R E S EA RCH AR T I C L E

Protection against cisplatin in calorie-restricted Saccharomycescerevisiae is mediated by the nutrient-sensor proteins Ras2,

Tor1, or Sch9 through its target Glutathione

Diana Mariani1, Frederico A.V. Castro1, Luciana G. Almeida1, Fernanda L. Fonseca2,3

& Marcos D. Pereira1

1Departamento de Bioqu�ımica, Instituto de Qu�ımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2Centro de

Desenvolvimento Tecnol�ogico em Sa�ude, CDTS Fiocruz, Rio de Janeiro, RJ, Brazil; and 3Instituto de Microbiologia Professor Paulo de G�oes,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Correspondence: Marcos D. Pereira,

Avenida Athos da Silveira Ramos, 149, Bloco

A - 5° andar, laborat�orio 547, Cidade

Universit�aria, Rio de Janeiro, RJ, Brasil, CEP:

21941-909. Tel.: +55 21 2562 7735;

fax: +55 21 2562 7266;

e-mail: marcosdp@iq.ufrj.br

Received 2 April 2014; revised 5 August

2014; accepted 2 September 2014. Final

version published online 21 October 2014.

DOI: 10.1111/1567-1364.12214

Editor: Ian Dawes

Keywords

calorie restriction; cisplatin; Ras2, Sch9, or

Tor1; glutathione; Saccharomyces cerevisiae.

Abstract

There is substantial interest in developing alternative strategies for cancer che-

motherapy aiming to increase drug specificity and prevent tumor resistance.

Calorie restriction (CR) has been shown to render human cancer cells more

susceptible to drugs than normal cells. Indeed, deficiency of nutrient signaling

proteins mimics CR, which is sufficient to improve oxidative stress response

and life expectancy only in healthy cells. Thus, although CR and reduction of

nutrient signaling may play an important role in cellular response to chemo-

therapy, the full underlying mechanisms are still not completely understood.

Here, we investigate the relationship between the nutrient sensor proteins Ras2,

Sch9, or Tor1 and the response of calorie-restricted Saccharomyces cerevisiae

cells to cisplatin. Using wild-type and nutrient-sensing mutant strains, we show

that deletion of any of these proteins mimics CR and is sufficient to increase

cell protection. Moreover, we show that glutathione (GSH) is essential for

proper CR protection of yeast cells under cisplatin chemotherapy. By measur-

ing the survival rates and GSH levels, we found that cisplatin cytotoxicity leads

to a decrease in GSH content reflecting in an increase of oxidative damage.

Finally, investigating DNA fragmentation and apoptosis, we conclude that GSH

contributes to CR-mediated cell survival.

Introduction

The search for alternative strategies during cancer chemo-

therapy aiming to increase drug specificity and prevent

tumor resistance has generated substantial interest. One

of these approaches, calorie restriction (CR), makes

human cancer cells more susceptible to drugs if compared

with normal cells (Lee et al., 1998, 2012; Raffaghello

et al., 2008). For instance, the combination of a chemo-

therapy drug, for example, cisplatin, with 2-deoxy-D-glu-

cose (2DG), a glucose analog which cannot be further

metabolized by cells, enhances cytotoxicity of cancer cells

by a mechanism related to oxidative stress (Ahmad et al.,

2005; Andringa et al., 2006; Simons et al., 2007). Such

findings indicate a new horizon for cancer treatment

using CR as an alternative for sensitizing cancer rather

than normal cells.

The process of CR is characterized by the reduction of

calorie intake, generally, c. 20–40%, without compromis-

ing the normal daily diet. It has been suggested that CR

is the best alternative intervention, which improves cellu-

lar function of many species (Dilova et al., 2007; Fontana

et al., 2010). From yeast to humans, CR triggers complex

signaling pathways that promote healthy extension of life

due to the enhancement of reproductive time, oxidative

stress response, and resistance to abnormal toxic proteins

(Roth et al., 2002; Bordone & Guarente, 2005; Dilova

et al., 2007). Recently, CR has also been related to pre-

vention of several human pathologies such as obesity, dia-

betes, hypertension, and atherosclerosis, protection and

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

YEA

ST R

ESEA

RC

H

reduction risk for factors for cancer and cardiovascular

disease, and last but not least, reduce neurodegeneration

(Gross & Dreyfuss, 1990; Roth et al., 2002; Hursting

et al., 2003; Bordone & Guarente, 2005; Dilova et al.,

2007). Although much attention has been dedicated to

understanding the effects of CR, the exact mechanism by

which it exerts such benefits is not completely clear. It is

possible that the lower availability of nutrients during CR

is detected by sensors and effectors, triggering signaling

cascades that account for beneficial effects. Indeed, previ-

ous studies indicated that Ras2, Tor1, and Sch9 are nutri-

ent-responsive proteins related to signaling cascades that

modulate cell metabolism in response to nutrients

(Thevelein et al., 2000; Pedruzzi et al., 2003; Roosen

et al., 2005). These regulatory proteins positively regulate

the expression of common effectors involved in several

processes related to cell growth, such as nutrient uptake,

biogenesis of ribosomes, and protein synthesis (Thevelein

et al., 2000; Martin et al., 2004; Roosen et al., 2005;

Wullschleger et al., 2006). Moreover, they negatively reg-

ulate other important processes, such as stress response

and autophagy (Rolland et al., 2002; Minina et al., 2013).

Interestingly, the decrease (or deletion) of these protein

sensor activities increases replicative life span as well as

oxidative stress response and has been proposed as a

model of CR intervention (Rolland et al., 2002; Dilova

et al., 2007; Fontana et al., 2010; Minina et al., 2013).

Although it has been suggested that CR and reduction of

signaling mediated by nutrient-sensing proteins may play

an important role in cellular response to chemotherapy

(Lee et al., 1998, 2012; Raffaghello et al., 2008), the full

underlying mechanisms are still not completely under-

stood.

Here, we investigate the relationship between nutrient

sensor proteins and the response of yeast cells to chemo-

therapy drugs, for instance cisplatin. Our results show

that deletion of any single nutrient sensor protein tested,

that is, Ras2, Tor1, or Sch9, mimics CR and is sufficient

to increase cell protection against cisplatin. Analysis of

protein–protein interaction (PPI) networks demonstrates

that several transcription factors (TFs) involved in stress

response are negatively regulated by direct or indirect

interaction with Ras2, Tor1, and Sch9. Furthermore,

among the protective factors positively controlled by the

TFs found in PPI, the tripeptide glutathione (c-glutamyl-

cysteinyl-glycine, GSH) has a pivotal role in cells sub-

jected to the effects of cisplatin, possibly acting in the

control of redox status, with consequences to oxidative

stress balance and apoptosis.

Material and methods

Yeast strains and growth conditions

Saccharomyces cerevisiae strains used in this work are

listed in Table 1. Stocks of all strains were maintained on

solid 2% YPD (2% glucose, 1% yeast extract, 2% pep-

tone, and 2% agar) at 4 °C to avoid the selection of

petites or suppressors. For all experiments using the

BY4741 background, cells were grown up to the early

exponential phase (0.8 mg dry weight mL�1) in either

liquid 2% YPD (2% glucose, 1% yeast extract, and 2%

peptone) or 0.5% YPD (1% yeast extract, 0.5% glucose,

and 2% peptone) medium using an orbital shaker at

28 °C and 160 r.p.m., with the ratio of flask volume:

medium of 5 : 1. The wild-type and isogenic strains from

DBY476 background were also grown up to the early

exponential phase but in liquid 2% SD (2% glucose,

0.67% nitrogen base without amino acids) or 0.5% SD

(2% glucose and 0.67% nitrogen base without amino

acids) supplemented with required amino acids (see

genotypes in Table 1).

Evaluation of cisplatin toxicity

Cytotoxicity was analyzed by direct addition of cisplatin

(80 or 450 lM) to 10 mL of the cell culture for 1, 2, and

24 h at 28 °C/160 r.p.m. Cellular viability was monitored

before and after cisplatin exposition by plating, after

proper dilution (10009 in distilled water) in triplicate,

on solidified 2% YPD medium. Then, the plates

were incubated at 28 °C for 72 h and the colonies

counted. Cellular viability was expressed as percentage of

survival.

Table 1. Strains of the yeast Saccharomyces cerevisiae used in this work

Strains Genotype Phenotype Source

DBY476 MATa, leu 2-3.112, his 3Δ1, trp 1-289, ura 3-52, GAL+ Wild type *

sch9D Isogenic to DBY746 except SCH9::URA Sch9 deficient *

ras2D Isogenic to DBY746 except RAS2::LEU Ras2 deficient *

tor1D Isogenic to DBY746 except TOR1::HIS Tor1 deficient *

BY4741 MATa; his3; leu2; met15; ura3 Wild type †

gsh1D Isogenic to BY4741 except GSH1::KanMX4 GSH deficient †

*Andrus Gerontology Center, Department of Biological Sciences, and Norris Cancer Center, University of Southern California, Los Angeles, USA.†Euroscarf, Institute of Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany.

FEMS Yeast Res 14 (2014) 1147–1159ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

1148 D. Mariani et al.

Protein–protein interaction (PPI) network

design and gene ontology (GO) analysis

PPI network between the nutrient-sensing proteins Ras2,

Tor1, and Sch9 and stress responsive proteins as well as

GO was essentially performed as described by Bonatto

(2007). For PPI network analysis, a multiple name search

was performed using STRING 9.05 database (http://

string-db.org/) with limited number of interactors and

high confidence. The obtained PPI networks were

recorded and analyzed using the freeware CYTOSCAPE ver-

sion 3.0.1 available at http://www.cytoscape.org/plugins2.

php. Analysis of GO was carried out with all proteins

obtained in the nutrient-sensing protein PPI network

designed by CYTOSCAPE. Statistical evaluation of GO was

determined by Biological Network Gene Ontology (BINGO)

using the actual annotations of the Gene Ontology Con-

sortium (http://www.geneontology.org).

Determination of glutathione contents

Glutathione, reduced (GSH) or oxidized (GSSG) forms,

was determined spectrophotometrically, in neutralized

10% trichloroacetic acid (TCA) extracts obtained from

cells subjected or not to cisplatin (80 or 450 lM). GSH

and GSSG were measured according to Bernt & Bergmey-

er (1974).

Analysis of lipid peroxidation

For lipid peroxidation determination, 50 mg of cells sub-

jected to cisplatin (450 lM/2 h) at 28 °C was cooled on

ice, harvested by centrifugation, and washed twice with

20 mM Tris-HCl buffer, pH 7.4. Cells were resuspended

in 500 lL of the same buffer, and 1.5 g of glass beads

was added. The samples were lysed by three cycles of 1-

min agitation on a vortex mixer followed by 1 min on

ice and the extracts were used for detection of malondial-

dehyde-MDA as described by Steels et al. (1994).

Evaluation of aconitase inactivation

Aconitase activity was determined spectrophotometrically

before and after exposition of cells to 450 lM cisplatin

for 2 h using an isocitrate dehydrogenase NADPH-depen-

dent coupled reaction, as previously reported by Gardner

et al. (1995).

Analysis of chromatin fragmentation and

apoptosis

Determination of chromatin fragmentation and apoptosis

was performed before and after exposition to cisplatin

(450 lM/2 h). For chromatin fragmentation, cells were

washed two times with PBS, incubated with 5.0 lg mL�1

DAPI (Sigma-Aldrich, St. Louis) in PBS for 30 min and

then washed three times with PBS. Apoptosis was ana-

lyzed by exposition of phosphatidylserine, which was

detected by the reaction with FITC-coupled annexin V

(Annexin V-FITC Apoptosis Detection Kit – APOAF,

Sigma-Aldrich), essentially as described by Madeo et al.

(1997). In both assays, the yeast cells were applied to

microscope slides and observed using an Axioplan 2 fluo-

rescence microscope (Zeiss, Germany). Images were

acquired using a Color View SX digital camera and were

processed with the ANALYSIS software system (Soft Imaging

System) prior to processing with IMAGEJ software (pro-

vided by NIH, http://rsb.info.nih.gov/ij/). To determine

the frequencies of morphological phenotypes (DAPI and

annexinV), at least 300 cells of three independent experi-

ments were evaluated.

Data analysis

The results represent the mean � SD of at least three

independent experiments. Statistical differences were

tested using Student’s t-test. The latter denotes homoge-

neity between experimental groups at P < 0.05.

Results and discussion

Nutrient-sensing proteins affect the response

of S. cerevisiae to chemotherapy by interacting

with TFs

In several biological systems, the nutrient-sensing signal-

ing pathways modulate life span extension and resistance

to oxidative stress (Droge, 2005; Powers et al., 2006; Wei

et al., 2009). In fact, deficiency in a single nutrient-sens-

ing protein such as Ras2, Sch9, or Tor1 is sufficient to

improve oxidative stress response and life expectancy

(Kale & Jazwinski, 1996; Fabrizio et al., 2003; Powers

et al., 2006). Interestingly, deletion of genes that code for

these proteins mimics CR, which explains life span exten-

sion in several species (Fabrizio et al., 2003; Hursting

et al., 2003; Bordone & Guarente, 2005; Powers et al.,

2006; Dilova et al., 2007; Wei et al., 2009).

To investigate whether nutrient-sensing proteins are

related to chemotherapy response, we subjected the wild-

type, DBY746, and Ras2, Tor1, and Sch9 mutant cells to

cisplatin. The absence of one of the nutrient sensor proteins

increased survival of cells after cisplatin treatment (Fig. 1a).

We also tested the performance of these mutant cells to

other chemotherapy drugs such as carboplatin, doxorubi-

cin, etoposide, and 5-fluorouracil. Similarly, all mutant

cells were extremely resistant against chemotherapy

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Protection against cisplatin 1149

(Supporting Information, Fig. S1). Our results indicate that

a common protective mechanism against these chemother-

apeutic drugs seems to be triggered due to the reduction or

impairment of nutrient-sensing signaling through Ras2,

Tor1, and Sch9. The physical interaction between these

nutrient sensors may explain why the absence of a single

nutrient sensor can disrupt nutrient signaling. We have

also confirmed that mutation in these nutrient sensors can

mimic CR. Accordingly, calorie-restricted wild-type strain

presented the same survival profile of mutants when

exposed to cisplatin (Fig. 1b). Taken together, these results

indicate that CR and mutations in nutrient-sensing path-

ways share the same survival mechanisms during cisplatin

chemotherapy.

System biology is a powerful tool widely used to inte-

grate the high amount of physical protein–protein inter-

actions (PPPI) of biological systems solidly grounded on

molecular-level understanding. We performed PPI net-

work analysis between the nutrient-sensing proteins

Ras2, Tor1, and Sch9 and responsive stress targets. As

expected, PPPI network revealed a direct association

between these nutrient-sensing proteins (Fig. 2a and b).

In addition, GO clustering also showed that the most

relevant biological categories were mostly related to sig-

naling transduction, response to nutrient signals, cellular

metabolism related to cell proliferation, and response to

stress (Table 2).

At a molecular level, analysis of PPPI network indicates

that although nutrient-sensing proteins act through dis-

tinct signaling pathways, they overlap regarding stress-

related TFs controlled by Ras2, Tor1, and Sch9 (Fig. 2a

and b). For example, Tor and PKA signaling cascades

negatively regulate Rim15 contributing to repression of

stress genes and stimulating cellular proliferation (Ped-

ruzzi et al., 2003; Swinnen et al., 2006). Rim15 is a ser-

ine/threonine protein kinase repressed by glucose that

plays a crucial function in the upregulation of stress

responsive genes as well as G0 entrance by activating

Msn2/4 and Gis1 (Pedruzzi et al., 2003; Cameroni et al.,

2004; Swinnen et al., 2006;). Furthermore, MCODE analysis

of our PPPI network revealed three subnetworks with

high score and strong significance (5.533; 2.923; and

1.917). These three networks were merged using the CYTO-

SCAPE core Merge Networks plug-in, and the results

obtained indicated the presence of the most relevant fac-

tors related to stress response: heat shock factor 1 (Hsf1),

multicopy suppressor of SNF1 mutation 2 and 4 (Msn2

and Msn4, respectively), suppressor of Kre Null 7 (Skn7),

Glg1-2 suppressor (Gis1), UREidosuccinate transport

(Ure2), and the regulator of IME2 (Rim15) (Fig. 2b).

To find new insights regarding the involvement of

nutrient-sensing proteins and response to stress, we fur-

ther explored within the PPPI network the possible inter-

actions between Ras2, Tor1, and Sch9 and stress-related

TFs. Among the thirteen TFs obtained in our PPPI net-

work, six factors presented high relevance for stress sig-

naling: the well-studied Hsf1, Msn2 and Msn4, Yap1,

Skn7, and Gis1 (Table 3). TFs regulate the expression of

several genes including some coding for major protective

factors (heat shock proteins, antioxidant enzymes, and

glutathione) involved in cell maintenance during stress

conditions (Schmitt & McEntee, 1996; Lee et al., 1999;

Raitt et al., 2000; Hahn et al., 2004; Harbison et al.,

2004). A cross-talk between some of these TFs and nutri-

ent sensors has been reported, strengthening the connec-

tion between them and the appropriate cellular response

under stress conditions (Powers et al., 2006; Swinnen

et al., 2006).

To the best of our knowledge, protective factors such

as superoxide dismutases, catalases, and GSH are under

regulation by the TFs obtained in our PPPI networks

(Table 4). Although those proteins are highly relevant for

cell maintenance under stress conditions, only the absence

of GSH was critical for cell survival under cisplatin expo-

sure (Fig. S2). GSH, synthesized by a coupled reaction

(a)

(b)

Fig. 1. Nutrient-sensing genes modulate survival of Saccharomyces

cerevisiae subjected to cisplatin. (a) The wild-type (DBY746) and

nutrient-sensing mutants (ras2D, tor1D, and sch9D) cells were grown

to early exponential phase in 2% SD media, subjected directly to

80 lM cisplatin for 24 h and then plated on solid 2% YPD. (b) The

wild-type cells were grown to early exponential phase in 2% SD or

0.5% SD media, subjected directly to 80 lM cisplatin for 24 h and

then plated on solid 2% YPD. Cellular survival was determined as the

percentage of viable cells after cisplatin exposition. The results

represent the mean � SD of at least three independent experiments

(*means different results at P < 0.05).

FEMS Yeast Res 14 (2014) 1147–1159ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

1150 D. Mariani et al.

(a)

(b)

Fig. 2. A protein–protein interaction network between the nutrient-sensing proteins Ras2, Tor1, and Sch9. These three initial proteins (black)

were used to create a network of physical protein–protein interactions (60 interactors) whose information was obtained from Saccharomyces

cerevisiae database (a). MCODE analysis of PPPI network revealed three subnetwork merged using the CYTOSCAPE core Merge Networks plug-in (b).

TFs involved in stress response are also highlighted (gray circles). All edges represent first (one-way) interactions.

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Protection against cisplatin 1151

catalyzed by c-glutamylcysteine synthetase (Gsh1) and

glutathione synthetase (Gsh2), is the most abundant

intracellular low molecular weight thiol within cells. It

may have an outstanding function because it is involved

in several physiological processes such as the control of

intracellular redox status by reacting with ROS or bind-

ing to sulfhydryl groups on proteins (glutathiolation),

drug metabolism, DNA synthesis, protein stabilization,

mitochondrial function and integrity, cell proliferation,

and apoptosis (Grant et al., 1996; Ballatori et al., 2009;

Pallard�o et al., 2009). GSH is also a substrate for enzymes

such as glutathione transferases, which catalyze the nucle-

ophilic attack on several electrophilic xenobiotics favoring

the efflux of conjugates from cells (Hayes et al., 2005).

Interestingly, cisplatin can be detoxified through this

mechanism of drug efflux as glutathione-Pt complex, Pt

Table 3. TFs obtained from the PPI network under the regulation of Ras2, Tor1, and Sch9 nutrient protein sensors

Name Biochemical function*

Negatively regulated

Gln3 Positively regulating genes that are subject to nitrogen catabolite repression

Skn7 Activates gene expression in response to heat, oxidative, osmotic, and hypoxic stress conditions

Msn2 Activates cellular general stress response under adverse conditions

Msn4 Activates cellular general stress response under adverse conditions

Hsf1 Activates multiple genes in response to highly diverse stresses, mainly heat stress

Gis1 Activator or repressor involved in the expression of genes involved in postdiauxic shift

Yap1 Activator involved in oxidative stress response and redox homeostasis

Positively regulated

Mbp1 Stimulates proliferation by inducing the expression of genes related to transition from G1 to S phase

Gcn4 Transcriptional activator of amino acid biosynthetic genes. Activated by amino acid starvation prior to entering cell cycle progression

Sfp1 Positive regulator of genes related to ribosome biogenesis

Tec1 Positively controls cell division and vegetative adhesion

Maf1 Negative regulator of RNA polymerase III (pol III) transcription

Ste12 Induces gene expression to mating or pseudohyphal/invasive growth pathways

*Biochemical function was extracted from S. cerevisiae genome database http://www.yeastgenome.org/.

Table 2. Analysis of GO performed using all proteins from the protein–protein network generated by CYTOSCAPE

Biological process (GO category) GO No. P value Corrected P value* k† f‡

Signal transmission 23060 6.51 9 10�25 4.96 9 10�22 33 262

Biological regulation 65007 1.91 9 10�20 2.56 9 10�18 58 1792

Aging 7568 1.93 9 10�18 1.42 9 10�16 19 74

Ras protein signal transduction 7265 2.76 9 10�12 1.85 9 10�10 12 34

Response to stimulus 50896 7.88 9 10�9 4.53 9 10�7 35 965

Response to chemical stimulus 42221 3.86 9 10�8 1.83 9 10�6 23 399

Second-messenger-mediated signaling 19932 7.28 9 10�8 3.25 9 10�6 9 29

Regulation of cell size 8361 3.06 9 10�7 1.30 9 10�5 13 104

Regulation of cAMP biosynthetic process 30817 1.08 9 10�6 3.48 9 10�5 5 5

Regulation of cyclic nucleotide biosynthetic process 30802 1.08 9 10�6 3.48 9 10�5 5 5

Developmental process 32502 2.45 9 10�6 7.04 9 10�5 21 399

Regulation of molecular function 65009 3.49 9 10�6 9.03 9 10�5 14 153

Regulation of catalytic activity 50790 3.59 9 10�6 9.03 9 10�5 13 126

Regulation of cellular metabolic process 31323 1.40 9 10�5 2.89 9 10�4 32 1045

Protein amino acid phosphorylation 6468 1.61 9 10�5 3.17 9 10�4 13 142

Regulation of transcription, DNA-dependent 6355 6.72 9 10�5 1.13 9 10�3 21 478

TOR signaling cascade 31929 7.71 9 10�5 1.27 9 10�3 6 16

Regulation of RNA metabolic process 51252 1.01 9 10�4 1.61 9 10�3 21 489

Response to abiotic stimulus 9628 1.03 9 10�4 1.61 9 10�3 13 165

Regulation of cell growth 1558 4.75 9 10�4 6.48 9 10�3 5 11

Response to stress 6950 5.99 9 10�4 7.65 9 10�3 24 704

Cellular response to nutrient levels 31669 8.15 9 10�3 8.48 9 10�2 8 74

*P values calculated after FDR application.†Number of proteins found in the network that integrate a specific GO.‡Total number of proteins belonging to a specific GO.

FEMS Yeast Res 14 (2014) 1147–1159ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

1152 D. Mariani et al.

(GS)2 (Hayes et al., 2005; Kuo et al., 2007), which may

be of relevance in understanding the relationship between

GSH and the protection it confers to this chemotherapy

drug. Indeed, analyzing the levels of GSH in the wild-type

DBY746, we have observed that cisplatin chemotherapy

caused a significant reduction (�fourfold) in the levels of

GSH (Fig. 3a). On the other hand, in the nutrient-sensing

mutant strains Tor1 and Sch9, the same treatment with

cisplatin did not affect the levels of GSH (Fig. 3a).

Regarding to Ras2 mutant strain, the 1.5-fold reduction

did not seem to be relevant to promote cell susceptibility

during cisplatin exposure. Furthermore, analyzing the

GSH levels in calorie-restricted wild-type (DBY746)

strain, we can observe the same GSH profile as presented

by the Ras2 mutant (Fig. 4). This result combined with

survival of Ras2 and calorie-restricted wild type indicates

that may exist a threshold in GSH levels necessary to pro-

tect cells during cisplatin chemotherapy. In accordance,

the treatment of cells with BSO (buthionine sulfoximine),

an inhibitor of GSH synthesis, conferred a strong sensitiv-

ity in all cells studied (Fig. S3) Interestingly, GSSG levels

remained unchanged in all strains (Figs 3b and 4b), indi-

cating that the decrease of GSH levels is mainly due to Pt

(GS)2 generation. In addition, these results also show that

Ras2, Tor1, and Sch9 single deletions may be advanta-

geous for GSH metabolism.

Table 4. Transcriptional regulators obtained in our PPPI networks related to stress-protective genes

Stress-related genes

SOD1 SOD2 CTT1 CTA1 GSH1

Documented Skn71, Yap12,

Msn23 and Msn43Skn71, Yap14, Msn25,

Msn46 and Hsf17Skn71,8, Yap11,8, Msn25,8,

Msn45,8, Hsf17 and Gis19Yap14,8 and Msn210 Skn71,2,6, Yap11,2,6,8,10,

Msn26 and Msn46

Potential Gis1 Gis1 Hsf1 Gis1 and Hsf1

1Lee et al. (1999)); 2Lee et al. (2002); 3Cameroni et al. (2004); 4Dubaq et al. (2006); 5Berry & Gasch (2008); 6Kelley & Ideker (2009; e1000488);7Eastmond & Nelson (2006); 8Gasch et al. (2000); 9Zhang & Oliver (2010); 10Harbison et al. (2004).

(a)

(b)

Fig. 3. Effect of cisplatin on the glutathione levels of Saccharomyces

cerevisiae mutants in nutrient-sensing proteins. The wild-type

(DBY746) and nutrient-sensing mutants (ras2D, tor1D, and sch9D)

cells were grown to early exponential phase in 2% SD media.

Intracellular (a) GSH and (b) GSSG, levels were determined

spectrophotometrically, in neutralized 10% TCA cell extracts, before

and after exposition of cells to 80 lM cisplatin for 24 h. The results

represent the mean � SD of at least three independent experiments

(*means different results at P < 0.05 compared with control cells).

(a)

(b)

Fig. 4. Effect of cisplatin on the glutathione levels of calorie-

restricted wild-type Saccharomyces cerevisiae. The wild-type (DBY746)

was grown to early exponential phase in 0.5% SD media. Intracellular

(a) GSH and (b) GSSG, levels were determined spectrophotometrically,

in neutralized 10% TCA cell extracts, before and after exposition of

cells to 80 lM cisplatin for 24 h. The results represent the

mean � SD of at least three independent experiments (*means

different results at P < 0.05 compared with control cells).

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Protection against cisplatin 1153

GSH is important for the fully operating CR

protection mechanism against cisplatin in

S. cerevisiae

Given the observed connection between nutrient-sensing

signaling pathways and GSH, we decided to further investi-

gate the involvement of GSH in CR benefits under cisplatin

treatment. Thus, using the wild-type BY4741 and the iso-

genic mutant strain deficient in Gsh1, we analyzed the

effect of GSH absence in calorie-restricted cell response

under cisplatin chemotherapy. As expected, cells of the

wild-type strain BY4741, growing on 2% glucose medium,

showed increased cisplatin susceptibility over time

(Fig. 5a). On the other hand, when calorie-restricted cells

were subjected to cisplatin treatment, resistance was

observed (Fig. 5b). This result confirms that regulating the

calorie uptake, by nutrient sensor modulation or even

nutrient intake, benefits cell response against cisplatin che-

motherapy. Accordingly, previous findings described that

dietary restriction protects from toxicities of

chemotherapeutic agents (Raffaghello et al., 2008; Lee

et al., 2012). However, the possibility of regulating calorie

intake to protect humans against the harmful effects of che-

motherapeutic agents is still under investigation (Safdie

et al., 2009). Interestingly, unlike the wild-type strain, the

gsh1 mutant lost the ability to respond to cisplatin even

when cells were cultured under CR (Fig. 5a and b). The

maintenance of GSH content in calorie-restricted cells

exposed to cisplatin, as revealed in Fig. 6, seems to account

for this improved survival. Indeed, the preservation of GSH

levels in calorie-restricted cells, after cisplatin exposure,

contributed to their high survival, whereas cells cultured in

high-glucose culture experienced a drop in GSH content

and decreased survival. These results point to a central role

of GSH for survival and cellular response during cisplatin

chemotherapy. Although there are several findings showing

the contribution of GSH for cellular protection, this is the

first evidence for a role of GSH in cellular protection con-

ferred by the CR regimen against cisplatin.

As cisplatin exposure induced a decrease in the GSH

content in cells grown in media containing 2% glucose,

we measured the extent of oxidative damage generated by(a)

(b)

Fig. 5. Intracellular glutathione is important for survival of

Saccharomyces cerevisiae cells subjected to cisplatin. The wild-type

(BY4741) and its isogenic mutant gsh1D were grown to early

exponential phase in 2% YPD (a) or 0.5% YPD (b) media, subjected

directly to 450 lM cisplatin for 24 h and then plated on solid 2%

YPD. Cellular survival was determined, at appropriate time, as the

percentage of viable cells after cisplatin exposition. The results

represent the mean � SD of at least three independent experiments

(*means different results at P < 0.05 compared with the wild-type

strain; **means different results at P < 0.05 compared with cells

cultured in 2% SD).

(a)

(b)

Fig. 6. Effect of cisplatin exposition on glutathione levels in

Saccharomyces cerevisiae cells growing on rich (2% YPD) or calorie-

restricted (0.5% YPD) media composition. The wild-type (BY4741)

was grown to early exponential phase in 2% YPD (a) or 0.5% YPD

(b) media. Intracellular GSH and GSSG, levels were determined

spectrophotometrically, in neutralized 10% TCA cell extracts, before

and after exposition of cells to 450 lM cisplatin for 2 h. The results

represent the mean � SD of at least three independent experiments

(*means different results at P < 0.05 compared with control cells;

**means different results at P < 0.05 compared with cells cultured in

2% SD).

FEMS Yeast Res 14 (2014) 1147–1159ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

1154 D. Mariani et al.

cisplatin treatment. The relevance of GSH-dependent CR

protection was also investigated. During oxidative stress,

lipids and intracellular proteins are the main targets of

ROS. While lipid peroxidation is a chain reaction causing

the loss of membrane properties and the production of

more toxic by-products, such as MDA (Steels et al.,

1994), the oxidative inactivation of proteins, such as

aconitase, occurs mainly due to the presence of iron–sul-fur (Fe-S) cluster that is susceptible to inactivation by

superoxide radicals (Gardner et al., 1995). Although cis-

platin halved the levels of GSH in the wild-type strain

(cultured in high glucose), this reduction was not fol-

lowed by damage to oxidative stress markers (Fig. 7a and

b). However, when the GSH synthesis was abolished in

the mutant strain gsh1D, we observed increased damage

in lipid peroxidation and inactivation of aconitase

(Fig. 7a and b). On the other hand, when calorie-

restricted cells were subjected to cisplatin, two distinct

aspects were observed; (1) an increase in the levels of

lipid peroxidation in the mutant gsh1D; and (2) a four-

fold increase in aconitase activity in the wild type in rela-

tion to the GSH-deficient strain. Indeed, the oxidative

damages were mainly observed in the gsh1D strain. It has

been described that depletion of GSH is related to defects

in iron homeostasis leading to an impairment of cellular

function. Furthermore, the authors stated that GSH is

not effective by itself to control cellular thiol-redox func-

tions contributing to enhanced cytotoxicity observed in

the mutant gsh1D (Kumar et al., 2011). Taken together,

besides cisplatin cytotoxicity, the abolishment of GSH

synthesis causes cellular disorders that put in check the

fully operating CR mechanism of protection.

Several data highlight the essential role of GSH for reg-

ulation of apoptosis signals (Rudin et al., 2003; P�ocsi

et al., 2004; Anathy et al., 2012). In addition, it has been

described that enhancement of intracellular ROS is deci-

sive to trigger apoptosis signals in yeast (P�ocsi et al.,

2004). Thus, an efficient antioxidant system is required

for the accurate balance between generation and elimina-

tion of intracellular ROS preventing the induction of the

hallmarks of programmed cell death. To investigate

whether cisplatin activates apoptosis in gsh1 mutant calo-

rie-restricted cells, we analyzed the levels of DNA frag-

mentation and the phosphatidylserine externalization by

DAPI and annexin staining, respectively. As expected, cis-

platin exposure induced DNA fragmentation in both yeast

strains growing in high-glucose medium; however, the

extent of DNA damage was significantly higher in cells

harboring GSH deficiency (Fig. 8a and b). This result is

consistent with certain data from the literature showing

that the lack of GSH, caused mainly by chemical stresses,

leads to the activation of apoptosis (Rudin et al., 2003;

P�ocsi et al., 2004; Anathy et al., 2012). The induction of

apoptosis has multiple features but the most outstanding

event during apoptosis is the strikingly depletion of GSH

levels without any dependence of ROS generation (Franco

(a) (b)

(c) (d)

Fig. 7. Cisplatin induces oxidative stress in glutathione-deficient Saccharomyces cerevisiae cells. Lipid peroxidation was measured by the TBARS

method in the early exponential wild type and its isogenic gsh1D mutant cells grown in 2% YPD (a and b) or 0.5% YPD (c and d) and then

subjected to 450 lM cisplatin for 2 h. Controls are cells that were not subjected to cisplatin. Aconitase activity was determined

spectrophotometrically in S. cerevisiae cells subjected to the same conditions described in lipid peroxidation. The results represent the mean � SD

of at least three independent experiments (*means different results at P < 0.05 compared with control; **means different results at P < 0.05

compared with 2% SD).

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Protection against cisplatin 1155

et al., 2007). Interestingly, the DNA damage observed in

the mutant strain was associated with the increase in

cytotoxicity and oxidative stress markers indicating that

during cisplatin exposure cells might be affected by both

alkylation and oxidative stress-induced apoptosis. Regard-

ing the effects of CR in DNA fragmentation, we can

observe an increase in the number of wild-type cells pre-

senting DAPI staining, contrasting with the reduction in

the percentage of DNA fragmentation in cells harboring

deficiency in GSH synthesis. This result suggests that CR

could be activating a self-defense system to protect the

cell population through apoptosis. As shown in Fig. 8c,

the higher number of annexin-positive cells in the gsh1Dcells suggests that deficiency in GSH metabolism contrib-

utes to apoptosis. On the other hand, due to the reduc-

tion in annexin staining, GSH seems to be essential for

activating apoptosis in calorie-restricted cells. Remarkably,

in calorie-restricted gsh1 mutant cells, the apoptotic pro-

file was quite different from the wild-type cells, suggesting

that for apoptosis activation in CR regimen, the presence

of GSH is vital.

At the best of our knowledge, cancer cells show altered

metabolism in comparison with its healthy counterparts.

Overall, cancer cells display increased rates of fermenta-

tive metabolism with enhancement of lactate production

(Warburg, 1956). Indeed, the reduced rates of respiration

associated with high rates of glycolysis suggest that cancer

cells depend mainly on ‘aerobic fermentation’, also

known as ‘Warburg effect’ (Brand, 1997; Gatenby & Gil-

lies, 2004). Interestingly, although the exact mechanism

by how cancer cells undergo this metabolic shift are still

unclear, this phenomenon may indicate an advantage for

cancer cells allowing them to proliferate in microenviron-

ments (hypoxic areas) usually encountered in solid

tumors (Gatenby & Gillies, 2004). As a Crabtree-positive

cell, S. cerevisiae has the capacity to alter its metabolism

according to nutrients availability. Thus, when glucose is

abundant S. cerevisiae uses fermentative metabolism as its

main energy supply source; however, when this nutrient

is depleted this yeast has the ability to shift to respiratory

metabolism (Entian et al., 1984; Thevelein, 1994). There-

fore, due to similar metabolic features of cancer cells,

S. cerevisiae has proved to be an excellent cellular

model for the screening of metabolism-targeted drugs

and alternative anticancer therapy (D�ıaz-Ruiz et al., 2009,

2011).

(a)

(b) (c)

Fig. 8. Impact on the chromatin fragmentation and apoptosis of glutathione-deficient S. cerevisiae cells subjected to cisplatin. (a) Representative

photographs of the wild-type (BY4741) and its isogenic mutant gsh1D grown to early exponential phase in 2% YPD or 0.5% YPD media,

subjected directly to 450 lM cisplatin for indicated 2 h and then stained with DAPI for chromatin fragmentation. Percentage of chromatin

fragmentation (b) and annexin V (c) in S. cerevisiae cells following exposition with cisplatin (450 lM/2 h) (*means different results at P < 0.05

between cells cultured in 2% SD and 0.5% SD ; **means different results at P < 0.05 between gsh1D cells cultured in 2% SD and 0.5% SD).

FEMS Yeast Res 14 (2014) 1147–1159ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

1156 D. Mariani et al.

CR or glucose deprivation has proven to generate a

metabolic shift from fermentative to respiratory pathways

(Lin et al., 2002; Oliveira et al., 2008). Several studies

have proved that CR makes human cancer cells more sus-

ceptible to drugs if compared with normal (healthy) cells

due to the induction of ROS generation and subse-

quently, oxidative stress (Lee et al., 1998, 2012; Raffag-

hello et al., 2008). In this context, the combination of CR

with chemotherapy agents (e.g. cisplatin) seems to be a

potential alternative intervention to induce cancer cell

death. The rational of this therapy is to promote a dual

mode of cytotoxicity, which leads to the induction of oxi-

dative damages through CR or glucose deprivation (2DG

administration) and by cisplatin (chemotherapy agents)

mode of action, both acting together to enhance cell

death (Simons et al., 2007). Unfortunately, the efficacy of

this therapy falls on the need to evaluate the redox state

of healthy cells before the start of the therapy; otherwise

this alternative treatment tends to be also highly cytotoxic

for normal cells. In this work using S. cerevisiae as a

model of healthy cells, we showed that CR induces

cellular protection against chemotherapy but it strikingly

dependent of GSH contents. Our results showed that in

healthy calorie-restricted cells, the absence of GSH

imposed critical dysfunctions leading to a dramatic

decrease in survival associated with oxidative damages

(e.g. lipid peroxidation and aconitase) and a significant

reduction in apoptosis. Furthermore, the critical role

of GSH for CR benefits is clearly shown when nutrient-

sensing mutants, which mimic CR, were treated with

BSO.

Taken together, the benefits of controlling calorie

intake for cells subjected to chemotherapy drugs are evi-

dent, but in this work, we highlight for the first time a

role of GSH for cellular protection against cisplatin. In

fact, the lack of GSH is critical for proper cellular

response, even if cells are in calorie-restrictive conditions.

Although improvement of cell response can be achieved

by CR, it seems to be very important to continue the

efforts to evaluate the real redox status of biological

systems before starting a CR-related chemotherapy.

Acknowledgement

This work was supported by grants from FAPERJ,

CAPES, and CNPq.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Nutrient-sensing genes modulate survival of Sac-

charomyces cerevisiae subjected to chemotherapy drugs.

Fig. S2. Survival of Saccharomyces cerevisiae cells sub-

jected to cisplatin.

Fig. S3. Dependence of GSH for nutrient-sensing proteins

modulate survival of Saccharomyces cerevisiae subjected to

cisplatin.

FEMS Yeast Res 14 (2014) 1147–1159 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Protection against cisplatin 1159