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ArticleTitle Cells derived from normal or cancer breast tissue exhibit different growth properties when deprived of arginineArticle Sub-Title

Article CopyRight Springer Science+Business Media, LLC(This will be the copyright line in the final PDF)

Journal Name Medical Oncology

Corresponding Author Family Name AlojParticle

Given Name Salvatore M.Suffix

Division Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”

Organization University of Naples Federico II

Address Naples, 80131, Italy

Division Istituto per la Endocrinologia ed Oncologia Sperimentale, National ResearchCouncil


Address Naples, Italy

Email [email protected]

Author Family Name ChiavielloParticle

Given Name AngelaSuffix








Author Family Name PacielloParticle

Given Name IdaSuffix








Author Family Name VenezianiParticle

Given Name Bianca MariaSuffix








Author Family Name PalumboParticle

Given Name GiuseppeSuffix








Schedule

Received 22 July 2011

Revised

Accepted 28 November 2011

Abstract Arginine deprivation impairs cell proliferation more strong in cancer than in normal cells; thus, it has beenproposed that such an effect could be exploited for cancer therapy. We have compared the effect of argininedeprivation on normal and cancer cells, studying growth rate, morphology, and protein expression patternsin immortalized human MCF10a cells and in MCF7 cells. Arginine deprivation forces MCF10a cells intoirreversible senescence while the vast majority of MCF7 cells become quiescent and resume normal growthfollowing arginine re-addition. Arginine deprivation induced a significant burst of p21cip1 in both cell linesthat were reversible in MCF7 and irreversible in MCF10 cells. In the latter cells, p21cip1 increase wasaccompanied by a time-dependent increase of p16INK4A. Similar effects could be obtained by treating bothcell types with α-difluoro-methyl-ornithine, but not with Nω-hydroxy-L-arginine, drugs that interferespecifically but differently with the major pathways of arginine metabolism. Our data suggest thatderangement in polyamine synthesis is the main consequence of arginine starvation.

Keywords (separated by '-') Breast cancer - Arginine - Senescence - MCF10a - MCF7 - α-Difluoro-methyl-ornithine - Nω-hydroxy-L-arginine

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UNCORRECTEDPROOF

ORIGINAL PAPER1

2 Cells derived from normal or cancer breast tissue exhibit different

3 growth properties when deprived of arginine

4 Angela Chiaviello • Ida Paciello • Bianca Maria Veneziani •

5 Giuseppe Palumbo • Salvatore M. Aloj

6 Received: 22 July 2011 / Accepted: 28 November 20117 � Springer Science+Business Media, LLC 2011

8 Abstract Arginine deprivation impairs cell proliferation

9 more strong in cancer than in normal cells; thus, it has been

10 proposed that such an effect could be exploited for cancer

11 therapy. We have compared the effect of arginine depri-

12 vation on normal and cancer cells, studying growth rate,

13 morphology, and protein expression patterns in immortal-

14 ized human MCF10a cells and in MCF7 cells. Arginine

15 deprivation forces MCF10a cells into irreversible senes-

16 cence while the vast majority of MCF7 cells become qui-

17 escent and resume normal growth following arginine

18 re-addition. Arginine deprivation induced a significant

19 burst of p21cip1 in both cell lines that were reversible in

20 MCF7 and irreversible in MCF10 cells. In the latter cells,

21 p21cip1 increase was accompanied by a time-dependent

22 increase of p16INK4A. Similar effects could be obtained by

23treating both cell types with a-difluoro-methyl-ornithine,

24but not with Nx-hydroxy-L-arginine, drugs that interfere

25specifically but differently with the major pathways of

26arginine metabolism. Our data suggest that derangement in

27polyamine synthesis is the main consequence of arginine

28starvation.

29

30Keywords Breast cancer � Arginine � Senescence �

31MCF10a � MCF7 � a-Difluoro-methyl-ornithine �

32Nx-hydroxy-L-arginine

33Introduction

34Several studies have investigated the effects of the amino

35acid arginine on the in vitro proliferation of cells derived

36from normal or cancer tissue. There is a consensus that

37arginine is not an essential amino acid, but it is rated as

38‘‘semi-essential’’ or ‘‘conditionally non-essential’’ amino

39acid. Indeed, arginine could be synthesized from citrulline

40through the sequential action of the cytosolic enzymes

41argininosuccinate synthetase (ASS) and argininosuccinate

42lyase; however, citrulline itself is made, to a large extent

43from arginine, as a by-product of the reaction catalyzed by

44the nitric oxide synthetase (NOS) family of enzymes. Thus,

45it is conceivable that minimal metabolic derangements and/

46or the developmental stage can turn the cell into a strict

47arginine auxotroph.

48In addition to its role as a building block in protein

49synthesis, arginine is the precursor of several metabolic

50pathways leading to the biosynthesis of molecules relevant

51to cell proliferation such as nitric oxide (NO) and poly-

52amines (spermidine, spermine, and putrescine) as well as

53the polyamine analog agmatine. All these molecules are

54relevant to the process of tumorigenesis as shown by

A1 A. Chiaviello � I. Paciello � B. M. Veneziani � G. Palumbo �

A2 S. M. Aloj (&)

A3 Dipartimento di Biologia e Patologia Cellulare e Molecolare

A4 ‘‘L. Califano’’, University of Naples Federico II, 80131 Naples,

A5 Italy

A6 e-mail: [email protected]

A7 A. Chiaviello


A9 I. Paciello


A11 B. M. Veneziani


A13 G. Palumbo


A15 A. Chiaviello � I. Paciello � B. M. Veneziani � G. Palumbo �

A16 S. M. Aloj

A17 Istituto per la Endocrinologia ed Oncologia Sperimentale,

A18 National Research Council, University of Naples Federico II,

A19 Naples, Italy

123Journal : Large 12032 Dispatch : 2-12-2011 Pages : 9

Article No. : 130h LE h TYPESET

MS Code : MEDO-2815 h CP h DISK4 4

Med Oncol

DOI 10.1007/s12032-011-0130-7

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55 arginine deprivation [1]. It has been reported that L-argi-

56 nine removal from the culture medium causes different

57 fates spanning from growth arrest to cell death [2]

58 depending, to a large extent, on the level of expression of

59 ASS [1]. Exposing cells to arginase, which causes relative

60 arginine deficiency, induces similar effects [3].

61 Arginine deprivation seems to affect cancer cells more

62 severely, which die more quickly than cells with a non-

63 malignant phenotype [2]; thus, it is not surprising that

64 several attempts have been made to exploit arginine

65 deprivation as an anticancer therapy. However, this

66 approach has not significant success. Indeed, the role

67 played by arginine in cell metabolism and growth is rather

68 complicated by the observation that excess arginine in the

69 diet produces metabolic imbalance. It has been proposed

70 that combining an arginine-rich diet with cancer chemo-

71 therapy has an enhanced therapeutic effect [4]. Szende

72 et al. [5] have reported that D-arginine, administered orally

73 at a daily dose of 500 mg/kg to rats bearing Yoshida’s

74 sarcoma, significantly inhibited tumor growth and intra-

75 peritoneal injection of a similar dose into mice bearing

76 Ehrlich carcinoma inhibited growth of the tumor, albeit not

77 significantly.

78 L-arginine metabolism follows at least two major path-

79 ways: the oxidative deaminase pathway and the NADPH-

80 dependent enzyme nitric oxide synthase (NOS) generating

81 nitric oxide (NO) pathway. The arginase pathway yields

82 ornithine and polyamines that interact with the genomic

83 DNA, regulating its transcription among other effects on

84 proliferation [6]. The availability of intracellular L-arginine

85 is also a rate-limiting factor in nitric oxide (NO) production

86 [7]. NO participates in many cellular metabolic processes,

87 including cell proliferation.

88 In this study, we have attempted to explore the effect of

89 arginine deprivation on the proliferation of two related cell

90 lines: MCF10a and MCF7. MCF10a cells are diploid,

91 normal-like human breast cells that spontaneously became

92 immortal in culture [8]. MCF7 is a well-established human

93 breast tumor cell line that was originally developed in 1973

94 [9]. The aim was to determine whether arginine deprivation

95 would have any selective growth inhibition of normal-like,

96 versus malignant cells of similar tissue origin and which of

97 the major pathways of arginine metabolism is affected by

98 such deprivation.

99 Materials and methods

100 Cell cultures and media

101 MCF7 and MCF10a human mammary cell lines were

102 obtained from the American Type Culture Collection

103 (Rockville, MD) and were both grown in the presence of

104L-glutamine (2 mM), penicillin (100 units/mL), strepto-

105mycin (100 lg/mL), and 10% fetal calf serum. The

106chemically modified media were DMEM for MCF7 cells

107and a mixture (1:1) of DMEM and Coon’s modified F12

108medium for MCF10a cells. Both cell lines were p53?/?.

109The MCF7 cells are p16INK4 null. All media and cell cul-

110ture reagents were purchased from Life Technologies

111(Milan, Italy). Arginine-free medium was prepared

112according to Tesseraud et al. [10].

113Drugs

114a-Difluoromethylornithine (DFMO), an inhibitor of ornithine

115decarboxylase (ODC), was supplied by Alexis (UK). DFMO

116solution was prepared in water to obtain 10 mg/mL stock.

117Final concentration of DFMO was 5 mM in all experiments.

118The arginase inhibitor Nx-hydroxy-L-arginine (NOHA)

119was purchased from Calbiochem, UK. It was dissolved in

120water to obtain 5 mg/mL stock solution. Final concentra-

121tion of NOHA was 1 mM in all experiments.

122During incubations with drugs, cell media containing

123either DFMO or NOHA (5 and 1 mM, respectively) were

124replaced every second day.

125Sample treatment schedules

126Experiments performed in arginine-free media

127Usually, 5 9 104 MCF7 or MCF10a cells were seeded in

12860-mm dishes in complete medium. Twenty-four hours

129later, they were washed and incubated in arginine-free

130medium (-R). After 5 days, cells were washed and

131released into fresh complete medium (?R) or maintained

132into arginine-free medium for the needed time.

133Experiments performed in the presence of DFMO

134or NOHA

135The effect 5 mM DFMO or 1 mM NOHA (5 days incu-

136bation) on cell proliferation was performed on 1 9 105

137cells seeded in 6 wells plates.

138Cell-cycle profiles, protein expression, and onset of

139premature senescence were analyzed either in cells incu-

140bated for 5 days with the drugs or after cells released into

141drug-free media.

142The data from triplicate samples are reported as the

143mean ± SD.

144Thymidine incorporation

145[3H]-thymidine incorporation experiments were performed

146in triplicates in 24 multi-well plates according to Love-

147Schimenti et al. [11]. Measured volumes of samples were

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148 used to quantify the protein concentrations [12], while

149 appropriate aliquots were employed to measure the effi-

150 ciency of thymidine incorporation by Harvester 96

151 (Skatron Instruments, Lier, Norway).

152 Flow cytometry

153 MCF10a and MCF7 cells (4 9 105) were seeded in 10-cm

154 plates and cultivated in arginine-free media (-R) or with

155 the drugs as reported above. Data have been obtained from

156 triplicate samples and reported as the mean ± SD. Cyto-

157 fluorimetric analyses were done according to Crescenzi

158 et al. [13] at established times. The orange fluorescence of

159 propidium iodide was detected in a linear scale using a

160 CyAn ADP Flow Cytometer (DAKO Cytomation, UK) and

161 Summit Software. About 30,000 events were collected and

162 recorded for each sample.

163 Apoptosis assays

164 Annexin V test: Detection of apoptotic cells with mem-

165 brane alterations (phosphatidylserine translocation) was

166 performed cytofluorimetrically using the Annexin-V-Alexa

167 568 kit (Roche, Milan Italy) according to the manufac-

168 turer’s instructions.

169 Caspases 3 and 7: The activity of Caspase 3 and 7 was

170 monitored by using the Caspase-Glo� 3/7 Assay kit

171 (Promega, Milan Italy).

172 Senescence-associated b-galactosidase (SA-b-Gal)

173 assay

174 Cell senescence was evaluated by assessing senescence-

175 associated-b-galactosidase activity [14]. To measure the

176 sudden appearance of premature senescence in our condi-

177 tions, 7 9 104 (either MCF7 or MCF10a) cells were see-

178 ded, deprived of arginine, or incubated with drugs as

179 reported above. At established times, cells were fixed for

180 5 min (room temperature) in 3% formaldehyde, washed,

181 and incubated at 37�C with fresh senescence-associated-b-

182 Gal-staining solution constituted of 1 mg/mL 5-bromo-4-

183 chloro-3-indolyl b-D-galactoside, 40 mM citric acid/

184 sodium phosphate pH 6.0, 5 mM potassium ferrocyanide,

185 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM

186 MgCl2. In such conditions, staining was maximal at

187 12–16 h. As expected, all b-galactosidase positive (senes-

188 cent) cells presented flat morphology. However, not all

189 flattened cells were positive to staining.

190 Western blot analysis

191 Cell extracts were obtained as previously described [13]

192 and analyzed by polyacrylamide gels [15]. Molecular

193weight standards were from New England Biolabs

194(Beverly, MA). Separated proteins were blotted onto

195nitrocellulose (Hybond-C pure, Amersham Italy), washed,

196stained with specific primary antibodies (all from Santa

197Cruz, CA), and incubated with horseradish peroxidase-

198conjugated secondary antisera (Biorad, Italy). Filters were

199developed using an electro-chemiluminescent Western

200blotting detection reagent (Amersham, Italy); profiles were

201acquired and quantified by scanning with a Discover

202Pharmacia scanner equipped with a Sun Spark Classic

203Workstation. The anti-Bcl-2 (100SC509), anti-p21cip1

204(C-19), anti-p53 (DO-1), anti-HSP70 (K-20), anti-p16

205INK4A (C-20), and actin (C-2) antibodies were from Santa

206Cruz Biotechnology (Santa Cruz, CA, USA). Anti-tubulin

207(MCA77G) was from Serotec (Kidlington, UK). Gel

208loadings were internally normalized by probing the filters

209with anti-tubulin or anti-actin antibodies.

210Statistical analysis

211All data are expressed as mean ± SD. Significance was

212assessed by the Student’s t test for unpaired data for

213comparisons between two means. Statistical significance

214was defined as *P\ 0.01; **P\ 0.001; ***P\ 0.0001.

215Results and discussion

216Exposure of growing MCF10a cells or MCF7 cells to

217arginine-free medium for up to 5 days caused growth arrest

218in both cell lines (Fig. 1, upper panels). These cells nor-

219mally grow in media containing different levels of arginine

220or arginine precursors (e.g., proline). In fact, MCF10a grow

221in a medium that contains high levels of arginine (about

222250 mg/L and about 15 mg/L of proline), whereas MCF7

223cells, which express high levels ASS [16], require far less

224arginine (about 85 mg/L and no proline). The upper panels

225of Fig. 1 suggest that after 5 days of arginine deprivation,

226MCF10a cells were decreased, and MCF7 slightly aug-

227mented. However, the differences are not significant. A few

228MCF7 cells were dead, as suggested by the appearance of a

229small but reproducible sub-G1 fraction (see below)

230amounting to\3% at 5 days and\10% at 10 days. Cell

231death could be attributed to a non-apoptotic mechanism

232since neither caspase 3 nor caspase 7 were activated. The

233cytofluorimetric Annexin V test, performed at day 5, was

234negative in both cell lines (data not shown).

235Both cell lines showed different, yet profound, mor-

236phological changes (Fig. 1, lower panel): MCF10a cells

237acquired a flat phenotype and became positive for

238SA-b-galactosidase staining, consistent with premature

239senescence, whereas MCF7 cells remained negative to

240SA-b-galactosidase staining. MCF7 cells killed by arginine

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241 deprivation appeared significantly micronucleated while

242 displaying remarkable nuclear fragmentation (indicated by

243 the arrow in the lower panel of Fig. 1). This observation,

244 together with the lack of apoptotic signals, is consistent

245 with cell death by mitotic catastrophe [17]. The impact of

246 an identical metabolic stress generates different responses

247 in normal-like cells and in malignant cells of similar tissue

248 origin.

249 Following re-addition of arginine (?R) to the media of

250 the two cell lines, their behavior was different; thus, cell

251 proliferation, as measured by cell number (Fig. 1, upper

252 panel), resumed in MCF7 cells, but did not change

253 appreciably in MCF10a. Consistent with this observation,

254 arginine re-addition determined a significant increase of

255DNA synthesis (40%) in MCF7 but not in MCF10a cells

256(1–2%).

257In order to investigate the molecular mechanisms under-

258lying these differences, we studied cell-cycle profiles of the

259two cell lines after arginine depletion (-R) and

260re-addition (?R). As reported in Table 1, cell-cycle patterns

261indicate a time-dependent increase in the number of both

262MCF7 and MCF10a cells arrested in G0/G1 during arginine

263starvation. The modest reduction of MCF7 cells in G0/G1 at

26410 days of arginine starvation reflects, most likely, the frac-

265tionof dead/dying cells.Resumption of proliferation ofMCF7

266cells after arginine re-addition is accompanied by progression

267along the cell cycle. This was not the case for MCF10a cells

268that remained essentially arrested in G0/G1 (not shown).

0 2 4 6 8 10 12 14

Cell

s x

10

-3

MCF10a

days

1000

100

10

1

+R

-R

0 2 4 6 8 10 12 14

MCF7

Cell

s x

10

-3

days

1000

100

10

1

+R

-R

MCF10a

MCF7

a b

c d

Fig. 1 Upper growth curves. Cells were starved in arginine-free

medium (-R) for 5 days, washed, and released into complete

medium (?R) or maintained into medium without arginine (-R),

as indicated. The points represent the average of three sets of

duplicates. When analyzed statistically, differences in growth (?R

and -R) were not significant in MCF10a and highly significant

(P\ 0.0001) in MCF7. Lower arginine starvation, premature senes-

cence, and cell morphology. MCF10a: Proliferating (a) and 10 day-

starved cells (b). Several flattened, SA-b-galactosidase-positive cells

are clearly visible in b. MCF7: proliferating (c) and 10 day-starved

cells: The arrow points to a micronucleated cell enlarged in the insert

of d

Table 1 Distribution of cell populations in the cell cycle in arginine-free media

MCF7 MCF10a

Arginine-free medium (days) 0 1 3 5 10 0 1 3 5 10

G0/G1 54 ± 3 69 ± 2 75 ± 5 81 ± 3 75 ± 4 56 ± 5 76 ± 4 79 ± 5 83 ± 5 80 ± 4

S 31 ± 5 21 ± 3 5 ± 4 9 ± 2 9 ± 1 28 ± 3 15 ± 2 11 ± 4 8 ± 5 6 ± 2

G2/M 15 ± 4 10 ± 3 20 ± 1 7 ± 1 8 ± 2 16 ± 4 9 ± 3 9 ± 2 9 ± 3 14 ± 1

Sub—G1 0 0 0 3 ± 3 9 ± 4 0 0 0 0 0

MCF7 and MCF10a were incubated for different times (1, 3, 5 and 10 days) in an arginine-free medium then analyzed by flow cytometry. Data

have been obtained from triplicate samples and reported as the mean ± SD

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269 Mechanisms of cell-cycle arrest and senescence

270 Differences in the response to arginine deprivation

271 prompted us to investigate the molecular mechanisms

272 triggered in the two cell lines. Attention has been focused

273 on the senescent phenotype, which indicates a major dif-

274 ference between MCF10a and MCF7 cells in response to

275 metabolic stress. DNA-damaging stresses and oncogene

276 activation, as well as agents that induce telomere shorten-

277 ing, can also channel a cell toward a senescence program.

278 This is initiated by the activation of various cell-cycle

279 inhibitors and requires the functions of several proteins,

280 including p53, the CDKN1A gene product WAF1 (also

281 known as p21cip1), the CDKN2A gene product, INK4A

282 (also known as p16INK4A), and others.

283 Analysis of proteins involved in transduction of senes-

284 cence [18] inducing signals has focused on cyclin depen-

285 dent kinase (CDK) inhibitors. The expression of p21cip1 has

286 been measured in MCF10a and MCF7 cells exposed to

287 medium without and with arginine (Fig. 2). Exposure to

288 arginine-free medium (-R) caused a significant burst of

289 p21cip1 in both cell lines, which declined rapidly while

290 arginine starvation continued and growth was arrested.

291 Previous reports showed that during the decline of p21cip1,

292 the expression levels of p16INK4A, another CDK inhibitor,

293 gradually increases in senescent cells assuming, with time,

294 a pivotal role in the maintenance of the senescence pro-

295 gram [18]. Consistent with these observations, the levels of

296 p16INK4A increased in a time-dependent manner in

297 MCF10a cells (Fig. 2). In addition, the finding that the

298 p21cip1 decline could not be reversed is illustrated by

299 the sustained increase of p16INK4A that prevents the loss of

300 the senescent phenotype upon arginine restoration.

301 Protein p21cip1 is a downstream effector of p53. During

302 the first 5 days of starvation, the expression of p53 in

303 MCF10a cells resembles that of p21cip1, with the notable

304 difference that p53 did not disappear at longer times.

305Arginine starvation also induced up-regulation of Bcl-2 in

306MCF10a, which was reversed after restoration of arginine.

307This finding is in line with the fate of MCF10a cells that

308proceed to premature senescence upon arginine starvation

309and is in agreement with recent studies showing that

310senescence may be accompanied by Bcl-2 up-regulation.

311Indeed, Bcl-2 protein expression is increased in naturally

312senescent human fibroblasts as well as in cells in which a

313senescence-like growth arrest is induced by different

314stresses [19]. Finally, we observed that arginine starvation

315caused a slightly delayed expression of heat shock protein

31670 (HSP70), which is known to protect cells against

317apoptosis [20]. Possibly, the sustained over-expression of

318this protein may be considered as an indicator of cell auto-

319protective activity against errors induced by different

320stimuli, including nutrient deficiency. This fact agrees with

321the view that HSP70, a protein that supports p53 stability

322under stress conditions contributes to the recovery of

323damaged proteins [21]. It is noteworthy that the HSP70

324expression, increased by prolonged arginine absence, was

325not reversed by arginine restoration. This is not at all

326surprising since the senescent phenotype of MCF10a cells

327is not reversed by arginine addition to the culture medium.

328In contrast to the MCF10a cells, senescent features did

329not appear in MCF7cells (Fig. 1, panel d and Table 2),

330although they undergo growth arrest when exposed to

331arginine-free medium (-R). Expression profiles of p21cip1

332and p53 diverged from those observed in MCF10a cells in

333that they appear to increase continuously up to 10 days of

334starvation (Fig. 2, right panel). If starvation was limited to

3355 days only, the addition of arginine promptly reversed this

336behavior.

337These results are in line with cytofluorimetric data

338showing that arginine starvation induced a reversible,

339p21cip1-mediated, G0/G1 arrest (Table 1). It is to be noted

340that the effects caused by arginine withdrawal on p16INK4

341null-MCF7 cells are not limited to (reversible) growth

days days

Bcl-2

MCF7

p53

HSP 70

p21 cip1

Actin

- R

3

510510

3

510510

p16 INK4 A

MCF10a

+ R

- R

3

51050+ R 3

51050

null

Actin

Fig. 2 Protein expression

following subtraction (-R) and

re-addition of arginine (?R).

Expression levels of p21cip1,

p53, HSP70 (upper panel),

Bcl-2, and p16INK4A (lower

panel) in MCF10a and MCF7

cells. The rightmost lanes refer

to extracts of cells starved of

arginine for 5 days and

re-exposed to arginine for 3

following days. Actin was used

to normalize gels loading

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342 arrest but include also the appearance of a sub-G1 fraction

343 (dead cells and/or fragments thereof) which, albeit small, is

344 measurable and reproducible in MCF7 and not evident in

345 MCF10a. The size of this fraction increases prolonging

346 starvation to 10 days (Table 1). Arginine starvation

347 dependent apoptosis in MCF7 cells appears to be ruled out

348 since Annexin test was negative (not shown). Necrosis

349 could not be ruled out; however, the occurrence of multi-

350 nucleated cells (inset of Fig. 1) would suggest that the

351 small fraction (10% or less of total cells) of damaged

352 MCF7 cells may have undergone mitotic catastrophe [22].

353 a-Difluoromethyl-ornithine (DFMO) and Nx-hydroxy-

354 L-arginine (NOHA) inhibit MCF10a and MCF7 cells

355 proliferation

356 The observation of the effects of arginine depletion reported

357 above prompted us to investigate whether or not these effects

358 could be mimicked by drugs that interfere with the major

359 arginine catabolic pathways summarized in the following

360 simplified scheme:

361

362

Arginine

NOHA

Ornithine

NO

Arginase

NOS NOS

ODCPolyamine

363

364 DFMO is an inhibitor of ornithine decarboxylase (ODC)

365 known to cause cytostatic effects by reducing the cellular

366 polyamines pool [23]. The drug has been also used to treat

367 malignancies and found to be successful only in patients

368 expressing high ODC levels [24].

369 Arginine metabolism may also proceed to NO biosyn-

370 thesis. This biosynthetic pathway in mammals involves the

371 oxidation of arginine to NO and citrulline. This reaction is

372 catalyzed by a particular class of nitric oxide synthases

373(NOS) and requires the formation of an intermediate,

374namely Nx-hydroxy-L-arginine (NOHA). This compound

375negatively regulates arginase activity so that it can inhibit

376cell proliferation of cancer cells characterized by high

377levels of this enzyme. In addition, it has been shown that

378treatment of various tumor cell lines with NOHA causes

379significant cellular effects including arrest of cell cycle,

380changes in mitochondrial permeability, and even cell death

381[25]. Besides the inhibitory action of NOHA on arginase

382activity, this compound potently regulates the production

383of NO since a massive presence of this intermediate may

384exert positive pressure on NO synthesis by mass action.

385We have studied the effects of DFMO and NOHA, on the

386proliferation rates of MCF10a and MCF7 cells. The con-

387centration of DFMO (5 mM) and NOHA (1 mM) has been

388chosen on the basis of our own experience and literature

389data [23, 25]. Both drugs inhibited cell proliferation, albeit

390with minor differences (Fig. 3, upper panels). These data

391were also confirmed by studying cell-cycle profiles. Treat-

392ment with NOHA for 5 days caused G0/G1 arrest in both

393cell lines. The accumulation amounted to *12 and *9%

394over the controls for MCF7 and MCF10a, respectively, and

395was accompanied by a concomitant loss of cells from the S

396phase (about 10% in both cell lines, Table 3).

397DFMO (5 days) appeared a more potent inhibitor of cell-

398cycle progression for both cell lines. Thus, theODC inhibitor

399forced a greater proportion (*25% over control) of MCF7

400cells into the G0/G1 phase while the number of cells in the S

401phase was reduced by a factor *4 (Table 3). Similarly, a

402larger fraction (about 15% over the control) of MCF10 cells

403treated with DFMO was arrested in the G0/G1 phase with a

404concomitant decrease in the S population (Table 3).

405This finding is in agreement with recent data showing

406that DFMO may induce G0/G1 arrest in both normal and

407transformed cells [23]. Switching cells incubated for

4085 days with NOHA to drug-free media for 3 days resulted

409in a full recovery of growth rate for both cell lines. DFMO

410treatment and release, following the identical sequence,

411had a similar effect on MCF7 cells; however, this was not

412the case for MCF10a cells, which were unable to recover

413from the DFMO-induced cell-cycle arrest (not shown).

414Wedid not use a direct inhibitor ofNOproduction, such as

415the L-arginine derivatives NG-nitro-L-arginine (L-NOARG)

416or NG-nitro-L-arginine methyl ester (L-NAME), which have

417been used to inhibit constitutive NO synthase (NOS) in dif-

418ferent biological systems [26]. Indeed, Reisser et al. [27]

419have shown that L-NAME also inhibited the activity of

420arginase of tumor cells in culture. More important, they

421showed that L-NAME inhibition of arginase activity per-

422sisted after treatment ceased. This irreversible effect on

423arginase commands caution when L-NAME is used on the

424assumption that it inhibits only NO synthase.

Table 2 SA-b-galactosidase activity in MCF7 and MCF10a cells

induced by arginine deprivation and re-addition

MCF7 MCF10a

Arginine-free medium

(days)

10 10 10 10

Arginine re-addition

(days)

– 10 – 10

Ability to grow – ? – –

Senescent Cells (%) 4 ± 0.5 1 ± 0.1 20 ± 2.8 18 ± 3.5

Both cell lines were incubated for 10 days in an arginine-free medium

or washed and released into fresh complete medium (?arginine) for

additional 10 days then analyzed for SA-b-galactosidase staining

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425 Effects of inhibitors on premature senescence

426 To study the effect of DFMO or NOHA on the onset of

427 premature senescence in MCF7 and MCF10a, cells were

428 incubated, as above, with the inhibitor. Cells were analyzed

429 after 5 days of drugs exposure or after 3 days of recovery

430 in drug-free medium. Neither NOHA nor DFMO caused

431 premature senescence in MCF7 cells incubated in the

432presence of the drug for 5 days. In contrast, MCF10a cells

433proved to be quite sensitive to DFMO treatments but were

434unaffected by NOHA treatment (Fig. 3, lower left).

435Exposure for 5 days to DFMO induced a 5-6-fold increase

436in the activity of SA-b-galactosidase and led to the

437senescent phenotype. More importantly, this phenotype

438persisted when the cells were transferred to a drug-free

439medium for an additional 3 days (Fig. 3, lower left panel).

440The behavior of MCF10a cells toward DFMO treatment

441very closely resembles that of exposure to arginine-free

442medium with a measurable increase in the proportion of

443senescent cells over the control (*25% in DFMO, *20%

444in arginine-free medium).

445To gain further insight in the molecular mechanisms

446causing this different behavior, we also evaluated the

447expression profile of some key proteins involved in pre-

448mature senescence, using proteins extracted from MCF7 or

449MCF10a cells after NOHA or DFMO treatment as indi-

450cated above. First, we analyzed the p21cip1 expression

451levels after NOHA treatment in MCF7 cells. Treatment for

4525 days induced a significant up-regulation of p21cip1 as

453well as p53 expression (Fig. 3 lower right panel). These

454changes were not evident in MCF10a cells. Also the

455p16INK4A level in MCF10a was not modified as compared

MCF10a

Time (days)

1 2 3 4

20

40

60

80

100

120

00

Cell

nu

mb

er

10

-4 CNT

DFMO

NOHA

MCF7

Time (days)

1 2 3 4

CNT

DFMO

NOHA

0

20

40

60

80

100

120

0

Cell

nu

mb

er

10

-4

Controls

DFMO

NOHA

5 days 5+3days

MCF10a

10

20

30

**

***

** *

**

Co

nt

5 d

5+

3d

NOHA

MCF10a

p16 INK4A

Actinp21cip1

p53

Actin

null

MCF7

Tubulin

Tubulin

p53

DFMO

p21 cip1

p16 INK4A

Co

n

5 d

5+

3d

null

Se

ne

sc

en

t c

ell

s (

%)

Fig. 3 Upper panels growth curves in presence of DFMO and

NOHA. Each point represents the average of three sets of duplicates.

Results are reported as the mean ± SD. Statistical significance was

assessed by the Student’s t test for unpaired data for comparisons

between two means and was defined as *P\ 0.01; **P\ 0.001;

***P\ 0.0001. Lower, left Premature senescence induced by DFMO

or NOHA in MFC10a cells. Staining of SA-b-galactosidase was

performed after 5 days of incubation or after 5 days of incubation

followed by 3 days of release in drug-free media. Each point

represents the average of three sets of triplicates. Results are reported

as the mean ± SD. Lower, right Protein expression induced by

DFMO or NOHA treatments. Expression levels of p21cip1, p53, and

p16INK4A in MCF10a and p16INK4A null-MCF7) cells. Filters were

probed for individual proteins as indicated. Actin and Tubulin were

used to normalize the two gels loading

Table 3 Distribution of cell populations in the cell cycle in MCF7

and MCF10a induced by treatment with DFMO or NOHA

MCF7 MCF10a

0 DFMO

5 mM

NOHA

1 mM

0 DFMO

5 mM

NOHA

1 mM

G0/G1 54 ± 3 80 ± 5 66 ± 2 56 ± 1 71 ± 4 65 ± 2

S 31 ± 3 8 ± 3 20 ± 4 28 ± 2 17 ± 1 19 ± 4

G2/M 15 ± 1 12 ± 1 14 ± 2 16 ± 4 12 ± 2 16 ± 3

MCF7 and MCF10a cells were incubated in the presence of DFMO or

NOHA (5 and 1 mM, respectively). The media were replaced every

second day. At day 5, cells were analyzed by flow cytometry. Data

have been obtained from triplicate samples and reported as the

mean ± SD

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456 to controls (MCF7 are p16INK4A null). It is noteworthy that

457 all changes observed in MCF7 protein expression disap-

458 peared after switching cells to drug-free media for a min-

459 imum of 3 days (Fig. 3, lower right panel). This divergent

460 effect is not easy to explain but can be ascribed to the

461 dissimilar arginase and ODC activities described previ-

462 ously in various cells including breast cancer cells [28].

463 DFMO treatment appeared to have only a moderate

464 effect on p21cip1 and p53 profiles in both MCF7 and

465 MCF10a cells and of p16 INK4A in MCF10a cells. It appears

466 that all changes observed in the normal-like MCF10a cells

467 when cultivated in the absence of arginine could also be

468 obtained by treatment with a specific inhibitor of poly-

469 amine synthesis, suggesting that inhibition of this pathway

470 is responsible for the divergent behavior of normal and

471 transformed cells.

472 In conclusion, we have observed that in two cell lines,

473 whose arginine requirement for normal growth is sub-

474 stantially different, prolonged arginine deprivation causes

475 growth arrest. Re-addition of arginine to the culture med-

476 ium elicits quite different response in the two cell lines;

477 thus, whereas MCF7 cancer cells resume growth, MCF10a

478 cells do not. It is to be noted that for optimal growth

479 MCF10a cells require much higher concentration of argi-

480 nine in the culture medium (see above) than MCF7 cells. It

481 is conceivable that the different dependence upon arginine

482 of the two cell lines stems from the fact that MCF7 cells

483 express high levels of ASS. The observation that pharma-

484 cological inhibition of ODC elicits effects similar to argi-

485 nine deprivation suggests that derangement of polyamine

486 synthesis is the critical arm of arginine metabolism altered

487 in both cell lines.

488 The assumption that arginine deprivation has a strong

489 inhibitory effect on proliferation of cancer cells may not be

490 always justified. In fact, this effect can be less dramatic on

491 cancer cells expressing high levels of ASS (1), which are

492 less dependent upon arginine supply, as well as on cancer

493 cells expressing active estrogen receptors (such as breast

494 MCF7 or lung A549 cells). The presence of estrogen-

495 responsive elements in the promoter region of the bcl-2

496 gene [29] makes these cells refractory to pro-apoptotic

497 stresses [29, 30] in vivo and in vitro when grown in serum-

498 containing media. Although the results of this study refer to

499 cell lines grown in vitro, they may be construed as a caveat

500 if arginine metabolism is considered a target for cancer

501 therapy.

502 Acknowledgments The project was supported by MERIT/MIUR503 project (cod. E61J10000200001) and in part by an Industrial Italian504 Research plan (PON01_02433). We are deeply indebted to doctors505 Jan and Edie Wolff (National Institutes of Health, Bethesda, MD,506 USA) for critical review of the manuscript.

507References

5081. Delage B, Fennel DA, Nicholson L, McNeish I, Lemoine NR,509Crook T, Szolarek PW. Arginine deprivation and argininosucci-510nate synthetase expression in the treatment of cancer. Int J511Cancer. 2010;126:2762–72.5122. Scott L, Lamb J, Smith S, Wheatley DN. Single amino acid513(arginine) deprivation: rapid and selective death of cultured514transformed, malignant cells. Br J Cancer. 2000;83:800–10.5153. Umeda M, Diringer D, Heidelberger C. Inhibition of the growth516of cultured cells by arginase and soluble proteins from mouse517skin. Isr J Med Sci. 1968;4:1216–22.5184. Brittenden J, Heys SD, Eremin O. L-arginine and malignant519disease: a potential therapeutic role? Eur J Surg Oncol. 1994;20:520189–92.5215. Szende B, Tyihak E, Trezl L. Role of arginine and its methylated522derivatives in cancer biology and treatment. Cancer Cell Int.5232001;1:3–7.5246. Pegg AE, McCann PP. Polyamine metabolism and function. Am525J Physiol. 1982;243:C212–21.5267. Mori M, Gotoh T. Regulation of nitric oxide production by527arginine metabolic enzymes. Biochem Biophys Res Commun.5282000;275:715–9.5298. Soule HD, Maloney TM, Wolman SR, Peterso WDJ, Brent R,530McGrath CM, Russo J, Pauley RJ, Jones RF, Brooks SC. Isolation531and characterization of a spontaneously immortalized human532breast epithelial cell line, MCF-10. Cancer Res. 1990;50:6075–86.5339. Soule HD, Vazquez J, Long A, Albert S, Brennan M. A human534cell line from a pleural effusion derived from a breast carcinoma.535J Nat Cancer Inst. 1973;51:1409–16.53610. Tesseraud S, Bigot K, Taouis M. Amino acid availability regu-537lates S6K1 and protein synthesis in avian insulin-insensitive QM7538myoblasts. FEBS Lett. 2003;540:176–80.53911. Love-Schimenti CD, Gibson DF, Ratnam AV, Bikle DD. Anti-540estrogen potentiation of anti-proliferative effects of vitamin D3541analogues in breast cancer cells. Cancer Res. 1996;56:2789–94.54212. Bradford MM. A rapid and sensitive method for the quantitation543of microgram quantities of protein utilizing the principle of544protein-dye binding. Anal Biochem. 1976;72:248–54.54513. Crescenzi E, Chiaviello A, Canti G, Reddi E, Veneziani BM,546Palumbo G. Low doses of cisplatin or gemcitabine plus Photo-547frin/photodynamic therapy: disjointed cell cycle phase-related548activity accounts for synergistic outcome in metastatic non-small549cell lung cancer cells:(H1299). Mol Cancer Ther. 2006;5:776–85.55014. Crescenzi E, Palumbo G, Brady HJ. Roscovitine modulates DNA551repair and senescence: implications for combination chemother-552apy. Clin Cancer Res. 2005;11:8158–71.55315. Laemmli UK. Cleavage of structural proteins during the assembly554of the head of bacteriophage T4. Nature. 1971;227:680–5.55516. Lee JB, Shim YJ, Shin YJ, Jeong SY, Oh J, Park GH, Lee KH,556Min BH. Arginine deiminase enhances MCF-7 cell radiosensi-557tivity by inducing changes in the expression of cell cycle-related558proteins. Mol Cells. 2008;25:305–11.55917. Okada H, Mak TW. Pathways of apoptotic and non-apoptotic560death in tumour cells. Nat Rev Cancer. 2004;4:592–603.56118. Roninson IB, Broude EV, Chang BD. If not apoptosis, then what?562Treatment- induced senescence and mitotic catastrophe in tumor563cells. Drug Resist Updat. 2001;4:303–13.56419. Tombor B, Rundell K, Oltvai ZN. Bcl-2 promotes premature565senescence induced by oncogenic Ras. Biochem Biophys Res566Commun. 2003;303:800–7.56720. Bivik C, Rosdahl I, Ollinger K. Hsp70 protects against UVB568induced apoptosis by preventing release of cathepsins and

Med Oncol




Au

tho

r P

ro

of

UNCORRECTEDPROOF

569 cytochrome c in human melanocytes. Carcinogenesis. 2007;28:570 537–44.571 21. Walerych D, Olszewski MB, Gutkowska M, Helwak A, Zylicz572 M, Zylicz A. Hsp70 molecular chaperones are required to support573 p53 tumor suppressor activity under stress conditions. Oncogene.574 2009;28:4284–94.575 22. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R,576 Kroemer G. Cell death by mitotic catastrophe: a molecular def-577 inition. Oncogene. 2004;23:2825–37.578 23. Koomoa DL, Yco LP, Borsics T, Wallick CJ, Bachmann AS.579 Ornithine decarboxylase inhibition by alpha-difluor-580 omethylornithine activates opposing signaling pathways via581 phosphorylation of both Akt/protein kinase B and p27Kip1 in582 neuroblastoma. Cancer Res. 2008;68:9825–31.583 24. Shantz LM, Levin VA. Regulation of ornithine decarboxylase584 during oncogenic transformation: mechanisms and therapeutic585 potential. Aminoacids. 2007;33:213–23.586 25. Singh R, Pervin S, Chaudhuri G. Caspase-8-mediated BID587 cleavage and release of mitochondrial cytochrome c during588 Nomega-hydroxy-L-arginine-induced apoptosis in MDA-MB-468589 cells. antagonistic effects of L-ornithine. J Biol Chem. 2002;277:590 37630–6.

59126. Pfeiffer S, Leopold E, Schmidt K, Brunner F, Mayer B. Inhibition592of nitric oxide synthesis by NG-nitro-L-arginine methyl ester593(L-NAME): requirement for bioactivation to the free acid,594NG-nitro-L-arginine. Br J Pharmacol. 1996;118:1433–40.59527. Reisser D, Onier-Cherix N, Jean-Francois J. Arginase activity is596inhibited by l -NAME, both in vitro and in vivo. J Enzyme Inhib597Med Chem. 2002;4:267–70.59828. Hu X, Washington S, Verderame MF, Manni A. Interaction599between polyamines and the mitogen-activated protein kinase600pathway in the regulation of cell cycle variables in breast cancer601cells. Cancer Res. 2005;65:11026–133.60229. Perillo B, Sasso A, Abbondanza C, Palumbo G. 17beta-estradiol603inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via604two estrogen-responsive elements present in the coding sequence.605Mol Cell Biol. 2000;20:2890–901.60630. Stabile LP, Davis AL, Gubish CT, Hopkins TM, Luketich JD,607Christie N, Finkelstein S, Siegfried JM. Human non-small cell608lung tumors and cells (A549) derived from normal lung express609both estrogen receptor a and b and show biological responses to610estrogen. Cancer Res. 2002;62:2141–50.

611

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