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

COMMENTARYEndocrine-Related Cancer (2008) 15 1–10

Modulation of proteomic profile in H295Radrenocortical cell line induced by mitotane

A Stigliano1,2, L Cerquetti1,2, M Borro3, G Gentile3, B Bucci 2, S Misiti1,2,P Piergrossi2, E Brunetti2, M Simmaco3 and V Toscano1

1Endocrinology, II Faculty of Medicine, S Andrea Hospital, ‘Sapienza’ University of Rome, 00189 Rome, Italy2Research Center, S Pietro Hospital, Rome, Italy3Diagnostica Molecolare Avanzata, II Faculty of Medicine, S Andrea Hospital, ‘Sapienza’ University of Rome, 00189 Rome, Italy

(Correspondence should be addressed to A Stigliano Endocrinology, II Faculty of Medicine, Research Center, ‘Sapienza’ University

of Rome, 00189 Rome, Italy; Email: [email protected])

A Stigliano and L Cerquetti equally contributed to this work

Abstract

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p0-DDD), is a compound thatrepresents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to blockcortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remainsstill unknown. In this study, we have already shown a greater impairment in the first steps of thesteroidogenesis and recognizeda little effecton cell cycle.We also evaluated the variation of proteomicprofile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction aftertreatment 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 themitochondrial fraction, the following proteins appeared to bedown regulated: aldolase A, peroxiredoxinI, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-b isoform II, heat shock cognate 71 kDaprotein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heatshock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic studyon the mitotane effectson ACC. It permits to identifysome protein classes affected by the drug involvedin 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


1351–0088/08/015–001 q 2008 Society for Endocrinology Printed in Great

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

Britain

DOI: 10.1677/ERC-07-0003

Online version via http://www.endocrinology-journals.org

http://dx.doi.org/10.1677/ERC-07-0003

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 10K5 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 mg/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).

Figure 1 Effects of mitotane treatment on cell proliferation.

2

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 K20 8C. 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 mg 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

Figure 2 Inhibition percentage of steroid hormones synthesis inmitotane-treated H295R cells assayed by the determination ofhormone concentration in cell culture medium after 24 h (whitebars), 48 h (grey bars) and 72 h (black bars) following drugaddition. Androgens, glucocorticoids, and mineralocorticoidslevels were respectively inhibited.

www.endocrinology-journals.org


NH4HCO3, dehydrated with 100% acetonitrile and in

gel digested with 0.5 ng/ml 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 K80 8C. 70 mg 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 b-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.

Figure 3 Proteomic map obtained by the two-dimensional gel elemarked by arrows correspond to the identified proteins listed in Ta


Results

Cell proliferation and treatment

Schteingart et al. (1993) reported that mitotane at

10K5 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

ctrophoresis of the total protein extract of H295R cells. Spotsble 1.

3

Table 1 List of identified proteins in two-dimensional proteomic maps of H295R cells

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

5c, 6c D-3-phosphoglycerate dehydrogenase O43175 56 519.31/6.31 86

7 Retinal dehydrogenase 1 P00352 54 730/6.30 68

8c, 9c 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

21c Prohibitin P35232 29 804/5.57 87

22c, 23c, 24c, 25c Triosephosphate isomerase P60174 26 823/6.51 104

26 Rho-GDP-dissociation inhibitor 1 P52565 23 207/5.03 67

27c Heat shock protein b-1 P04792 22 782/5.98 127

28c 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

37c Histidine triad nucleotide binding protein 1 P49773 13 670/6.46 60

38c, 39c, 40c Profilin-1 P07737 14 923/8.47 125

–d Tubulin b-2 chain P07437 49 670/4.78 102

–d Heat shock cognate 71 kDa protein P11142 70 898/5.37 78

–d Peroxiredoxin-1 Q06830 22 110/8.27 82

–d Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/hnRNP B1) P22626 37 429/8.97 94

–d Nucleoside diphosphate kinase B P22392 17 298/8.5 97

–d 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 O60 are significant (P!0.05).aEntry name and accession number according to Swiss-Prot (http://expasy.org).bProtein score represents the probability that the observed match is a random event.cProteins showing differential expression in proteomic profiles of H295R total extracts following mitotane treatment.dProteins showing differential expression in proteomic profiles of H295R extracts enriched in mitochondria following mitotane treatment.

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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

Table 2 Differentially expressed proteins after mitotane treatment

Total cellular extracts 15 m

Triose phosphate isomerase (spot 23) NE

Triose phosphate isomerase (spot 24) NE

a-enolase (spot 8) 4a-enolase (spot 9) YD-3-phosphglicerate dehydrogenase (spot 5) 4D-3-phosphglicerate dehydrogenase (spot 6) 4Peroxiredoxin VI 4Peroxiredoxin II [Heat shock protein b1 (HSP27) [Histidine triad nucleotide binding protein [Profilin (spot 38) [Profilin (spot 39) NE

Profilin (spot 40) [Prohibitin Y

Mitochondria-enriched fractions 24 h

Tubulin (b isoform II) 4Heat shock cognate 71 kDa protein 4Peroxiredoxin I YHeterogenous nuclear ribonucleoprotein

isoforms A2/B1

NE

Nucleotide diphosphate kinase NE

Cathepsin D 4Heat shock 70 kDa protein 1A [Adrenodoxin reductase [

The arrow [ indicates up-regulated proteins (R1.5-fold), the arrow4 indicates the unchanged level expression proteins. NE acronymshow multiple isoforms.


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

Time

in 1 h 5 h 24 h

[ [ Y[ [ YY 4 44 Y 4[ Y Y4 Y 4[ [ 4[ [ Y4 4 Y4 [ YNE [ 4[ [ [

NE [ 4Y [ [

48 h

YYY

NE

Y[

NE

4

Y indicates down-regulated proteins (%1.5-fold), and the arrowindicates unexpressed proteins. Some proteins listed in the table

5

Figure 4 Time-dependent expression profile of prohibitin inuntreated (white bars) and mitotane-treated (black bars) H295Rtotal cell extracts. Spot quantity is reported as the spot volumenormalized to the total density in all valid spots. The relativewestern blot analysis is given nearby.


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

6

with control cells. The least acidic isoform (spot 6)

showed a K2.0-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-b1 (HSP27; spot 27) underwent an

over-expression (C2.2-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 withC8-fold change after

15 min and C3-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-bisoform 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 K3-fold change at 48 h in

treated cells (Fig. 5B). The drug strongly repressed

the peroxiredoxin I (Prx I) of a K2.5-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 withK2.5-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 C12-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-b


Figure 5 (A) The expression profile of tubulin-b isoform II and (B) heat shock cognate 71 kDa proteins in untreated (white bars) andmitotane-treated (black bars) H295R total cell extracts enriched in mitochondria. Spot quantity is reported as the spot volumenormalized to the total density in all valid spots. The relative western blot analysis is given nearby.


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

10K5 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 cycleG2 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).

7

Figure 6 Expression profile of adrenodoxin reductase inuntreated (white bars) and mitotane-treated (black bars)H295R total cell extracts enriched in mitochondria. Spotquantity is reported as the spot volume normalized to the totaldensity in all valid spots. The relative western blot analysis isgiven nearby.


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-regulationof 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-bisoform II, an intrinsic component of mitochondrial

membranes (Carreet al. 2002), and profilin-1, an actin-

binding factor able to promote actin-filament poly-

merization, suggests an important role of mitotane in

8

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-b-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 amitochondrial 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) fromMinistero

dell’Universita e della Ricerca Scientifica e Tecnologica

(MURST) and ‘Sapienza’ Universita di Roma. The

authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

References

Beacauregard C, Dickstein G & Lacroix A 2002 Classic and

recent etiologies of Cushing’s syndrome: diagnosis and

therapy. Treatments in Endocrinology 1 79–94.

Bourne HR 1988 Do GTPases direct membrane traffic in

secretion? Cell 53 669–671.

Cai W, Counsell RE, Schteingart DE, Sinsheimer JE,

Vaz AD & Wotring LL 1997 Adrenal proteins bound

by a reactive intermediate of mitotane. Cancer

Chemotherapy and Pharmacology 39 537–540.

Carre M, Andre N, Carles G, Borghi H, Brichese L,

Briand C & Braguer D 2002 Tubulin is an inherent

component of mitochondrial membranes that interacts

with the voltage-dependent anion channel. Journal of

Biological Chemistry 277 33664–33669.

Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH,

Hall PA&Wright EG 2001Mammalian prohibitin proteins

respond tomitochondrial stress and decrease during cellular

senescence. Experimental Cell Research 265 262–273.

Fang VS 1979 Cytotoxic activity of 1-(o-chlorophenyl)-1-

(p-chlorophenyl)-2,2-dichloroethane (mitotane) and its

analogs on feminizing adrenal neoplastic cells in culture.

Cancer Research 39 139–145.

Fujii J & Ikeda Y 2002 Advances in our understanding of

peroxiredoxin, a multifunctional, mammalian redox

protein. Redox Report 7 123–130.

Gorg A, Postel W&Gunther S 1988 The current state of two-

dimensional electrophoresis with immobilized pH

gradients. Electrophoresis 9 531–546.

Hahner S & Fassnacht M 2005 Mitotane for adrenocortical

carcinoma treatment. Current Opinion in Investigational

Drugs 6 386–394.

Hanash S 2003 Disease proteomics. Nature 13 226–232.

Krecic AM & Swanson MS 1999 hnRNP complexes:

composition, structure, and function. Current Opinion in

Cell Biology 11 363–371.

Lindhe O, Skogseid B & Brandt I 2002 Cytochrome P450-

catalyzed binding of 3- methylsulfonyl-DE and o,p 0-DDD

in human adrenal zona fasciculata/reticularis. Journal of

Clinical Endocrinology and Metabolism 87 1319–1326.


Martz F & Straw JA 1980Metabolism and covalent binding of

1-(o-chlorophenyl)-1-(p- chlorophenyl)-2,2-dichloroethane

(o,p0-DDD). Correlation between adrenocorticolytic

activity and metabolic activation by adrenocortical mito-

chondria. Drug Metabolism and Disposition 8 127–130.

McClung JK, Jupe ER, Liu XT & Dell’Orco RT 1995

Prohibitin: potential role in senescence, development, and

tumor suppression. Experimental Gerontology 30 99–124.

Miller WL 2005 Minireview: regulation of steroidogenesis

by electron transfer. Endocrinology 146 2544–2550.

Di Nardo A, Gareus R, Kwiatkowski D & Witke W 2000

Alternative splicing of the mouse profilin II gene

generates functionally different profilin isoforms.

Journal of Cell Science 113 3795–3803.

NijtmansLG,NijtmansLG, de JongL,ArtalSanzM,CoatesPJ,

Berden JA, Back JW, Muijsers AO, van der Spek H &

Grivell LA 2000 Prohibitins act as a membrane-bound

chaperone for the stabilization of mitochondrial proteins.

EMBO Journal 19 2444–2451.

Pancholi V 2001 Multifunctional alpha-enolase: its role in

diseases.Cellular andMolecular Life Sciences 58 902–920.

Pignatelli D, Ferriera J, Soares P, Costa MJ &Magalhaes MC

2003 Immunohistochemical study of heat shock proteins

27, 60 and 70 in the normal human adrenal and in adrenal

tumors with suppressed ACTH production. Microscopy

Research and Technique 15 315–323.

RaineyWE,Bird IM&Mason JI 1994TheNCI-H295 cell line:

a pluripotent model for human adrenocortical studies.

Molecular and Cellular Endocrinology 100 45–50.

Richo G & Conner GE 1991 Proteolytic activation of human

procathepsin D. Advances in Experimental Medicine and

Biology 306 289–296.

Roberg K 2001 Relocalization of cathepsin D and cyto-

chrome c early in apoptosis revealed by immunoelectron

microscopy. Laboratory Investigation 81 149–158.

Schteingart DE, Sinsheimer JE, Counsell RE, Abrams GD, Mc

Clellan N, Djanegara T, Hines J, Ruangwises N, Benitez R

&Wotring LL 1993 Comparison of the adrenalytic activity

of mitotane and a methylated homolog on normal adrenal

cortex and adrenal cortical carcinoma. Cancer Chemo-

therapy and Pharmacology 31 459–466.

Seraphin B 1992 The HIT protein family: a new family of

proteins present in prokaryotes, yeast and mammals. DNA

Sequence 3 177–179.

Shevchenko A, Wilm M, Vorm O & Mann M 1996 Mass

spectrometric sequencing of proteins silver-stained poly-

acrylamide gels. Analytical Chemistry 68 850–858.

Thompson JR, Bell JK, Bratt J, Grant GA & Banaszak LJ

2005 Vmax regulation through domain and subunit

changes. The active form of phosphoglycerate dehydro-

genase. Biochemistry 44 5763–5773.

Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G,

Hagnere AM, Rene-Corail F, Jullian E, Gicquel C,

Bertagna X et al. 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 7622–7627.

9


Trainer PJ & Besser M 1994 Cushing’s syndrome.

Therapy directed at the adrenal glands. Endocrinology

and Metabolism Clinics of North America 23

571–584.

Tuckey RC & Sadleir J 1999 The concentration of

adrenodoxin reductase limits cytochrome p450scc

activity in the human placenta. European Journal of

Biochemistry 263 319–325.

Venkatesh S, Hickey RC, Sellin RV, Fernandez JF &

Samaan NA 1989 Adrenal cortical carcinoma. Cancer

64 765–769.

Weiske J & Huber O 2005 The histidine triad protein Hint1

interacts with Pontin and Reptin and inhibits TCF-b-catenin-mediated transcription. Journal of Cell Science

118 3117–3129.

10

Wittenmayer N, Rothkegel M, Jockusch BM & Schluter K

2000 Functional characterization of green fluorescent

protein-profilin fusion proteins. European Journal of

Biochemistry 267 5247–5256.

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 53 487–495.

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 66 217–224.


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

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