21-fold change at 5 h in treated samples. The expression profile of spot 39 showed a higher expression at 1 h, whereas it was undetectable at 15 min. At last, the spot 40 of profilin-1 showed a higher expression at 15 min. A 1.6-fold reduction of PHB; (spot 21) was observed in treated cells at 15 min and a 3-fold increase at 5 h as the results in western blot analysis (Fig. 4).

Figure 4
Figure 4

Time-dependent expression profile of prohibitin in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Table 2

Differentially expressed proteins after mitotane treatment

Time
Total cellular extracts15 min1 h5 h24 h
Triose phosphate isomerase (spot 23)NE
Triose phosphate isomerase (spot 24)NE
α-enolase (spot 8)
α-enolase (spot 9)
D-3-phosphglicerate dehydrogenase (spot 5)
D-3-phosphglicerate dehydrogenase (spot 6)
Peroxiredoxin VI
Peroxiredoxin II
Heat shock protein β1 (HSP27)
Histidine triad nucleotide binding protein
Profilin (spot 38)NE
Profilin (spot 39)NE
Profilin (spot 40)NE
Prohibitin
Mitochondria-enriched fractions24 h48 h
Tubulin (β isoform II)
Heat shock cognate 71 kDa protein
Peroxiredoxin I
Heterogenous nuclear ribonucleoprotein isoforms A2/B1NENE
Nucleotide diphosphate kinaseNE
Cathepsin D
Heat shock 70 kDa protein 1ANE
Adrenodoxin reductase

The arrow ↑ indicates up-regulated proteins (≥1.5-fold), the arrow ↓ indicates down-regulated proteins (≤1.5-fold), and the arrow ↔ indicates the unchanged level expression proteins. NE acronym indicates unexpressed proteins. Some proteins listed in the table show multiple isoforms.

Protein expression changes following mitotane treatment in mitochondria-enriched fractions

Comparative analysis of two-dimensional maps derived from mitochondria-enriched samples evidenced expression changes of 13 spots following mitotane treatment, 8 of which have been unambiguously identified (Table 2). Most of these proteins are down regulated by mitotane treatment. Tubulin-β isoform II resulted in a 3-fold down expression at 48 h after treatment (Fig. 5A). Heat shock cognate 71 kDa (HSP71) protein expression showed a progressive decrease until a −3-fold change at 48 h in treated cells (Fig. 5B). The drug strongly repressed the peroxiredoxin I (Prx I) of a −2.5-fold change after 24 and 48 h, and completely depleted the heterogenous nuclear ribonucleoprotein isoforms A2/B1 (hRNP A2/B1). Nucleotide diphosphate kinase (NDPK) appeared with −2.5-fold change with respect to control only after 48 h. Instead the proteins showed an increased expression at different times after the following treatments: the cathepsin D with +12-fold change at 48 h, heat shock cognate 70 kDa protein-1A (HSP70) detectable only at 24 h, and the adrenodoxin reductase (AdR) increased 2-fold at 24 h by drug treatment (Fig. 6). The proteomic results of tubulin-β isoform II, heat shock cognate 71 kDa, and adrenodoxin reductase have been confirmed by western blot analysis.

Figure 5
Figure 5

(A) The expression profile of tubulin-β isoform II and (B) heat shock cognate 71 kDa proteins in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts enriched in mitochondria. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Figure 6
Figure 6

Expression profile of adrenodoxin reductase in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts enriched in mitochondria. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

Citation: Endocrine-Related Cancer 15, 1; 10.1677/ERC-07-0003

Discussion

Mitotane is widely used for the treatment of patients affected by ACC (Trainer & Besser 1994, Beacauregard et al. 2002), which represents a rare and very aggressive neoplasm with poor prognosis (Venkatesh et al. 1989). The main benefit is represented by the reduction in symptoms and clinical signs due to steroid excess. In H295R adrenocortical functional cells, the mitotane 10−5 M concentration indeed was able to inhibit glucocorticoids, mineralocorticoids, and androgens secretion affecting upstream in steroidogenic cascade, as it results due to progesterone-reduced level. The observation of a small antiproliferative effect and the reversible reduction in cell cycle G2 phase in treated cells suggests that mitotane does not influence cell growth significantly and does not induce perturbation of cell cycle. Proteomic approach allowed to show the changes in the protein expression pattern in adrenocortical mitotane-treated H295R cells. On the basis of functional characteristics, the proteins involved in drug response can be divided into different classes.

Modulation of proteins involved in energetic metabolism

The drug interferes with the expressions of D-3-PGDH isoforms, enzymes responsible for NAPDH production (Thompson et al. 2005), involved in a crucial mechanism regarding redox potential. The temporal accumulation of AdR, an enzyme involved in the electron transfer from NADPH to the ferredoxin, which in turn donates electrons to the mitochondrial P450 (CYP) cytochromes (Miller 2005), could be explained by a drug interference in the mitochondrial molar ratio between AR/Adx and cytochrome. It leads to a changed electron flow in CYP11A1 and CYP11B1 systems (Tuckey & Sadleir 1999), involved in glucocorticoids and mineralocorticoids biosynthesis. This mechanism could explain the mitotane-mediated inhibition of CYP11B1 activity described by Lindhe et al. (2002). The NDPK reduction is involved in cholesterol trafficking, in agreement with Bourne (1988), who attributed to it a role in cholesterol transport to inner mitochondrial membrane. Finally, the modulation of triose phosphate isomerase and α-enolase probably reflects the adjustment of cellular metabolism to the perturbing stimuli introduced by the drug (Pancholi 2001).

Modulation of proteins involved in stress response

Hahner & Fassnacht (2005) suggested that mitotane affects the oxidative stress through the production of free radicals. The enhancement of Prx II and VI, a superfamily of Se-independent peroxidases, is involved in antioxidant activities and the down-regulation of proteins belonging to the HSP as HSP27 and HSP71 kDa, whose role is to protect cells against oxidative stress, and cytotoxic effects of some chemicals (Fujii & Ikada 2002, Pignatelli et al. 2003) seem to confirm this data. Moreover, the induction of HSP70 1A suggests a more specific implication in drug resistance.

Modulation of cytoskeleton proteins

The modulation of cytoskeleton proteins as tubulin-β isoform II, an intrinsic component of mitochondrial membranes (Carrèet al. 2002), and profilin-1, an actin-binding factor able to promote actin-filament polymerization, suggests an important role of mitotane in the mitochondrial machinery, by altering the membrane permeability and the cholesterol trafficking in H295R cells. These data are in agreement with those of some authors who report the involvement of microfilaments in steroidogenesis (Di Nardo et al. 2000, Wittenmayer et al. 2000).

Modulation of proteins involved in tumorigenesis

Several proteins affected by mitotane in H295R cells have a crucial role in cellular processes correlated with growth control, aging, transcription, RNA splicing, etc. Hint, a hydrolyzing enzyme, may function indeed as a tumor suppressor and be involved in apoptosis, leading to the inhibition of TCF-β-catenin-mediated transcription (Seraphin 1992, Weiske & Huber 2005). Interestingly, H295R cells display a constitutive activation of transactivation of T cell factor (TCF)-dependent transcription, due to the presence of an activating mutation (Tissier et al. 2005). PHB protein is essential in normal mitochondrial development and aging processes (Nijtmans et al. 2000, Coates et al. 2001). The nuclear co-localization with p53, retinoblastoma protein (pRb), and E2F induces to hypothesize a function as tumor suppressor for PHB (McClung et al. 1995). In mitotane-treated H295R cells, its modulation could be attributable to mitochondrial injury, confirming a predominant role of oxidative damage as a mediator of drug action. In regard to hnRNP, a protein implicated in some stages of mRNA metabolism (Krecic & Swanson 1999) and in tumorigenic process (Zhou et al. 2001), an interesting recent study describes a different expression pattern of its isoforms A2 and B1 in human adrenal tissue (Wu et al. 2005). Moreover, B1 expression was found to be increased in various adrenocortical secreting tumors, with a correlation between B1 expression and steroidogenesis. In H295R cells, the depletion of mitotane-induced hnRNPA2/B1 could be explained by a double mechanism leading to the impairment either of steroidogenesis or tumorigenesis. Finally, the increase of cathepsin D, a lysosomal protease involved in cell death (Richo & Conner 1991, Roberg 2001), could still suggest a mitochondrial injury as a main effect of mitotane.

Concluding remarks

These data represent an in-depth approach toward H295R cells in order to define the mechanism of the action of mitotane in ACC. The results show that the drug effects the steroidogenesis cascade upstream. The proteome profiling allowed us to identify some proteins related to different cellular functions, whose expressions were altered by mitotane treatment. Even if further studies are needed in order to improve the understanding of mitotane action in ACC therapy, the identified proteins might represent good targets for the development of strategies directed to contrast ACC.

Acknowledgements

This work was partially financed by research grants (progetti di rilevante interesse nazionale) from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) and ‘Sapienza’ Università di Roma. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagnere AM, Rene-Corail F, Jullian E, Gicquel C & Bertagna X 2005 Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Research 65 76227627.

    • Search Google Scholar
    • Export Citation
  • Trainer PJ & Besser M 1994 Cushing's syndrome. Therapy directed at the adrenal glands. Endocrinology and Metabolism Clinics of North America 23 571584.

    • Search Google Scholar
    • Export Citation
  • Tuckey RC & Sadleir J 1999 The concentration of adrenodoxin reductase limits cytochrome p450scc activity in the human placenta. European Journal of Biochemistry 263 319325.

    • Search Google Scholar
    • Export Citation
  • Venkatesh S, Hickey RC, Sellin RV, Fernandez JF & Samaan NA 1989 Adrenal cortical carcinoma. Cancer 64 765769.

  • Weiske J & Huber O 2005 The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-β-catenin-mediated transcription. Journal of Cell Science 118 31173129.

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    • Export Citation
  • Wittenmayer N, Rothkegel M, Jockusch BM & Schluter K 2000 Functional characterization of green fluorescent protein-profilin fusion proteins. European Journal of Biochemistry 267 52475256.

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    • Export Citation
  • Wu W, Kamma H, Fujiwara M, Yano Y, Satoh H, Hara H, Yashiro T, Ueno E & Aiyoshi Y 2005 Altered expression patterns of heterogeneous nuclear ribonucleoproteins A2 and B1 in the adrenal cortex. Journal of Histochemistry and Cytochemistry 53 487495.

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    • Export Citation
  • Zhou J, Allred DC, Avis I, Martinez A, Vos MD, Smith L, Treston AM & Mulshine JL 2001 Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP- A2/B1) in normal breast and neoplastic breast cancer. Breast Cancer Research and Treatment 66 217224.

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A Stigliano and L Cerquetti equally contributed to this work

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    Effects of mitotane treatment on cell proliferation.

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    Inhibition percentage of steroid hormones synthesis in mitotane-treated H295R cells assayed by the determination of hormone concentration in cell culture medium after 24 h (white bars), 48 h (grey bars) and 72 h (black bars) following drug addition. Androgens, glucocorticoids, and mineralocorticoids levels were respectively inhibited.

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    Proteomic map obtained by the two-dimensional gel electrophoresis of the total protein extract of H295R cells. Spots marked by arrows correspond to the identified proteins listed in Table 1.

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    Time-dependent expression profile of prohibitin in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

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    (A) The expression profile of tubulin-β isoform II and (B) heat shock cognate 71 kDa proteins in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts enriched in mitochondria. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

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    Expression profile of adrenodoxin reductase in untreated (white bars) and mitotane-treated (black bars) H295R total cell extracts enriched in mitochondria. Spot quantity is reported as the spot volume normalized to the total density in all valid spots. The relative western blot analysis is given nearby.

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    • Search Google Scholar
    • Export Citation
  • Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagnere AM, Rene-Corail F, Jullian E, Gicquel C & Bertagna X 2005 Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Research 65 76227627.

    • Search Google Scholar
    • Export Citation
  • Trainer PJ & Besser M 1994 Cushing's syndrome. Therapy directed at the adrenal glands. Endocrinology and Metabolism Clinics of North America 23 571584.

    • Search Google Scholar
    • Export Citation
  • Tuckey RC & Sadleir J 1999 The concentration of adrenodoxin reductase limits cytochrome p450scc activity in the human placenta. European Journal of Biochemistry 263 319325.

    • Search Google Scholar
    • Export Citation
  • Venkatesh S, Hickey RC, Sellin RV, Fernandez JF & Samaan NA 1989 Adrenal cortical carcinoma. Cancer 64 765769.

  • Weiske J & Huber O 2005 The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-β-catenin-mediated transcription. Journal of Cell Science 118 31173129.

    • Search Google Scholar
    • Export Citation
  • Wittenmayer N, Rothkegel M, Jockusch BM & Schluter K 2000 Functional characterization of green fluorescent protein-profilin fusion proteins. European Journal of Biochemistry 267 52475256.

    • Search Google Scholar
    • Export Citation
  • Wu W, Kamma H, Fujiwara M, Yano Y, Satoh H, Hara H, Yashiro T, Ueno E & Aiyoshi Y 2005 Altered expression patterns of heterogeneous nuclear ribonucleoproteins A2 and B1 in the adrenal cortex. Journal of Histochemistry and Cytochemistry 53 487495.

    • Search Google Scholar
    • Export Citation
  • Zhou J, Allred DC, Avis I, Martinez A, Vos MD, Smith L, Treston AM & Mulshine JL 2001 Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP- A2/B1) in normal breast and neoplastic breast cancer. Breast Cancer Research and Treatment 66 217224.

    • Search Google Scholar
    • Export Citation