Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane
A Stigliano1,2, L Cerquetti1,2, M Borro3, G Gentile3, B Bucci2, S Misiti1,2, P Piergrossi2, E Brunetti2, M Simmaco3 and V Toscano1
1Endocrinology, Il Faculty of Medicine, S Andrea Hospital, ‘Sapienza’ University of Rome, 00189 Rome, Italy
2Research Center, S Pietro Hospital, Rome, Italy
3Diagnostica Molecolare Avanzata, Il Faculty of Medicine, S Andrea Hospital, ‘Sapienza’ University of Rome, 00189 Rome, Italy
(Correspondence should be addressed to A Stigliano Endocrinology, Il Faculty of Medicine, Research Center, ‘Sapienza’ University of Rome, 00189 Rome, Italy; Email: antonio.stigliano@uniroma1.it)
A Stigliano and L Cerquetti equally contributed to this work
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, a-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-ß 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.
Endocrine-Related Cancer (2008) 15 1-10
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 neuro- logical 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 pharma- cological 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
DOI: 10.1677/ERC-07-0003 Online version via http://www.endocrinology-journals.org
A Stigliano, L Cerquetti et al .: Proteomic profile in H295R by mitotane induction
(Rainey et al. 1994). Mitotane-induced different expression of proteins involved in energetic meta- bolism, 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, Sunny- vale, CA, USA).
Steroid determination
Hormone levels were determined in the cell super- natant. Progesterone, testosterone and cortisol were measured by ECLIA (Roche). Aldosterone was measured by immunoenzymatic assay (DiaMetra, Milan, Italy).
35
30
No cells x 105
25
20
15
10
C
5
Mitotane
0
0
24
48
72
96
120
144
Time (h)
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 ℃. Protein pellets were dissolved in 8 M urea/4% CHAPS solution. A mitochondria-enriched fraction was obtained using the Mitochondria/Cytosol fractionation Kit (MBL Inter- national 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 electro- phoresis was run on 9-16% linear gradient polyacry- lamide 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
80
Mitotane 24
70
T
Mitotane 48
60
Mitotane 72
% Inhibition
50
T
T
T
40
T
T
30
20
10
0
Progesterone
Testosterone
Cortisol
Aldosterone
NH4HCO3, dehydrated with 100% acetonitrile and in gel digested with 0.5 ng/ul 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℃. 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), B-actin (AC-15 Sigma), HSP71 and ß-tubulin (Upstate Biotechnology), and adrenodoxin reductase (C-15 Santa Cruz Biotechnology Inc.) antibodies. Immuno- blots 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).
Impairment of steroid hormones secretion in mitotane-treated H295R cells
Since H295R cells are able to secrete several steroid hormones, the measurements of progesterone, tes- tosterone, 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
pH3
kDa
150
5
6
K
3 4
8
9
1
15 16 17
4
VVV
10
11 12 13
11 1
18 19 20
21
1
22 23 24 25
26
27
28
30
29
31
1 1/11
32
33 34 35 36
37
38 39 40
6.5
| Spot number | Protein name | Swiss-Prot ANa | Theoretical Mr/pI | Scoreb |
|---|---|---|---|---|
| 1 | Calreticulin | P27797 | 46 466/4.29 | 130 |
| 2, 3, 4 | 60 kDa heat shock protein | P10809 | 57 962/5.24 | 66 |
| 5°, 6° | D-3-phosphoglycerate dehydrogenase | O43175 | 56 519.31/6.31 | 86 |
| 7 | Retinal dehydrogenase 1 | P00352 | 54 730/6.30 | 68 |
| 8º, 9º | a-enolase | P06733 | 47 037/6.99 | 71 |
| 10 | 40S ribosomal protein SA | P08865 | 32 722/4.79 | 105 |
| 11, 12, 13 | Actin, cytoplasmic 1 | P60709 | 41 736/5.29 | 132 |
| 14 | L-lactate dehydrogenase B chain | P07195 | 36 507/5.72 | 78 |
| 15, 16, 17 | Fructose-bisphosphate aldolase A | P04075 | 39 288/8.39 | 87 |
| 18, 19, 20 | Glyceraldehyde-3-phosphate dehydrogenase | P04406 | 35 922/8.58 | 116 |
| 21℃ | Prohibitin | P35232 | 29 804/5.57 | 87 |
| 22°, 23°, 24°, 25℃ | Triosephosphate isomerase | P60174 | 26 823/6.51 | 104 |
| 26 | Rho-GDP-dissociation inhibitor 1 | P52565 | 23 207/5.03 | 67 |
| 27° | Heat shock protein B-1 | P04792 | 22 782/5.98 | 127 |
| 28℃ | Peroxiredoxin 6 | P30041 | 24 903/6.02 | 106 |
| 29c | Peroxiredoxin 2 | P32119 | 21 891/5.66 | 93 |
| 30 | Phosphatidylethanolamine binding protein | P30086 | 20 925/7.43 | 82 |
| 31 | Cofilin-1 | P23528 | 18 371/8.26 | 93 |
| 32, 33, 34, 35, 36 | Peptidyl-prolyl cis-trans isomerase A | P62937 | 18 109/ | 99 |
| 37° | Histidine triad nucleotide binding protein 1 | P49773 | 13 670/6.46 | 60 |
| 38°, 39°, 40℃ | Profilin-1 | P07737 | 14 923/8.47 | 125 |
| – | Tubulin B-2 chain | P07437 | 49 670/4.78 | 102 |
| – | Heat shock cognate 71 kDa protein | P11142 | 70 898/5.37 | 78 |
| d – | Peroxiredoxin-1 | Q06830 | 22 110/8.27 | 82 |
| – | Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/hnRNP B1) | P22626 | 37 429/8.97 | 94 |
| – | Nucleoside diphosphate kinase B | P22392 | 17 298/8.5 | 97 |
| – | Cathepsin D heavy chain | P07339 | 26 628/5.56 | 72 |
| d – | Heat shock 70 kDa protein 1A | Q5SP17 | 70 038/5.48 | 10 |
| d – | Adrenodoxin reductase | P22570 | 53 836/8.72 | 90 |
Protein scores > 60 are significant (P<0.05).
ªEntry 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.
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.
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.
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-depen- dent 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
| Total cellular extracts | Time | |||
|---|---|---|---|---|
| 15 min | 1 h | 5 h | 24 h | |
| Triose phosphate isomerase (spot 23) | NE | [ | 1 | 1 |
| Triose phosphate isomerase (spot 24) | NE | [ | 1 | 1 |
| a-enolase (spot 8) | 4 | Y | « | « |
| a-enolase (spot 9) | Y | 4 | 4 | « |
| D-3-phosphglicerate dehydrogenase (spot 5) | 4 | [ | 4 | 4 |
| D-3-phosphglicerate dehydrogenase (spot 6) | 4 | 4 | 1 | « |
| Peroxiredoxin VI | 4 | [ | 1 | H |
| Peroxiredoxin II | [ | [ | 1 | 1 |
| Heat shock protein ß1 (HSP27) | [ | 4 | H | 4 |
| Histidine triad nucleotide binding protein | [ | 4 | 1 | 1 |
| Profilin (spot 38) | [ | NE | [ | 4 |
| Profilin (spot 39) | NE | [ | 1 | 1 |
| Profilin (spot 40) | [ | NE | [ | 4 |
| Prohibitin | Y | Y | 1 | 1 |
| Mitochondria-enriched fractions | 24 h | 48 h | ||
| Tubulin (ß isoform II) | 4 | Y | ||
| Heat shock cognate 71 kDa protein | 4 | Y | ||
| Peroxiredoxin I | Y | Y | ||
| Heterogenous nuclear ribonucleoprotein isoforms A2/B1 | NE | NE | ||
| Nucleotide diphosphate kinase | NE | Y | ||
| Cathepsin D | 4 | [ | ||
| Heat shock 70 kDa protein 1A | [ | NE | ||
| Adrenodoxin reductase | [ | 4 | ||
The arrow t 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.
Prohibitin
Spot 21
2.0
T
T
T
T
% Spot volume
1.5
T
T
1.0
Z
-
0.5
0
1
15 min
1h
5h
24h
Time
C 15 min
Mitotane 15 min
Mitotane 1 h
Mitotane 5h
Mitotane 24h
C 1h
C 5h
C 24h
Prohibitin
32 KDa
ß-Actin
44 KDa
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. a-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-ß1 (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).
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 unambi- guously identified (Table 2). Most of these proteins are down regulated by mitotane treatment. Tubulin-B 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 pro- gressive 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-ß
Mitotane 24h
Mitotane 48 h
Tubulin-ß isoform II
A
0.5
C 24 h
C 48h
% Spot volume
0.4
T
Tubulin-ß
0.3
50.5 KDa
T
T
0.2
ß-Actin
44 KDa
0.1
0
1
24h
48h
Mitotane 24h
Mitotane 48 h
Heat shock cognate 71 kDa protein
B
0.8
% Spot volume
C 24 h
C 48h
0.6
T
0.4
T
Hsp71
71 KDa
T
0.2
-
ß-Actin
44 KDa
0
24h
48h
Time
isoform II, heat shock cognate 71 kDa, and adreno- doxin reductase have been confirmed by western blot analysis.
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 a-enolase probably reflects the adjustment of cellular metabolism to the perturbing stimuli introduced by the drug (Pancholi 2001).
Adrenodoxin reductase
0.4
0.3
T
T
% Spot volume
0.2
I
0.1
T
0
24h
48 h
Time
Mitotane 24h
Mitotane 48 h
C 24h
C 48h
Adrenodoxin reductase
51 KDa
B-Actin
44 KDa
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 super- family 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-ß isoform II, an intrinsic component of mitochondrial membranes (Carrèet al. 2002), and profilin-1, an actin- binding factor able to promote actin-filament poly- merization, suggests an important role of mitotane in
the mitochondrial machinery, by altering the mem- brane permeability and the cholesterol trafficking in H295R cells. These data are in agreement with those of some authors who report the involvement of micro- filaments 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-ß-catenin-mediated transcrip- tion (Seraphin 1992, Weiske & Huber 2005). Interestingly, H295R cells display a constitutive activation of transactivation of T cell factor (TCF)-de- pendent 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, retino- blastoma 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, confirm- ing 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 tumorigen- esis. 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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