Characterization of the protein ubiquitination responseinduced by DoxorubicinGiorgia Mandili1, Amina Khadjavi1, Valentina Gallo1, Valerio G. Minero2, Luca Bessone1,Franco Carta3, Giuliana Giribaldi1,* and Francesco Turrini1,*

1 Department of Genetics, Biology and Biochemistry, University of Turin, Turin, Italy

2 Department of Experimental Medicine and Oncology, University of Turin, Turin, Italy

3 Nurex SRL, Sassari, Italy

Keywords

cancer; Doxorubicin; neuroblastoma;

proteomics; ubiquitinated proteins

Correspondence

G. Mandili, Department of Genetics, Biology

and Biochemistry, University of Turin, via

Santena 5 bis, 10126 Torino, Italy

Fax: +39 11 6705845

Tel: +39 11 6705850

E-mail: giorgia.mandili@unito.it

*These authors contributed equally to this

work

(Received 4 January 2012, revised 13 March

2012, accepted 13 April 2012)

doi:10.1111/j.1742-4658.2012.08602.x

Doxorubicin is commonly considered to exert its anti-tumor activity by trig-

gering apoptosis of cancer cells through DNA damage. Recent reports have

shown that Doxorubicin elicits a marked heat shock response, and that either

inhibition or silencing of heat shock proteins enhance the Doxorubicin apop-

totic effect in neuroblastoma cells. In order to investigate whether Doxorubi-

cin may also act through protein modification, we performed a proteomic

analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin

treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitina-

tion of a specific set of proteins in the absence of measurable inhibition of

proteasome, (b) protein ubiquination patterns similar to those with Bortezo-

mib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated

enzymes such as lactate dehydrogenase and a-enolase, and (d) a decrease

in HSP27 solubility, probably a consequence of its binding to denatured

proteins. These data strongly reinforce the hypothesis that Doxorubicin may

also exert its effect by damaging proteins.

Introduction

Doxorubicin is an anti-tumor agent of the anthracy-

cline family that is widely used in the treatment of

solid tumors, including neuroblastoma (NB). NB is a

neoplasm that originates in the neural crest, and is the

most common extracranial solid tumor in children [1].

Advanced NB is still frequently fatal despite aggressive

management [2]: even though chemosensitive, it usually

recurs and resists further treatment [3]. Doxorubicin is

a well-known topoisomerase II inhibitor and also

inhibits DNA and RNA synthesis and produces single-

stranded DNA breaks [4,5].

Additional evidence suggests extra-nuclear action of

Doxorubicin. It is well-known that Doxorubicin

induces free radical formation [6], lipid peroxidation [7],

membrane damage [8,9], protein carbonylation and

morphological changes in the mitochondria [10]. More-

over, we previously demonstrated that Doxorubicin

treatment elicits a pronounced heat shock response [11].

It is commonly accepted that increased levels of heat

shock proteins HSPs indicate an accumulation of par-

tially denatured proteins [12]. Damaged proteins follow

two possible fates: repair by HSPs, or, if they are irre-

versibly damaged, degradation, mainly through the

ubiquitin–proteasome system [13].

Ubiquitination is a process during which ubiquitin, a

76 amino acid peptide, is covalently conjugated to the

protein substrate [14]. Substrates may be modified by

either a single ubiquitin, multiple single ubiquitins or a

polyubiquitin chain [15]. Ubiquitin conjugation may

occur on multiple lysine residues of the substrate or

Abbreviations

HSP, heat shock protein; LDH, lactate dehydrogenase; NB, neuroblastoma.

2182 FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS

ubiquitin itself (K6, K11, K48 and K63). The

polyubiquitin chain formed through the K48 residue of

ubiquitin with four or more ubiquitins is a recognition

signal for the 26S proteasome, and can therefore

mediate the degradation of substrates [14,16,17]. Ubi-

quitination has emerged as a central regulatory mecha-

nism, controlling not only protein stability but also

localization, interactions and functional activity of a

large number of protein substrates [15]. Protein degra-

dation via the ubiquitin–proteasome pathway may play

a critical role in the regulation of cell-cycle progression

and apoptosis [17]; thus, malignant cells, which often

show disruption to cell cycle, have an increased depen-

dency on proteasome-mediated degradation of aberrant

proteins, and consequently are more sensitive to pro-

teasome inhibitors than normal cells [18].

In the present work, we performed a comprehensive

study of the effect of nanomolar concentrations of

Doxorubicin on protein ubiquitination changes. The

protein ubiquitination patterns affected by Doxorubi-

cin displayed a marked resemblance to the patterns

obtained by inhibiting the proteasome using Bortezo-

mib. Mass spectrometry analysis confirmed that the

same proteins were ubiquitinated following Doxorubi-

cin or Bortezomib treatments, leading to the hypothe-

sis that the two drugs affect the same protein targets,

but with different mechanisms. As further evidence

that therapeutic Doxorubicin concentrations cause pro-

found protein modifications and may elicit the

observed heat shock response, the solubility of HSP27

was found to be reduced in a dose-dependent way,

suggesting that it binds to denatured proteins [19].

Results

Effect of Doxorubicin on protein ubiquitination

Given our previous data indicating that Doxorubicin

may induce cellular protein modifications [11], we per-

formed a comprehensive analysis of protein ubiquitina-

tion after treatment of NB cells with Doxorubicin

(1–100 nm). Figure 1 shows that, in the SJ-N-KP cell

line, Doxorubicin caused a measurable increase in pro-

tein ubiquitination even in the low nanomolar range

(statistically significant at 100 nm). Higher Doxorubi-

cin concentrations caused a progressive decrease in

protein ubiquitination, in accordance with the appear-

ance of strong cytotoxicity at concentrations above

1 lm (data not shown). Bortezomib, a proteasome

inhibitor, was used as a positive control because of its

known effect on protein ubiquitination [18]. Figure 1

also shows that the patterns of ubiquitinated proteins

were almost identical for both Doxorubicin and Borte-

zomib, indicating that the drugs caused ubiquitination

of the same proteins.

The analysis of ubiquitination levels in other NB cell

lines (SK-N-BE, SH-SY5Y, AF8 and IMR-5) reveals

different behavior, reflecting the heterogeneity of this

kind of tumor. SH-SY5Y and IMR-5 cells show over-

ubiquitination following Doxorubicin treatment that

is statistically significant at 100 nm and 1 lm for

SH-SY5Y cells and 10 lm for IMR-5 cells, but SK-N-BE

and AF8 do not respond to Doxorubicin treatment (data

not shown).

Effect of Doxorubicin on proteasome activities

In order to verify whether the similarities observed in

Doxorubicin and Bortezomib-induced protein ubiquiti-

Control

135 kDa

95 kDa72 kDa

52 kDa

34 kDa

26 kDa

17 kDa

Den

sito

met

ric

units

3.50

2.50

1.50

0.50

3.00

2.00

1.00

0.00

Wb anti-ubiquitin

Wb anti-actin

**

1 10 100 20

Doxorubicin (nM) Bortezomib (nM)

Fig. 1. Protein ubiquitination status in doxorubicin-treated NB cells.

SJ-N-KP cells were treated or not with 1, 10 and 100 nM doxorubicin

or 20 nM bortezomib for 24 h. Aliquots (30 lg) of whole-cell lysate

proteins were analyzed by Western blotting using antibodies against

ubiquitin and actin. The upper panel shows a representative

Western blot, and the lower panel shows data from densitometric

analysis. Values are means and SD of three independent experi-

ments. ANOVA was used to assess significance. *P £ 0.01 versus

control samples.

G. Mandili et al. Protein ubiquitination by Doxorubicin

FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS 2183

nation patterns were due to proteasome inhibition, we

measured proteasome activities. Figure 2 shows that

Doxorubicin did not inhibit any proteasome activities

(chymotrypsin-like, trypsin-like and peptidyl glutamyl

peptide hydrolase) but Bortezomib had a pronounced

inhibitory effect, as expected. These results indicate

that Doxorubicin may directly induce the observed

protein over-ubiquitination exerting a damaging effect

on cellular proteins.

Characterization of ubiquitinated proteins

To identify and characterize the ubiquitinated proteins

in NB cells treated with Doxorubicin or Bortezomib,

we purified them by immunoprecipitation. The purified

proteins were then separated by SDS ⁄PAGE (Fig. 3)

and identified by mass spectrometry. Identified

proteins and their ubiquitination sites are reported in

Table 1. We cannot exclude the possibility of ubiquiti-

nation of additional sites as a result of incomplete

protein sequence coverage. The majority of the ubiqui-

tinated proteins were HSPs, proteins engaged in fold-

ing mechanisms, metabolic enzymes such as lactate

dehydrogenase (LDH) and a-enolase, and cytoskeletal

proteins, indicating that such proteins represent prefer-

ential targets of both Doxorubicin and Bortezomib.

Loss of activity of LDH and a-enolase in

Doxorubicin-treated NB cells

To determine the fate of the identified proteins following

Doxorubicin-induced ubiquitination, LDH and a-eno-lase were chosen as representative ubiquitinated

0

0.2Pico

kata

l/mg

prot

ein

Doxorubicin (nM)

Bortezomib (nM)

–

– – – – 20

1 10 100

* * *

Chimotrypsine-likeTrypsine-likePGPH

–

0.4

0.6

0.8

1

1.2

1.4

Fig. 2. Effect of Doxorubicin and Bortezomib on proteasome activity in NB cells. SJ-N-KP cells were cultured in the presence or absence of

1, 10 and 100 nM Doxorubicin or 20 nM Bortezomib for 24 h. 40 mg of supernatant proteins from lysed cells were analyzed for proteasome

activities. Chymotrypsin-like, trypsin-like, and Peptidyl Glutamyl Peptide Hydrolase (PGPH) activities are shown. Results are expressed as

picokatal ⁄ mg proteins. Data are shown as means ± SD of three independent experiments. For significance ANOVA was used. *P £ 0.01 vs

control samples.

250 kDa

A B C

12345

678910111213141516171819

2021222324

252627

28

29

130 kDa

95 kDa

72 kDa

55 kDa

36 kDa

28 kDa

17 kDa

Fig. 3. Analysis of immunoprecipitated ubiquitinated proteins in NB

cells. Aliquots (100 lg) of proteins immunoprecipitated using anti-

body against ubiquitin from SJ-N-KP control cells (A) and cells trea-

ted with 100 nM doxorubicin (B) or 20 nM bortezomib (C) for 24 h

were analyzed by SDS ⁄ PAGE and stained using colloidal Coomas-

sie stain. Arrows indicate ubiquitinated proteins identified by

MALDI-TOF mass spectrometry and listed in Table 1.

Protein ubiquitination by Doxorubicin G. Mandili et al.

2184 FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS

Tab

le1.

Pro

tein

sid

entified

by

mass

spectr

om

etr

yanaly

sis

.B

ands

were

excis

ed

from

the

gelshow

nin

Fig

.3.

Band

num

ber

Pro

tein

nam

e

Sw

issP

rot

accessio

n

num

ber

Num

ber

of

matc

hin

g

peptides

Sequence

covera

ge

(%)

Mascot

score

Ubiq

uitin

yla

ted

peptides

Doxoru

bic

inB

ort

ezo

mib

1P

lectin-1

Q15149

21

⁄25

5104

2Fatt

yacid

synth

ase

P49327

13

⁄25

870

3R

as

GTP

ase-a

ctivating-lik

e

pro

tein

(IQ

GA

P1)

P46940

15

⁄25

11

96

81–93;

131–143;

267–274;

337–348;

358–368;

497–505;

769–774;

872–891;

902–916;

941–953;

956–963;

1084–1096;

1089–1111;

1166–1174;

1169–1185;

1434–1439;

1506–1516;

1533–1543

74–88;

131–143;

267–274;

349

357;

358–368;

872–891;9

43–955;9

89–997;

1089–1111;

1112–1128;

1166–1174

4C

lath

rin

heavy

chain

1Q

00610

15

⁄25

13

114

64–83;

99–112;

611–619;

1119–1130;

1255–1264

99–112;

158–163;

611–619;

924–941;

1102–1118;

1327–1333;

1435–1443;

1510–1516

5C

arb

am

oylphosphate

synth

ase

[am

monia

],

mitochondrial

P31327

13

⁄25

10

80

16–28;

177–197;

198–210;

229–237;

280–286;

308–316;

451–458;

524–532;

804–811;

881–889;

883–892;

909–919;

1150–1157;

1263–1269;

1349–1356

6E

longation

facto

r2

P13639

8⁄2

513

66

21–32;

151–159;

252–258;

310–318;

343–366;

387–400;

382–407;

599–605;

639–648

7H

eat

shock

pro

tein

HS

P90b

P08238

16

⁄25

23

150

181–196;

429–438;

553–559

181–196;

553–559

878

kD

aglu

cose-r

egula

ted

pro

tein

P11021

20

⁄25

40

254

337–352;

574–581

281–287;

325–336;

337–352;

447–464;

548–554;

574–581;

634–651

9H

eat

shock

cognate

71

kD

apro

tein

P11142

14

⁄25

28

141

89–108;

109–126;

127–137;

513–524;

518–526;

574–583;

590–601;

602–609

89–108;

113–126;

127–137;

501–507;

510–517;

518–526;

574–583

10

Heat

shock

70

kD

a

pro

tein

1

P08107

10

⁄25

24

102

77–88;

113–128;

323–328;

448–458;

498–507;

518–524;

518–526

11

Tra

nsketo

lase

P29401

7⁄2

520

60

12

Pyru

vate

kin

ase

isozy

mes

M1

⁄M2

P14618

10

⁄25

21

81

57–66

13

Pro

tein

dis

ulfi

de

isom

era

se

P07237

9⁄2

526

89

107–114;

327–338;

339–350;

425–436

31–42;

58–65;

196–207;

248–263;

301–308;

309–316;

327–338;

339–350;

387–401;

425–436

14

Pro

tein

dis

ulfi

de

isom

era

se

A3

P30101

7⁄2

519

64

336–447;

364–379

364–379;

396–410

G. Mandili et al. Protein ubiquitination by Doxorubicin

FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS 2185

Tab

le1.

(Continued).

Band

num

ber

Pro

tein

nam

e

Sw

issP

rot

accessio

n

num

ber

Num

ber

of

matc

hin

g

peptides

Sequence

covera

ge

(%)

Mascot

score

Ubiq

uitin

yla

ted

peptides

Doxoru

bic

inB

ort

ezo

mib

15

Mix

ture

217

Tubulin

bchain

P07437

12

⁄25

26

113

Tubulin

a-1

Cchain

Q9B

QE

38

⁄25

29

82

309–320;

312–326

312–326

16

Elo

ngation

facto

r1-c

P26641

6⁄2

521

58

17

a-enola

se

Q6G

MP

212

⁄25

33

102

72–81;

180–193

57–64;

93–105;

254–262;

257–262;

336–343

18

Actin,

cyto

pla

sm

ic1

Q96H

G5

8⁄2

529

95

Actin,

cyto

pla

sm

ic2

P63261

8⁄2

529

95

19

Fru

cto

se-b

isphosphate

ald

ola

se

A

P04075

10

⁄25

34

114

1–14

1–14

20

Gly

cera

ldehyde-3

-phosphate

dehydro

genase

P04406

7⁄2

530

89

140–162;

249–254

21

L-lacta

tedehydro

genase

Achain

P00338

9⁄2

528

82

74–81;

82–99;

107–118;

119–126;

223–228

22

Pro

hib

itin

-2Q

99623

6⁄2

526

64

89–97;

223–236;

237–250

23

Annexin

A5

P08758

8⁄2

536

105

291–301

291–301

24

Puta

tive

hete

rogeneous

nucle

ar

ribonucle

opro

tein

1-lik

epro

tein

3

P0C

7M

27

⁄25

22

67

Hete

rogeneous

nucle

ar

ribo

nucle

opro

tein

A1-lik

e

pro

tein

Q32P

51

7⁄2

522

67

Hete

rogeneous

nucle

ar

ribonucle

opro

tein

A1

P09651

7⁄2

519

64

79–87;

93–105

25

Pro

hib

itin

P35232

7⁄2

538

87

178–186;

203–208;

208–219

26

Phosphogly

cera

tem

uta

se

1P

18669

8⁄2

539

101

87–100;

142–157;

163–176

242–253

27

Triosephosphate

isom

era

se

P60174

8⁄2

532

97

7–18;

161–175

7–18

28

Glu

tath

ione

S-t

ransfe

rase

PP

09211

5⁄2

538

60

29

Phosphatidyle

thanola

min

e-

bin

din

gpro

tein

1

P30086

4⁄2

533

56

Protein ubiquitination by Doxorubicin G. Mandili et al.

2186 FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS

enzymes, and their activities were measured. Figure 4A

shows a loss of intracellular LDH activity following

Doxorubicin treatment, but extracellular LDH activity

was not impaired, demonstrating no evident cytotoxicity

of this Doxorubicin dose (data not shown). Similarly,

Doxorubicin caused a loss of a-enolase activity

(Fig. 4B). These data confirm that ubiquitination of the

two enzymes induced by Doxorubicin leads to their

impairment, suggesting possible protein damage.

HSP27 loss of solubility in Doxorubicin-treated

cells

As the solubility of HSP27 is known to decrease after

binding to denatured protein [19], we determined the

ratio between soluble and insoluble HSP27 in cells

treated with various concentrations of Doxorubicin.

Figure 5 shows Doxorubicin induced loss of solubility

of HSP27, which is recovered in the pellet and

disappears from the supernatants of whole-cell lysates

in a dose-dependent manner. This result suggests

that Doxorubicin can induce loss of folding of some

proteins and their binding to HSP27.

Discussion

Doxorubicin is one of the most widely used chemothera-

peutic agents. It is a well-known topoisomerase II inhibi-

tor, and also inhibits DNA and RNA synthesis and

produces single-stranded DNA breaks [4,5]. However,

evidence suggests additional mechanisms of action

through free radical formation and damage of multiple

cellular targets [6–10]. Moreover, our previous findings

suggested that, at nanomolar concentrations, Doxorubi-

cin induces a marked HSP activation, and that inhibition

or silencing of HSPs strongly enhanced the Doxorubicin

apoptotic effect, raising the possibility that protein dena-

turation plays a role in its anti-cancer activity [11].

We decided to study ubiquitination, one of the more

widespread modifications, to further investigate the

effects of Doxorubicin on protein status; Bortezomib,

a proteasome inhibitor, was used as a positive control.

The results obtained showed dose-dependent over-

ubiquitination of a specific set of protein with either

Doxorubicin or Bortezomib treatment. To exclude the

possibility that this action was mediated by a direct

effect on the proteasome as previously described

[4,9,20,21], proteasome activities following Bortezomib

and Doxorubicin treatment were measured. Doxorubi-

cin did not exert any inhibitory activity on protea-

somes in our study. These data support the possibility

that Doxorubicin may act on protein status.

All major ubiquitinated proteins and their ubiquitina-

tion sites were identified by mass spectrometry, showing

Control

*

*

LDH activity

α-ENOLASE activity

Doxorubicin 100 nM

Control Doxorubicin 100 nM

nmol

NA

DH

oxi

dise

d/to

tal

μg p

rote

ins

nmol

NA

DH

oxi

dise

d/to

tal

μg p

rote

ins

020406080

100120140

100200300400500600

0

700

A

B

Fig. 4. Effect of Doxorubicin on LDH and a-enolase activities. (A)

SJ-N-KP cells were incubated in the absence or presence of

100 nM Doxorubicin for 24 h. After treatment, cell lysates were

used in the LDH assay. Enzyme activity is expressed as nmol

NADH oxidized ⁄ total lg proteins. Values are means and SD of

three independent experiments. ANOVA was used to assess signif-

icance. *P < 0.05 versus control samples. (B) SJ-N-KP cells were

incubated in the absence or presence of 100 nM Doxorubicin for

24 h. After treatment, cell lysates were used in the a-enolase

assay. Enzyme activity is expressed as nmol NADH oxidized ⁄ total

lg proteins. Values are means and SD of three independent experi-

ments. ANOVA was used to assess significance. *P < 0.05 versus

control samples.

Control Doxorubicin (nM)

1 10

0

0.5Solu

ble/

unso

lubl

e

1

2

2.5

1.5

*

100

HSP27 (soluble)

HSP27 (unsoluble)

Fig. 5. Dose-dependent loss of HSP27 solubility. Whole-cell lysates

of SJ-N-KP cells treated or not with 1, 10 and 100 nM Doxorubicin

for 24 h were centrifuged at 1500 g. Aliquots (15 lg) of proteins

from lysate pellets (lower panel) and lysate supernatants (upper

panel) were analysed by Western blotting using antibody against

HSP27. Representative images from three independent experi-

ments are shown. Data from densitometric analysis presented as

the ratio between soluble and unsoluble HSP27. Values are

means ± SD of three independent experiments. ANOVA was used

to assess significance. *P £ 0.05 versus control samples.

G. Mandili et al. Protein ubiquitination by Doxorubicin

FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS 2187

a remarkable correspondence between over-ubiquitinat-

ed proteins after Doxorubicin or Bortezomib treat-

ments. The finding that both Bortezomib and

Doxorubicin treatments also induce over-expression of

HSP27 is in accordance with the accumulation of similar

patterns of modified proteins [11,22], and demonstrates

that Bortezomib and Doxorubicin act on the same tar-

gets, although through different mechanisms. Moreover,

this result suggests that these protein targets are more

likely to be ubiquitinated than other proteins. Most of

the identified proteins were metabolic enzymes, HSPs

and various proteins engaged in the folding of client

proteins. In addition to chaperone activity, HSPs have

been shown to facilitate the degradation of highly mis-

folded proteins by transferring them to the ubiquitin–

proteasome degradation system [23]. In particular,

HSP90b, the heat shock cognate 71 kDa protein and

heat shock 70 kDa protein are known to interact with

the ubiquitin ligase CHIP (carboxy terminus of HSP70-

interacting protein). Following ubiquitination by CHIP,

they cooperate in substrate targeting to the proteasome

[23–26]. A link between GRP78 (78 kDa glucose-regu-

lated protein) and the ubiquitination system has also

been proposed recently [27]. Among the identified pro-

teins, we also found actin and glyceraldehyde-3-phos-

phate dehydrogenase, which are degraded in a heat

shock cognate 71 kDa protein-dependent way [28].

To determine the effect of proteasome-independent

ubiquitination induced by Doxorubicin on protein

functionality, the fate of two of the identified ubiquiti-

nated proteins was monitored: LDH and a-enolase.Interestingly, consistent loss of activity of both

enzymes was observed following Doxorubicin treat-

ment, and this observation reinforces the idea of a

damaging effect of Doxorubicin even though we can-

not exclude a different significance for ubiquitination

induced by Doxorubicin in other proteins.

Finally, as HSP27 was one of most interesting HSPs

in our cellular system [11], and is known to link mis-

folded proteins [19], its solubility following Doxorubi-

cin treatment was investigated. The increased loss of

solubility with increasing Doxorubicin dose supports

the possibility that Doxorubicin may act on protein

folding, and that HSP27 may precipitate as a conse-

quence of binding to unfolded proteins.

In conclusion, our results provide the first evidence

that Doxorubicin, in the low nanomolar range, induces

a complex protein ubiquitination response without

inhibition of proteasome activities. They support the

hypothesis that Doxorubicin, in addition to its action

on nucleic acids, may exert its effect through intracel-

lular accumulation of damaged proteins, even though

the mechanism is not yet fully understood.

A very recent study [29] demonstrating that inducers

of protein misfolding such as hypertemia or Puromycin,

in combination with the proteasome inhibitor Bortezo-

mib, have a proteotoxic effect that is correlated with

increased cytotoxicity may provide support for a clinical

application of our data. Indeed, it has been demon-

strated that combined treatment of normal and chemo-

resistant NB cell lines using Bortezomib and other

chemotherapeutic agents, including Doxorubicin, has a

synergistic effect on the cells, considerably decreasing

the half-maximal effective concentration of Doxorubi-

cin, and combined treatment appears to be a promising

tool for a clinical trial [30]. Moreover, it was also dem-

onstrated by Zanini et al. [11] that combined treatment

with Quercetin and Doxorubicin causes sensitization of

NB cells at nanomolar concentrations of Doxorubicin,

suggesting a clinical interest. A better understanding of

the mechanism underlying the observed synergy may

improve NB treatment strategies in the future by provid-

ing a rationale for the design of novel therapeutic com-

binations with the aim of decreasing the effective dosage

of anticancer drugs and preventing ⁄overcoming the

development of drug resistance.

Experimental procedures

Materials

All materials were obtained from Sigma-Aldrich (St Louis,

MO, USA) unless otherwise stated. Doxorubicin was

obtained from Pharmacia (Milan, Italy); synthetic fluorogenic

peptide substrate Bz-VGR-AMC and monoclonal antibody

against ubiquitin were obtained from Biomol (Plymouth

Meeting, PA, USA); the DC protein assay kit, acrylamide and

enhanced chemiluminescence kit were obtained from Bio-Rad

(Hercules, CA, USA); fetal bovine serum (USA origin) was

obtained from EuroClone (Paignton, UK); the poly(vinyli-

dene fluoride) membrane Immobilon-P was obtained from

Millipore (Milan, Italy); antibodies against HSP27 and ubi-

quitin (polyclonal antibody) were obtained from Stressgen

(Ann Arbor, MI, USA); antibody against actin was obtained

from Santa Cruz Biotechnology (Santa Cruz, CA, USA);

horseradish-peroxidase labeled antibodies were obtained

fromGEHealthcare Bio-Sciences (Milan, Italy).

Cell cultures and treatments

The established NB cell line SJ-N-KP [31] was cultured at

37 �C, 5% CO2 in RPMI-1640 supplemented with 10%

heat-inactivated fetal bovine serum (USA origin),

100 lgÆmL)1 Streptomycin and 100 UÆmL)1 Penicillin. All

experiments were performed using sub-confluent cells. For

Western blotting experiments and measurement of protea-

some activities, cells were cultured in the presence or

Protein ubiquitination by Doxorubicin G. Mandili et al.

2188 FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS

absence of 1, 10 or 100 nm Doxorubicin or 20 nm Bortezo-

mib (a generous gift from Molinette Hospital, Turin, Italy)

for 24 h. For immunoprecipitation experiments, cells were

cultured in the presence or absence of 100 nm Doxorubicin

or 20 nm Bortezomib for 24 h. For measurement of lactate

dehydrogenase and a-enolase activities, cells were cultured

in the presence or absence of 100 nm Doxorubicin for 24 h.

Western blotting assays

SJ-N-KP cells were washed twice for few seconds in NaCl ⁄Pi

and solubilized in Laemmli buffer [32] containing protease

inhibitors and nuclease. After centrifugation at 17 000 g for

15 min at 4 �C, proteins were quantified using a DC protein

assay kit and supplemented with 60 mm dithiothreitol and

0.01% bromophenol blue. Lysates containing equal amounts

of proteins (30 lg) were subjected to SDS ⁄PAGE (10% gel)

using the Mini PROTEAN system (Bio-Rad).

The separated proteins were transferred to an Immo-bilon-

P membrane using a Bio-Rad transfer cell unit according to

the manufacturer’s instructions. The blot was blocked using

5% w ⁄ v BSA in NaCl ⁄Pi containing 0.1% Tween-20, and

probed using polyclonal rabbit antibody against HSP27

(diluted 1 : 5000), polyclonal rabbit antibody against ubiqui-

tin (diluted 1 : 5000) or polyclonal goat antibody against

actin (diluted 1 : 100) overnight at 4 �C. After washing using

NaCl/P containing 0.1% Tween-20 for 35 min, the blot was

incubated for 1 h with horseradish-peroxidase labeled anti-

bodies against rabbit or goat IgG (diluted 1 : 5000 and

1 : 20 000, respectively), and immunoreactivity was detected

using an enhanced chemilumine-scence kit (Bio-Rad).

Measurement of proteasome activities

Proteasome activities in SJ-N-KP cells were determined by

cleavage of specific fluorogenic substrates as previously

described [33]. Briefly, SJ-N-KP cells were washed twice with

NaCl ⁄Pi, solubilized in lysis buffer (20 mm Tris ⁄HCl, pH

7.2, containing 0.1 mm EDTA, 1 mm 2-mercaptoethanol,

5 mm ATP, 20% glycerol and 0.04% v ⁄ v Nonidet P-40) and

centrifuged at 17 000 g for 15 min at 4 �C, and the superna-

tant was collected. Protein concentration was determined

using a DC protein assay kit. Aliquots corresponding to

40 lg of proteins were incubated for 1 h at 37 �C in reaction

buffer (50 mm HEPES, pH 8.0, 5 mm EGTA) with the syn-

thetic fluorogenic peptide substrates Suc-LLVY-AMC, Bz-

VGR-AMC and Z-LLE-AMC at 40 lm concentrations, to

measure chymotrypsin-like, trypsin-like and peptidyl glutam-

yl peptide hydrolase activities, respectively. Following incu-

bation, the reaction was blocked using 0.01% ice-cold

trichloroacetic acid, and fluorescence was measured at an

excitation wavelength of 380 nm and an emission wavelength

of 460 nm, and read using an LS 55 spectrofluorometer (Per-

kin-Elmer, Waltham, MA, USA). Free aminomethyl couma-

rin (AMC) was used as a working standard.

Immunoprecipitation assays

To immunoprecipitate ubiquitinated proteins, cells were

washed twice with NaCl ⁄Pi and lysed in modified radioim-

munoprecipitation buffer (154 mm NaCl, 65 mm Tris ⁄HCl,

pH 7.4, 1% w ⁄ v Nonidet P-40, 0.25% w ⁄ v sodium deoxy-

cholate, 1 mm EDTA) containing protease inhibitors and

nuclease. Aliquots (10 mg) of whole-cell lysate were pre-

cleared using Protein A–agarose beads (3 mgÆmL)1) for 1 h

at 4 �C with rotation, and incubated with monoclonal

mouse antibody against ubiquitin (100 lg per sample) over-

night at 4 �C with rotation. Protein A–agarose beads were

then added for 3 h at 4 �C with rotation. Pellets were

washed three times with ice-cold radioimmunoprecipitation

buffer, solubilized in Laemmli buffer, and protein concen-

tration quantified using a DC protein assay kit. For protein

identification by mass spectrometry, 100 lg of immunopre-

cipitated proteins were run in a 10% polyacrylamide SDS

gel (big format gel, 18 · 20 · 0.1 cm) using a Bio-Rad XI

cell, and stained using colloidal Coomassie stain (18% v ⁄ vethanol, 15% w ⁄ v ammonium sulfate, 2% v ⁄ v phosphoric

acid, 0.2% w ⁄ v Coomassie G-250) for 48 h.

MS analysis and peptide mass fingerprinting

Gel slices from Coomassie-stained gels were excised and

destained by several washes in 50% v ⁄ v acetonitrile in 5 mm

NH4HCO3, and successively dried using pure acetonitrile.

The gel slices were rehydrated for 45 min at 4 �C in 20 lL of

5 mm NH4HCO3 digestion buffer containing 10 ngÆlL)1

trypsin. Excess protease solution was then removed, and the

volume was adjusted using 5 mm NH4HCO3 to cover the gel

slices. Digestion was allowed to proceed overnight at 37 �C[34]. MS analysis of peptides was performed using a

MALDI-TOF spectrometer (MALDI micro MX; Waters,

Milford, MA, USA) equipped with a delayed extraction unit,

according to the tuning procedures suggested by the manu-

facturer, operating on reflectron mode. Samples were loaded

onto the MALDI target using 1.5 lL of the tryptic digest

mixed 1 : 1 with a solution of a-cyanohydroxycinnamic acid

(10 mgÆmL)1) in 40% v ⁄ v acetonitrile, 60% v ⁄ v trifluoro-

acetic acid 0.1%. Peak lists were generated by ProteinLynx

(Waters, Milford, MA, USA) data preparation using the fol-

lowing parameters: external calibration with lock mass using

a mass of 2465.1989 Da for ACTH (adrenocorticotropic hor-

mone), background subtract type adaptive combining all

scans, and de-isotoping with a threshold of 1%. The 25 most

intense masses were used for database searches against the

SWISSPROT database using the free search program MAS-

COT (http://www.matrixscience.com). Peak lists created as

above described were used for a ProteinLynx Global Server

2.2.5 search using the SWISSPROT database (downloaded

from ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/; release

2011_08, 27 July 2011) and used for identification of ubiquiti-

nated sequences. In all cases, the search settings allowed one

G. Mandili et al. Protein ubiquitination by Doxorubicin

FEBS Journal 279 (2012) 2182–2191 ª 2012 The Authors Journal compilation ª 2012 FEBS 2189

missed cleavage with the trypsin enzyme selected, oxidation

of methionine as a potential variable modification, a peptide

tolerance of 100 ppm, taxon human. In the case of Protein-

Lynx Global Server searches, ubiquitination as potential var-

iable modification was added: peptides containing an

ubiquitinated site show an increase in molecular mass of

114 Da for each targeted lysine residue as trypsin digestion

cannot occur at the modified lysine and tandem glycines are

conjugated to the target lysine residue [14].

Enzymatic assays

Cell culture lysates were used in the LDH (lactate deydrogen-

ase) assay, as previously described [35], and in the

a-enolase assay (cells were lysed in 1 m Tris ⁄HCl, pH 8, and

mixed with 10 mm MgCl2, 1 m KCl, 100 lm 2-phosphogly-

ceric acid, 4 mm ADP, 6.8 UÆmL)1 pyruvate kinase,

9.9 UÆmL)1 LDH, 200 lm NADH). Activities for both

enzymes, measured spectrophotometrically using a Packard

EL340 microplate reader (Bio-Tek Instruments, Winooski,

VT, USA) as absorbance variation at 340 nm at 37 �C, wereexpressed as nmol NADH oxidized per total lg proteins.

Measurement of HSP27 solubility

SJ-N-KP cells were washed twice in NaCl ⁄Pi and solubi-

lized in radioimmunoprecipitation buffer containing prote-

ase inhibitors and nuclease. Whole-cell lysates were then

centrifuged at 1500 g for 10 min, and the protein content

of obtained pellets and supernatants was quantified using a

DC protein assay kit. Proteins (15 lg) from the pellet or

supernatant were then added to Laemmli buffer supple-

mented with 60 mm dithiothreitol and 0.01% w ⁄ v brom-

ophenol blue and subjected to SDS ⁄PAGE (12% gel) using

the Mini PROTEAN system (Bio-Rad) and Western blot-

ting with antibody against HSP27 (see above).

Statistical analysis

Experimental data are presented as means ± standard devi-

ation (SD) of three independent experiments. Data signi

ficance was determined by one-way analysis of variance

(ANOVA).

Acknowledgements

This work was supported by the ‘Oncology Special

Project’, Compagnia di San Paolo ⁄FIRMS (Fondazione

Internazionale di Ricerca inMedicina Sperimentale), and

by the ‘Ricerca Sanitaria Finalizzata’, Piedmont Region

(Italy). The authors thank Dr Mauro Prato (Department

of Genetics, Biology and Biochemistry, University of

Turin, Turin, Italy) andDrAndrea Bonetto (Department

of Cancer and Biology, Kimmel Cancer Center, Thomas

Jefferson University, Philadelphia, PA, USA) for critical

reading of themanuscript.

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