research article single amino acid arginine deprivation

Research ArticleSingle Amino Acid Arginine Deprivation Triggers ProsurvivalAutophagic Response in Ovarian Carcinoma SKOV3

Galyna Shuvayeva12 Yaroslav Bobak1 Natalia Igumentseva1 Rossella Titone2

Federica Morani2 Oleh Stasyk1 and Ciro Isidoro2

1 Institute of Cell Biology National Academy of Sciences of Ukraine Drahomanov Street 1416 Lviv 79005 Ukraine2 Laboratory of Molecular Pathology Department of Health Sciences Universita del Piemonte OrientaleVia P Solaroli 17 28100 Novara Italy

Correspondence should be addressed to Oleh Stasyk stasykcellbiollvivua and Ciro Isidoro ciroisidoromedunipmnit

Received 24 January 2014 Accepted 30 April 2014 Published 1 June 2014

Academic Editor Danny N Dhanasekaran

Copyright copy 2014 Galyna Shuvayeva et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Autophagy is a process of cytosol-to-lysosome vesicle trafficking of cellular constituents for degradation and recycling of theirbuilding blocks Autophagy becomes very important for cell viability under different stress conditions in particular under aminoacid limitation In this report we demonstrate that single amino acid arginine deprivation triggers profound prosurvival autophagicresponse in cultured human ovarian cancer SKOV3 cells In fact a significant drop in viability of arginine-starved SKOV3 cells wasobserved when autophagy was inhibited by either coadministration of chloroquine or transcriptional silencing of the essentialautophagy protein BECLIN 1 Enzymatic arginine deprivation is a novel anticancer therapy undergoing clinical trials This therapyis considered nontoxic and selective as it allows controlling the growth of malignant tumours deficient in arginine biosynthesisWe propose that arginine deprivation-based combinational treatments that include autophagy inhibitors (eg chloroquine) mayproduce a stronger anticancer effect as a second line therapy for a subset of chemoresistant ovarian cancers

1 Introduction

It is established that some types of tumours are deficient in thebiosynthesis of certain amino acids and often exhibit elevatedsensitivity to deprivation of a corresponding single aminoacid (such as arginine methionine and asparagine) both invitro and importantly in vivo (for recent reviews [1ndash5])Thisprovided a rational basis for the development of metabolicanticancer therapies based on the application of recombinantamino acid degrading enzymes such as asparaginase for thetreatment of leukemias and other tumours [2 5 6] Firstclinical trials with recombinant enzymes hydrolyzing aminoacid arginine human arginase I and Mycoplasma hominisarginine deiminase have demonstrated therapeutic efficacy incontrolling the growth of hepatocarcinomas and melanomas[7ndash9] Recent in vitro studies also suggested that other typesof cancers may be potentially sensitive to this therapy (pan-creatic prostate renal carcinomas and mesotheliomas) dueto the transcriptional silencing of arginine anabolic enzyme

of urea cycle argininosuccinate synthetase (ASS) [10ndash13](see Figure 2) It was also observed that the development ofchemoresistance to platinum compounds in ovarian carci-nomas leads to collateral appearance of arginine auxotrophydue to the downregulation of ASS [14] adding these tumoursto the list of potential targets of arginine deprivation-basedenzymotherapy

Although metabolic enzymotherapy based on argininedeprivation is considered as nontoxic and selective it is notfree of certain limitations One such limitation arises from theupregulation of ASS expression inmany tumours in responseto arginine starvation leading to the appearance of the ASS-positive tumour relapse insensitive to the therapy [2] Alsowe recently observed that tumour cells become profoundlymore resistant to arginine withdrawal in in vitro 3D spheroidmodels relative to respective monolayer cultures [15 16]This phenomenon is consistent with the results of animalstudies and ongoing clinical trials which showed that argininedeprivation is effective in inhibiting tumour growth but not

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 505041 10 pageshttpdxdoiorg1011552014505041

2 BioMed Research International

in inducing tumour regressionThe latter observation stimu-lates further search for more efficient rational combinationaltherapeutic approaches based on arginine deprivation

Arginine besides being required for protein biosynthesishas other versatile functions in the cell as a precursor of nitricoxide agmatine and polyamines [17] It was also demon-strated that arginine is an essential amino acid for culturedtumour cells due to their deficiency in arginine biosynthesisde novo [18] Thus arginine withdrawal profoundly affectstumour cell physiology In this work we show that argininedeprivation strongly induces the autophagic process in ovar-ian carcinoma cells in monolayer culture Autophagy theselective process of lysosomal recycling of cell constituentsis known to have a prosurvival role under different stressesin tumour cells [19] Therefore we addressed the questionwhether inhibition of autophagy affects tumour cell survivalupon arginine starvation Such a strategy could be appliedto enhance the therapeutic effect of enzymotherapy based onarginine deprivation

2 Materials and Methods

21 Reagents The following antibodies were used poly-clonal antibodies against MAP-LC3 (Novus Biologicals COUSA) and BECLIN 1 (BD Biosciences CA USA) mousemonoclonal anti-LAMP1 (BD) and anti-Golgin97 (SantaCruz Biotechnology Santa Cruz CA USA) monoclonalmouse anti-120573-actin (Sigma-Aldrich St Louis MO USA)ph-4E-BP1 and ph-p70-S6k (Cell Signaling TechnologyBeverly MA USA) FITC-conjugated polyclonal goat anti-rabbit (Santa Cruz) and Cy3-conjugated polyclonal goat anti-mouse (Santa Cruz) and horseradish peroxidise- (HRP-)conjugated polyclonal goat anti-mouse and anti-rabbit (bothfromMillipore Corporation Bedford MA USA)

Monodansylcadaverine (MDC) 3-methyladenine (3MA)chloroquine (CQ) asparagine (Asn) and other bench chem-icals were purchased from Sigma-Aldrich

22 Cell Line and Culture Conditions SKOV3 cells originat-ing from human ovarian carcinoma tissues were obtainedfrom ATCC (USA) The cells were grown in Dulbeccorsquosmodified Eaglersquos medium (DMEM HyClone LaboratoriesLogan Utah USA) with 10 foetal bovine serum (FBSPAA Laboratories GmbH Pasching Austria) 2mmolL glu-tamine and 50mgL gentamycin (Sigma-Aldrich SteinheimGermany) and maintained in the incubator at 37∘C with5 CO

2 Where indicated arginine-containing (04mM

HyClone Laboratories Logan UT USA) and arginine-freemedia were supplemented with 5 dialysed FBS (HyClone)To study the growth dynamics of ovarian carcinoma cellsunder standard and arginine-deprived conditions the cellswere seeded at a density of 20000 cells per well in regularmedium in 96-well plates and allowed to adhere for 24 h thenthemediumwas aspirated and the cellmonolayer waswashedtwo times with PBS and finally the cells were incubated withfresh complete medium or arginine-free medium (AFM)The cells were cultured for up to 96 h and cell growthwas assessed by counting the cells every 24 h in triple Cell

viability was assessed using the trypan blue (final concentra-tion 005) dye exclusion Viable (unstained) and nonviable(blue-stained) cells were counted on a haemocytometer bylight microscopy

23 RT-PCR Total RNA was isolated from cells by themethod of Chomczynski and Sacchi [20] First-strand cDNAsynthesis was performed using First Strand cDNA SynthesisKit (Fermentas Vilnius Lithuania) and an oligo-dT primeraccording to the manufacturerrsquos instructions PCR was per-formed using a High Fidelity PCR Enzyme Mix (Fermentas)with the following primer pairs

ASS-S 51015840-GGGGTCCCTGTGAAGGTGACC-31015840

ASS-AS 51015840-CGTTCATGCTCACCAGCTC-31015840

ASL-S 51015840-GAAGCGGATCAATGTCCTGC-31015840

ASL-AS 51015840-CTCTTGGTGAATCTGCAGCG-31015840

OTC-S 51015840-AATCTGAGGATCCTGTTAAACAATG-31015840

OTC-AS 51015840-CTTTTCCCCATAAACCAACTCA-31015840

GAPDH-S 51015840-CAAGGTCATCCATGACAACTT-TG-31015840

GAPDH-AS 51015840-GTCCACCACCCTGTTGCTGTA-G-31015840

PCR fragments were separated by electrophoresis on 15agarose gel and visualized by ethidium bromide stainingThe relative mRNA expression levels were estimated afternormalization with GAPDH The number of cycles waschosen at which PCR product amount was optimal andwithin the linear portion of the curve well before saturationpoint

ASS number of cyclesmdash26 size of the ampliconmdash448 bp

ASL number of cyclesmdash27 size of the ampliconmdash502 bp

OTC number of cyclesmdash40 size of the ampliconmdash1125 bp

GAPGH number of cyclesmdash25 size of the ampli-conmdash496 bp

24 Visualization of Monodansylcadaverine-Labelled Vac-uoles MDC is an autofluorescent weak base that accumu-lates in acidic lysosomal vacuoles and autophagolysosomes[21] Cells attached to glass coverslips were incubated with005mM MDC (Sigma-Aldrich) in PBS at 37∘C for 10minAfter incubation cells were washed three times with PBSand immediately analyzed with a fluorescence microscope(ZEISS Axio Imager A1) equippedwith Axio Vision Software(v 463) Images were captured with a CCD camera andimported into Photoshop Quantification of cell fluorescencewas conducted using ImageJ 148v Software

BioMed Research International 3

25 Immunofluorescence and Microscopy Analysis Immun-ofluorescence staining was performed as previously de-scribed [22] Essentially cells cultured on glass coverslipswere washed with PBS fixed with cold methanol andpermeabilized with 02 Triton X-100 in PBS The coverslipswere then incubated with the indicated primary antibodiesin PBS contained 01 Triton X-100 and 4 FBS overnight at4∘C and thereafter incubated with the appropriate secondaryantibodies for 1 hr at room temperature Nuclei were stainedwith DAPI Coverslips were mounted on microscope slidesandmonitored under a ZEISS fluorescencemicroscope (AxioImager A1) equipped with Axio Vision Software (v 463)Images were captured with a CCD camera and imported intoPhotoshop Pearson correlation coefficients were calculatedusing ImageJ 148v Software to assess the degree of colocal-ization of protein markers of autophagy

26 Immunoblotting The cell monolayer was washed withice-cold PBS and lysed in Extraction Buffer (10mmolL Tris-HCl pH 75 150mmolL NaCl 1 NP-40 5mmolL EDTA50mmolL NaF 1mmolL Na

3VO4 5mmolL benzamidine

1mmolL PMSF 10mgmL aprotinin 10mgmL Leupeptinand 1mgmL Pepstatin A) at 4∘C for 20min Cell extractswere obtained after centrifugation at 12000 g at 4∘C for30min and cellular proteins were quantified using Petersonrsquosmethod [23] Equal amounts of protein homogenates wereloaded separated by SDS-PAGE (concentration of acry-lamide varied depending on the size of the protein to bedetected) and transferred onto PVDF membrane (MilliporeCorp Billerica MA USA) The membranes were blockedwith 5 nonfat dried milk in PBS containing 005 Tween-20 and probed with the indicated primary and secondary(horseradish peroxidase-conjugated) antibodies 120573-Actin wasused for protein loading control The bands were visualizedusing the enhanced chemiluminescence reagent (MilliporeCorp) Band densitometry quantification was performedusing the Gel-Pro analyzer (Version 32)

27 Small Interfering RNA Transfection BECLIN 1 silencingwas achieved using RNA interference as previously described[24] Cells were transfected using Oligofectamine reagent(Invitrogen Carlsbad CA USA) according to the manu-facturerrsquos manual Protein knockdown was determined byimmunoblotting 48 hr after transfection As control (shamtransfection) a nonspecific scramble sequence siRNA wasused

28 Statistics In each individual experiment triplicate wellswere used for each treatment and control All experimentswere repeated at least three times Statistical analyses wereperformed using Studentrsquos t-test Results were expressed asmeans plusmn SD Significance was established when the P valuewas less than 005

3 Results31 SKOV3 Cells Retain Viability and Proliferative Potentialunder Long-Term Arginine Deprivation In Vitro Tumourcell lines in monolayer cell culture substantially differ in

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48 967224

Cel

l gro

wth

()

CMAFM

(h)

AFMrarrCM (72h)

Figure 1 Effect of arginine deprivation on SKOV3 cell proliferationThe growth of SKOV3 cells in arginine-sufficient complete medium(CM) in arginine-free medium (AFM) and the ability of SKOV3cells to rescue cell proliferation after different periods of argininestarvation was assessed by counting only the viable cells After theindicated periods of incubation in AFM cells were shifted to afresh CM and allowed to proliferate for additional 72 h (AFMrarrCM(72 h)) Viable cells were determined by the trypan blue exclusiontest The initial number of cells (time-point zero) was considered as100 Data from three independent experiments in triplicate

their sensitivity to a single amino acid withdrawal [18]Therefore we first analyzed whether and to what extentarginine deprivation affects the viability and the proliferativepotential of SKOV3 cells Upon shifting to a defined arginine-deficient medium (AFM) growth arrest and reduction inthe proportion of viable cells were observed It is to benoted that even after 4 days of arginine starvation SKOV3cells were still able to resume cell proliferation in responseto arginine resupplementation though regrowth potentialprogressively declined in the course of incubation in AFM(Figure 1) No signs of PARP fragmentation as a reporterof apoptosis in arginine-starved SKOV3 cells were observed(data not shown) This observation suggested that a sub-stantial fraction of SKOV3 cells remained viable even afterthe prolonged arginine withdrawal indicating that these cellsare rather resistant to this metabolic stress For comparisonhepatocellular carcinoma HepG2 cells lose their proliferativepotential already after 2 days of arginine starvation andexhibit concomitant apoptosis [18]

32 Arginine Is an Essential Amino Acid for Cultured SKOV3Cells The effect on growth arrest in AFM (Figure 1) sug-gested that arginine is an essential amino acid for SKOV3cells The RT-PCR analysis of arginine key anabolic enzymesof the urea cycle revealed that SKOV3 cells incubated incomplete medium do not express mitochondrial argininebiosynthetic enzyme ornithine transcarbamylase (OTC itconverts ornithine to citrulline) but do express cytosolic


CM Hep

G2

SKOV3

OTC

ASS

ASL

GAPDH

AFM

05

h

AFM

4h

AFM

8h

AFM

24h

(a)

Arginine

Arginine

OrnithineOTC

ASL

Fumarate

Urea

UreaARG 1

ARG 2

Cyto

plas

mM

itoch

ondr

ia

Carbamyl phosphateASS

Aspartate

Citrulline

Argininosuccinate

(b)

Figure 2 Expression of the key genes of arginine anabolism in SKOV3 cells (a) Specific mRNA levels determined by RT-PCR analysis asdescribed in Section 2 Cells were cultured in arginine-freemedium (AFM) for 24 h or in arginine-sufficient completemedium (CM) HumanhepatocarcinomaHepG2 cells (which express urea cycle enzymes) were used as a positive control GAPDH expression was used as an internalloading control (b) Scheme of arginine biosynthesis in the urea cycle ARG1 arginase I ARG2 arginase II OTC ornithine transcarbamylaseASS argininosuccinate synthetase and ASL argininosuccinate lyase

argininosuccinate synthetase (ASS it converts citrulline toargininosuccinate) and argininosuccinate lyase (ASL it con-verts argininosuccinate to arginine) (Figures 2(a) and 2(b))Arginine deprivation did not trigger an upregulation of ASSor ASL often observed in other tumour cell lines or induc-tion of OTC transcription (Figure 2(a)) Human hepatocellu-lar carcinomaHepG2 cells were used as a positive control [18]OTC deficiency in cultured SKOV3 cells implies that theyare deficient in endogenous arginine anabolism and argininecan only be synthesized via ASS-mediated conversion ofexogenously supplied citrulline (which is absent in standardDMEMmedium) Accordingly exogenous ornithine did notsupport proliferation of SKOV3 cells in AFM (data notshown) Therefore we can assume that incubation in AFMsurely induces arginine starvation in SKOV3 cells

33 Arginine Deprivation in SKOV3 Cells Triggers ProfoundAutophagic Response One of the prosurvival responses oftumour cells triggered upon amino acids limitation isthe elevated intracellular protein recycling via autophagy[19] In particular arginine deprivation has been shownto induce autophagy in melanomas [25] However it isknown that different tumour cells exhibit varying basaland stimulus-dependent inducible autophagic proficiency[26] We addressed the question whether low sensitivity (interms of cell survival and proliferation) of SKOV3 cells toarginine withdrawal as an essential amino acid (Figure 1)was associated with or causally linked to induction ofautophagy To monitor autophagy we first employed aclassical staining of acidic vacuoles which includes lyso-somes and autophagolysosomes with the vital fluorescentdye MDC [21] Arginine deprivation rapidly and profoundlyinduced the expansion of the autophagolysosomal com-partment in SKOV3 cells (Figure 3) We observed MDC-labelled intracellular vacuoles in SKOV3 cells already after30min of arginine starvation Treatment of the starved cells

with 3-methyladenine (3MA 10mM) a classic inhibitor ofautophagy that inhibits the formation of autophagosomes[22] strongly diminished the fluorescence signal (Figure 3)Upon treatment with chloroquine (CQ 25120583M) anotherknown inhibitor of autophagy [21] MDC staining of thecells further increased (Figure 3) This effect was expectedas CQ impairs the late stages of autophagy and leads tothe accumulation of autophagosomes and of autophagolyso-somes [21] By contrast a partial decrease inMDC staining inarginine-deprived SKOV3 cells was observed upon concomi-tant treatment with an excess of asparagine (Asn 50mM)which is known to downregulate autophagy through thestimulation of mTOR a negative regulator of autophagy [21]Quantification of cell fluorescence under arginine starvationand upon cotreatment with inhibitors using ImageJ 148vSoftware revealed an approximately four-time decrease in flu-orescence between control arginine-starved and CQ-treatedcells versus those treated with 3MA and Asn at 4 hours ofincubation (data not shown) Taken together the above dataindicate thatMDC staining is indeedmirroring the inductionof autophagolysosomes in arginine-deprived SKOV3 cellsTo definitively demonstrate the induction of autophagy ina parallel experiment the cells cultured on coverslips wereimmunostained for LC3 (a hallmark of autophagosomes) andLAMP1 (a marker of lysosomes) as well as for BECLIN 1(a component of the autophagy interactome) and Golgin97(which labels the Golgi complex) Colocalization of LC3and LAMP1 is a reliable indicator for the formation ofautophagolysosomes while formation of BECLIN 1 aggre-gates in the Golgi area is indicative of activation of theautophagy process [24]

We observed the colocalization of such signals (yel-low fluorescence) already at 30min of arginine deprivation(Figures 4(a) and 4(b)) indicating a fast upregulation ofthe autophagy process Colocalization of autophagosome-


AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

Figure 3MDC staining of acidic vacuoles in ovarian carcinoma SKOV3 cells subjected to arginine deprivation (AFM)The cells were labelledwith MDC as described in Section 2 at zero point and after 05 4 and 8 hours of arginine withdrawal and immediately monitored under afluorescence microscope Magnification 400x

and lysosome-associated proteins was evident during thewhole time course of our analysis (Figures 4(a) and 4(b))Importantly in agreement with the MDC data (Figure 3)3MA and Asn reduced while CQ increased the number andthe size of LC3-positive vacuoles (ie autophagosomes) andalso the aggregation of BECLIN 1 in the Golgi area (Figures4(a) and 4(b)) The calculated values of Pearson correlationcoefficient for the pairs LC3LAMP1 and BECLIN 1Golgin97were ge05 for arginine-starved cells whereas for cells treatedwith inhibitors (3MA CQ and Asn) the coefficients valueswere 0 to le02 From these data we conclude that argininedeprivation strongly induces an autophagic response in ovar-ian carcinoma SKOV3 cells

34 Effects of Arginine Deprivation and Autophagy Impair-ment on mTOR-Dependent Biosynthesis Pathway Aminoacid starvation is known to inhibit the biosynthetic pathwayand in parallel to induce the autophagy degradation ofredundant protein as an attempt to rescue the amino acidsneeded for the synthesis of vital proteins The mTOR kinaseis placed at the cross-point and is a master regulator of boththese pathways [27 28] To get an insight into the relationshipbetween the induction of autophagy and the biosyntheticpathway under arginine-deprivation conditions we assayedthe activation status of 4E-BP1 (eukaryotic initiation factor4E-binding protein 1) and of p70-S6K (ribosomal p70 S6kinase) two downstream substrates of mTOR that directprotein synthesis [29] in the presence of CQ that hampersthe last step of autophagy In fact in the presence of CQ the

consumption of autophagosomes is interrupted as witnessedby the induced accumulation of LC3II both in CM and inAFM conditions and this accumulation increases with timeof incubation (Figure 5) However in the cells subjected toarginine deprivation the level of LC3I decreases with timeof incubation despite the fact that no further increase inLC3II is observed indicating a rapid autophagy flux andconsumption of autophagosomes When these cells wereconcomitantly exposed to CQ the autophagosomes in factaccumulated with time (as indicated by increased LC3II at8 versus 2 h) Next we looked at the translational activityin SKOV3 cells cultivated under these conditions Whilephosphorylation of 4E-BP1 elicits its inhibition and thereforereliefs the inhibitory action of 4E-BP1 on protein synthesisthe phosphorylation of p70-S6K elicits the activation of thetranslational process [29] Thus the phosphorylation of both4E-BP1 and p70S6k converges on triggering the initiationof protein synthesis Both 4EBP and p70-S6K were highlyphosphorylated in the cells cultivated in CM in the presenceof CQ while their activation status was greatly reduced underAFM culture condition regardless of whether CQ was or wasnot present (Figure 5)

35 Effect of Autophagy Inhibition by Transcriptional Silencingof BECLIN 1 or with CQ on the Sensitivity of SKOV3 Cellsto Arginine Starvation To elucidate the role of autophagy inmaintaining SKOV3 cell viability under arginine deprivationwe manipulated the autophagy pathway by genetic and phar-macologic approaches Transient transfection with a specific


AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)

Figure 4 (a) Immunofluorescence staining of the autophagosomal protein LC3 (green fluorescence) and of the lysosomal protein LAMP1(red fluorescence) and (b) of BECLIN 1 (green fluorescence) and Golgi-associated Golgin97 (red fluorescence) in SKOV3 cells subjected toarginine starvation Nuclei were labelled with DAPI Images were captured with ZEISS fluorescence microscope (Axio Imager A1) equippedwith Axio Vision Software v 463 Magnification 1000x


CM AFM

LC3I

LC3II

ph-p70-S6K

Actin

+ + + +minus minus minus CQ0 2 8 2 8 2 8 (hrs)

ph-4E-BP1120572120573120574

Figure 5 Effect of arginine deprivation and CQ treatment on accu-mulation of autophagosomal protein LC3II and phosphorylation ofmTOR substrates After indicated periods of treatment the cellswere washed and harvested for WB analysis 50 120583g of total sampleprotein was loaded per lane 120573-Actin served as the loading standard

siRNA elicited the transcriptional silencing (approximately54) of the autophagy protein BECLIN 1 (Figure 6(a)) Afterthree days of incubation in AFM the number of viablecells in siRNA-BECLIN 1 transfected culture decreased byapproximately 40 as compared to the sham-transfectedculture (Figure 6(b)) Arginine-deprived BECLIN 1-silencedcells also exhibited a decrease in their proliferative poten-tial upon shift to the fresh CM relative to control cells(Figure 6(b)) thus suggesting a significant prosurvival role ofautophagy in the cell response to arginine starvation

Next we addressed the question whether cotreatmentwith CQ which impairs the late steps of the autophagy path-way affects the survival of SKOV3 cells under arginine depri-vation in a similar manner as observed with the silencing ofBECLIN 1 expression To this end wemonitored cell viabilityunder the combined treatment and cell proliferation uponarginine resupplementationThe treatment with CQ (25 120583M)dramatically decreased cell viability and the proliferativepotential under arginine starvation (Figure 7) ImportantlyCQ treatment rendered arginine-starved SKOV3 cells unableto resume proliferation in fresh CM already after two daysof the combined treatment (Figure 7 cf Figure 1) It is to benoted that a prolonged incubation with this same concentra-tion of CQwas cytotoxic for SKOV3 cells also in CMmedium(data not shown) No signs of apoptosis (as tested by westernblotting of PARP and Annexin V staining) and no signsof senescence (as tested by positivity for beta-galactosidasestaining) were detected in AFM plus CQ cotreated cells up to144 h However SKOV3 cells double staining with ethidiumbromide and Hoechst 33342 revealed that under this culturecondition the majority of cells died via necrosis (not shown)

4 Discussion

Autophagy a cytosol-to-lysosome membrane-traffickingprocess of degradation of cellular constituents is a house-keeping homeostatic pathway and also plays a fundamentalrole to preserve cell viability upon different stress conditions[19] One of such stresses is nutrient limitations in particularamino acid restriction [30] Autophagy is known to havedual function in cancerogenesis playing both negative andpositive roles in cancer progression and being implicatedin chemoresistance of certain tumour types (for review[26 31ndash33]) It was also established that even single aminoacid starvation triggers autophagic response in tumourcells [11 34] Previously reported [35] and our unpublisheddata suggest that the most profound autophagic responsein tumour cells is triggered by starvation for argininemethionine lysine and leucine relative to other amino acidsIt remains to be elucidated whether specific regulation bythis set of amino acids involves the mTORC1 complex orother mechanisms Our data indicate that single argininedeprivation early affects the mTOR-dependent biosyntheticpathway

Arginine besides being required for protein biosynthesishas other versatile functions in the cell as a precursor ofnitric oxide agmatine and polyamines and as a regulatorymolecule (for review [17]) For cultured tumour cells argi-nine is an essential amino acid due to their deficiency inarginine biosynthesis de novo [18] Ovarian carcinomas wererecently added to the growing list of tumour types poten-tially sensitive to the treatment with recombinant arginine-degrading enzymes In fact it was demonstrated that relapsesof ovarian carcinomas resistant to cisplatinum treatment con-comitantly become deficient in argininosuccinate synthetasea rate limiting enzyme of arginine biosynthesis and thuspotentially sensitive to arginine-degrading enzymes [14] Inthis work we investigated how modulation of autophagyaffects ovarian cancer cells viability under arginine depriva-tion Human ovarian carcinoma SKOV3 cells were used asan experimental model We found that SKOV3 cells exhibithigh resistance to the stress exerted by arginine deprivation(Figure 1) This fact potentially allows studying the physio-logical role of autophagy under arginine withdrawal withoutinterferingwith processes of programmed cell death (apopto-sis) that are often triggered to a different extent in cancer cellsunder such conditions [18] Although SKOV3 cells exhibithigh expression of argininosuccinate synthetase (as we showin Figure 2) they still are a suitable informative model forin vitro studies since SKOV3 cells are fully dependent onexogenous arginine supply due to the deficiency in ornithinetranscarbamylase (OTC) an upstream enzyme of arginineanabolism (Figure 2) In this report we demonstrate thatargininewithdrawal rapidly andmarkedly induces autophagyin SKOV3 cells (Figures 3 and 4) Under starvation thebiosynthetic pathway is impaired while basal autophagy risesup and both these pathways are controlled by mTOR [2736] Autophagy fuels the cytoplasm with the amino acidsderiving fromproteolysis andCQ is expected to interrupt thisprocess by impairing the formation of autophagolysosomesand by inhibiting the acid-dependent proteolysis mediated


BECLIN 1

Actin

Intensity ()

siRNA BECLIN 1 Control

466 plusmn 13 100

(a)

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()

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160

siRNA BECLIN 1 transfectionControl

AFM (96h)

lowast

AFM rarr CM (72h)

(b)

Figure 6 Transcriptional silencing of BECLIN 1 expression leads to decreased viability of SKOV3 cells under arginine starvation (a)Westernblot analysis of BECLIN 1 protein in SKOV3 cells transfected with BECLIN 1 siRNA or with nonsilencing control siRNA 120573-Actin served asthe loading standard Densitometry quantification and the calculation of the relative band intensity were performed as described in Section 2Intensity represents the mean value of three independent experiments and is expressed as means plusmn SD (b) histogram represents the effect ofBECLIN 1 silencing on the viability of arginine-starved cells and their proliferative potential after arginine resupplementation lowast119875 lt 005

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6 4 2

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grow

th aft

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)

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AFM rarr CM (AFM

72h)

(a)

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cell

grow

th aft

er tr

eatm

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)

6 4 2 Days

lowastlowast

AFM + CQ (25 120583M)AFM + CQ (25 120583M) rarr CM (72h)

(b)

Figure 7 Effect of the autophagy inhibitor chloroquine (CQ) on SKOV3 cells viability and proliferative potential Histograms showing cellsurvival and ability to resume proliferation after arginine resupplementation in cells deprived of arginine (AFM) (a) or cotreated with CQ(b) After the indicated periods of the treatment the medium was changed to CM and cells were allowed to grow for additional 72 h Viablecell numbers were determined by the trypan blue exclusion test 100 is the number of cells at zero time point lowastlowast119875 lt 001

by lysosomal cathepsins The mTORC1 complex senses theavailability of amino acids and phosphorylates downstreamsubstrates in order to switch on and off the pathways forautophagy degradation or for protein synthesis accordingly[28] Arginine deprivation in fact depressed the activationof the signalling kinases 4E-BP1 and p70-S6k that governthe protein synthesis pathway (Figure 5) These kinases aredownstream of mTOR [29] which also negatively controlsautophagy CQ which further reduces the availability of

autophagy-derived amino acids affected the signalling thatgoverns the biosynthetic pathway in the cells cultivated inCM indicating that despite the presence of amino acids inthe culture medium the block of the autophagy degradationimposed by chloroquine was sensed by mTOR By contrastin AFM the mTOR pathway was inactive since the first2 h of incubation and CQ did not reduce further the levelof phosphorylation of 4E-BP1 and p70-S6k indicating thatarginine deprivation per sewas sufficient to limit or inhibit the


activation of the protein synthesis pathway We also demon-strate that autophagy process is important for maintainingcell viability under arginine deprivation This conclusion issupported by the significant drop in viability of arginine-starved SKOV3 cells in which autophagy is inhibited Inthis respect either coadministration of CQ or transcriptionalsilencing of the essential autophagy protein BECLIN 1 pro-duced similar effects (Figures 6 and 7) Strikingly in thecase of BECLIN 1 siRNA silencing the observed decrease inviability and proliferative potential was roughly proportionalto the remaining level of BECLIN 1 protein in the transfectedculture (Figure 6) Preliminary data from our laboratoriesindicate that coapplication of taxane (taxol) at low doses mayfurther decrease viability of ovarian carcinoma SKOV3 cellsunder arginine deprivation (to be published elsewhere) Inthis context it is to be noted that taxol is a disruptor of thecytoskeleton and negatively impacts on the autophagosome-lysosome fusion step In conclusion our data support theconception that combinational treatment based on argininedeprivation and an autophagy inhibitor (eg chloroquine aknown nontoxic antimalarial drug) can potentially be appliedas a second line treatment for a subset of ovarian carcinomasdeficient in ASS

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Galyna Shuvayeva was awarded with a travel fellowship fromThe Company of Biologists (J Cell Science 2009) and wassupported by the Consorzio Interuniversitario Biotecnologie(Trieste Italy) during her stage in Novara Rossella Titonewas recipient of a ldquoM Di Lauro-Isidorordquo fellowship grantedby Associazione per la Ricerca Medica Ippocrate-Rhazi diNovara (Italy) Research in the laboratory of Ciro Isidoro issupported byComoli-Ferrari amp SpA (Novara Italy) Researchin the laboratory ofOVS was supported by statutory funds tothe ICB NASU and OVS was supported in part by Grant noN303318239 from the Polish Ministry of Science and HigherEducation The authors thank Olena Karacaj and LilianaLyniv (ICB NASU) for invaluable help in performing someof the experiments described in this paper

References

[1] L Feun M You C J Wu et al ldquoArginine deprivation as atargeted therapy for cancerrdquo Current Pharmaceutical Designvol 14 no 11 pp 1049ndash1057 2008

[2] M T Kuo N Savaraj and L G Feun ldquoTargeted cellu-lar metabolism for cancer chemotherapy with recombinantarginine-degrading enzymesrdquoOncotarget vol 1 no 4 pp 246ndash251 2010

[3] B Delage D A Fennell L Nicholson et al ldquoArginine depri-vation and argininosuccinate synthetase expression in thetreatment of cancerrdquo International Journal of Cancer vol 126no 12 pp 2762ndash2772 2010

[4] J R BertinoWRWaudWB Parker andM Lubin ldquoTargetingtumors that lack methylthioadenosine phosphorylase (MTAP)activity Current strategiesrdquoCancer Biology andTherapy vol 11no 7 pp 627ndash632 2011

[5] D Covini S Tardito O Bussolati et al ldquoExpanding targets fora metabolic therapy of cancer L-Asparaginaserdquo Recent Patentson Anti-Cancer Drug Discovery vol 7 no 1 pp 4ndash13 2012

[6] L G Feun AMarini GWalker et al ldquoNegative argininosucci-nate synthetase expression in melanoma tumours may predictclinical benefit from arginine-depleting therapy with pegylatedarginine deiminaserdquo British Journal of Cancer vol 106 no 9 pp1481ndash1485 2012

[7] T-S Yang S-N Lu Y Chao et al ldquoA randomised phase IIstudy of pegylated arginine deiminase (ADI-PEG 20) in Asianadvanced hepatocellular carcinoma patientsrdquo British Journal ofCancer vol 103 no 7 pp 954ndash960 2010

[8] P AOtt RD Carvajal N Pandit-Taskar et al ldquoPhase III studyof pegylated arginine deiminase (ADI-PEG 20) in patients withadvanced melanomardquo Investigational New Drugs vol 31 no 2pp 425ndash434 2013

[9] T Yau P N Cheng P Chan et al ldquoA phase 1 dose-escalatingstudy of pegylated recombinant human arginase 1 (Peg-rhArg1)in patients with advanced hepatocellular carcinomardquo Investiga-tional New Drugs vol 31 no 1 pp 99ndash107 2013

[10] T L Bowles R Kim J Galante et al ldquoPancreatic cancercell lines deficient in argininosuccinate synthetase are sensitiveto arginine deprivation by arginine deiminaserdquo InternationalJournal of Cancer vol 123 no 8 pp 1950ndash1955 2008

[11] R H Kim J M Coates T L Bowles et al ldquoArginine deiminaseas a novel therapy for prostate cancer induces autophagy andcaspase-independent apoptosisrdquo Cancer Research vol 69 no 2pp 700ndash708 2009

[12] C-Y Yoon Y-J Shim E-H Kim et al ldquoRenal cell carcinomadoes not express argininosuccinate synthetase and is highlysensitive to arginine deprivation via arginine deiminaserdquo Inter-national Journal of Cancer vol 120 no 4 pp 897ndash905 2007

[13] P W Szlosarek A Klabatsa A Pallaska et al ldquoIn vivo loss ofexpression of argininosuccinate synthetase inmalignant pleuralmesothelioma is a biomarker for susceptibility to argininedepletionrdquo Clinical Cancer Research vol 12 no 23 pp 7126ndash7131 2006

[14] L J Nicholson P R Smith L Hiller et al ldquoEpigenetic silencingof argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotro-phy in ovarian cancerrdquo International Journal of Cancer vol 125no 6 pp 1454ndash1463 2009

[15] B Vynnytska-Myronovska Y Bobak Y Garbe C Dittfeld OStasyk and L A Kunz-Schughart ldquoSingle amino acid argininestarvation efficiently sensitizes cancer cells to canavanine treat-ment and irradiationrdquo International Journal of Cancer vol 130no 9 pp 2164ndash2175 2012

[16] B Vynnytska-Myronovska Y Kurlishchuk Y Bobak C Dit-tfeld L A Kunz-Schughart and O Stasyk ldquoThree-dimensionalenvironment renders cancer cells profoundly less susceptible toa single amino acid starvationrdquo Amino Acids vol 45 no 5 pp1221ndash1230 2013

[17] S M Morris Jr ldquoArginine beyond proteinrdquo American Journalof Clinical Nutrition vol 83 no 2 pp 508Sndash512S 2006

[18] Y P Bobak B O Vynnytska Y V Kurlishchuk A A SibirnyandO V Stasyk ldquoCancer cell sensitivity to arginine deprivationin vitro is not determined by endogenous levels of arginine


metabolic enzymesrdquo Cell Biology International vol 34 no 11pp 1085ndash1089 2010

[19] G Kroemer G Marino and B Levine ldquoAutophagy and theIntegrated Stress Responserdquo Molecular Cell vol 40 no 2 pp280ndash293 2010

[20] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[21] D J Klionsky F C Abdalla H Abeliovich et al ldquoGuidelines forthe use and interpretation of assays for monitoring autophagyrdquoAutophagy vol 8 no 4 pp 445ndash544 2012

[22] R Castino N Bellio C Follo D Murphy and C IsidoroldquoInhibition of Pi3k class III-dependent autophagy preventsapoptosis and necrosis by oxidative stress in dopaminergicneuroblastoma cellsrdquo Toxicological Sciences vol 117 no 1 pp152ndash162 2010

[23] G L Peterson ldquoA simplification of the protein assay methodof Lowry et al Which is more generally applicablerdquo AnalyticalBiochemistry vol 83 no 2 pp 346ndash356 1977

[24] N F Trincheri C Follo G Nicotra C Peracchio R Castinoand C Isidoro ldquoResveratrol-induced apoptosis depends onthe lipid kinase activity of Vps34 and on the formation ofautophagolysosomesrdquoCarcinogenesis vol 29 no 2 pp 381ndash3892008

[25] N Savaraj M You C Wu M Wangpaichitr M T Kuo and LG Feun ldquoArginine deprivation autophagy apoptosis (aaa) forthe treatment of melanomardquo Current Molecular Medicine vol10 no 4 pp 405ndash412 2010

[26] J Reyjal K Cormier and S Turcotte ldquoAutophagy and cell deathto target cancer cells exploiting synthetic lethality as cancertherapiesrdquo Advances in Experimental Medicine and Biology vol772 pp 167ndash188 2014

[27] Y-Y Chang G Juhasz P Goraksha-Hicks et al ldquoNutrient-dependent regulation of autophagy through the target ofrapamycin pathwayrdquo Biochemical Society Transactions vol 37no 1 pp 232ndash236 2009

[28] L Yan and R F Lamb ldquoAmino acid sensing and regulation ofmTORC1rdquo Seminars in Cell and Developmental Biology vol 23no 6 pp 621ndash625 2012

[29] K Hara K Yonezawa Q-PWeng M T Kozlowski C Belhamand J Avruch ldquoAmino acid sufficiency and mTOR regulatep70 S6 kinase and eIF-4E BP1 through a common effectormechanismrdquo The Journal of Biological Chemistry vol 273 no23 pp 14484ndash14494 1998

[30] M H Stipanuk ldquoMacroautophagy and its role in nutrienthomeostasisrdquo Nutrition Reviews vol 67 no 12 pp 677ndash6892009

[31] F Morani R Titone L Pagano et al ldquoAutophagy and thyroidcarcinogenesis genetic and epigenetic linksrdquo Endocrine RelatedCancer vol 21 no 1 pp R13ndashR29 2013

[32] S Lorin A Hamaı M Mehrpour and P Codogno ldquoAutophagyregulation and its role in cancerrdquo Seminars in Cancer Biologyvol 23 no 5 pp 361ndash379 2013

[33] C Peracchio O Alabiso G Valente and C Isidoro ldquoInvolve-ment of autophagy in ovarian cancer a working hypothesisrdquoJournal of Ovarian Research vol 5 no 1 p 22 2012

[34] J-H Sheen R Zoncu D Kim and D M Sabatini ldquoDefectiveregulation of autophagy upon leucine deprivation reveals atargetable liability of human melanoma cells in vitro and invivordquo Cancer Cell vol 19 no 5 pp 613ndash628 2011

[35] E Shvets E Fass and Z Elazar ldquoUtilizing flow cytometry tomonitor autophagy in living mammalian cellsrdquo Autophagy vol4 no 5 pp 621ndash628 2008

[36] Z Feng H Zhang A J Levine and S Jin ldquoThe coordinateregulation of the p53 and mTOR pathways in cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 102 no 23 pp 8204ndash8209 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


in inducing tumour regressionThe latter observation stimu-lates further search for more efficient rational combinationaltherapeutic approaches based on arginine deprivation

Arginine besides being required for protein biosynthesishas other versatile functions in the cell as a precursor of nitricoxide agmatine and polyamines [17] It was also demon-strated that arginine is an essential amino acid for culturedtumour cells due to their deficiency in arginine biosynthesisde novo [18] Thus arginine withdrawal profoundly affectstumour cell physiology In this work we show that argininedeprivation strongly induces the autophagic process in ovar-ian carcinoma cells in monolayer culture Autophagy theselective process of lysosomal recycling of cell constituentsis known to have a prosurvival role under different stressesin tumour cells [19] Therefore we addressed the questionwhether inhibition of autophagy affects tumour cell survivalupon arginine starvation Such a strategy could be appliedto enhance the therapeutic effect of enzymotherapy based onarginine deprivation

2 Materials and Methods

21 Reagents The following antibodies were used poly-clonal antibodies against MAP-LC3 (Novus Biologicals COUSA) and BECLIN 1 (BD Biosciences CA USA) mousemonoclonal anti-LAMP1 (BD) and anti-Golgin97 (SantaCruz Biotechnology Santa Cruz CA USA) monoclonalmouse anti-120573-actin (Sigma-Aldrich St Louis MO USA)ph-4E-BP1 and ph-p70-S6k (Cell Signaling TechnologyBeverly MA USA) FITC-conjugated polyclonal goat anti-rabbit (Santa Cruz) and Cy3-conjugated polyclonal goat anti-mouse (Santa Cruz) and horseradish peroxidise- (HRP-)conjugated polyclonal goat anti-mouse and anti-rabbit (bothfromMillipore Corporation Bedford MA USA)

Monodansylcadaverine (MDC) 3-methyladenine (3MA)chloroquine (CQ) asparagine (Asn) and other bench chem-icals were purchased from Sigma-Aldrich

22 Cell Line and Culture Conditions SKOV3 cells originat-ing from human ovarian carcinoma tissues were obtainedfrom ATCC (USA) The cells were grown in Dulbeccorsquosmodified Eaglersquos medium (DMEM HyClone LaboratoriesLogan Utah USA) with 10 foetal bovine serum (FBSPAA Laboratories GmbH Pasching Austria) 2mmolL glu-tamine and 50mgL gentamycin (Sigma-Aldrich SteinheimGermany) and maintained in the incubator at 37∘C with5 CO

2 Where indicated arginine-containing (04mM

HyClone Laboratories Logan UT USA) and arginine-freemedia were supplemented with 5 dialysed FBS (HyClone)To study the growth dynamics of ovarian carcinoma cellsunder standard and arginine-deprived conditions the cellswere seeded at a density of 20000 cells per well in regularmedium in 96-well plates and allowed to adhere for 24 h thenthemediumwas aspirated and the cellmonolayer waswashedtwo times with PBS and finally the cells were incubated withfresh complete medium or arginine-free medium (AFM)The cells were cultured for up to 96 h and cell growthwas assessed by counting the cells every 24 h in triple Cell

viability was assessed using the trypan blue (final concentra-tion 005) dye exclusion Viable (unstained) and nonviable(blue-stained) cells were counted on a haemocytometer bylight microscopy

23 RT-PCR Total RNA was isolated from cells by themethod of Chomczynski and Sacchi [20] First-strand cDNAsynthesis was performed using First Strand cDNA SynthesisKit (Fermentas Vilnius Lithuania) and an oligo-dT primeraccording to the manufacturerrsquos instructions PCR was per-formed using a High Fidelity PCR Enzyme Mix (Fermentas)with the following primer pairs

ASS-S 51015840-GGGGTCCCTGTGAAGGTGACC-31015840

ASS-AS 51015840-CGTTCATGCTCACCAGCTC-31015840

ASL-S 51015840-GAAGCGGATCAATGTCCTGC-31015840

ASL-AS 51015840-CTCTTGGTGAATCTGCAGCG-31015840

OTC-S 51015840-AATCTGAGGATCCTGTTAAACAATG-31015840

OTC-AS 51015840-CTTTTCCCCATAAACCAACTCA-31015840

GAPDH-S 51015840-CAAGGTCATCCATGACAACTT-TG-31015840

GAPDH-AS 51015840-GTCCACCACCCTGTTGCTGTA-G-31015840

PCR fragments were separated by electrophoresis on 15agarose gel and visualized by ethidium bromide stainingThe relative mRNA expression levels were estimated afternormalization with GAPDH The number of cycles waschosen at which PCR product amount was optimal andwithin the linear portion of the curve well before saturationpoint

ASS number of cyclesmdash26 size of the ampliconmdash448 bp

ASL number of cyclesmdash27 size of the ampliconmdash502 bp

OTC number of cyclesmdash40 size of the ampliconmdash1125 bp

GAPGH number of cyclesmdash25 size of the ampli-conmdash496 bp

24 Visualization of Monodansylcadaverine-Labelled Vac-uoles MDC is an autofluorescent weak base that accumu-lates in acidic lysosomal vacuoles and autophagolysosomes[21] Cells attached to glass coverslips were incubated with005mM MDC (Sigma-Aldrich) in PBS at 37∘C for 10minAfter incubation cells were washed three times with PBSand immediately analyzed with a fluorescence microscope(ZEISS Axio Imager A1) equippedwith Axio Vision Software(v 463) Images were captured with a CCD camera andimported into Photoshop Quantification of cell fluorescencewas conducted using ImageJ 148v Software









0

100

200

300

400

500

600

700

800

48 967224

Cel

l gro

wth

()

CMAFM

(h)

AFMrarrCM (72h)





CM Hep

G2

SKOV3

OTC

ASS

ASL

GAPDH

AFM

05

h

AFM

4h

AFM

8h

AFM

24h

(a)

Arginine

Arginine

OrnithineOTC

ASL

Fumarate

Urea

UreaARG 1

ARG 2

Cyto

plas

mM

itoch

ondr

ia


Aspartate

Citrulline

Argininosuccinate

(b)







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)



CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























0

100

200

300

400

500

600

700

800

48 967224

Cel

l gro

wth

()

CMAFM

(h)

AFMrarrCM (72h)





CM Hep

G2

SKOV3

OTC

ASS

ASL

GAPDH

AFM

05

h

AFM

4h

AFM

8h

AFM

24h

(a)

Arginine

Arginine

OrnithineOTC

ASL

Fumarate

Urea

UreaARG 1

ARG 2

Cyto

plas

mM

itoch

ondr

ia


Aspartate

Citrulline

Argininosuccinate

(b)







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)



CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CM Hep

G2

SKOV3

OTC

ASS

ASL

GAPDH

AFM

05

h

AFM

4h

AFM

8h

AFM

24h

(a)

Arginine

Arginine

OrnithineOTC

ASL

Fumarate

Urea

UreaARG 1

ARG 2

Cyto

plas

mM

itoch

ondr

ia


Aspartate

Citrulline

Argininosuccinate

(b)







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)



CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h







AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)



CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(a)

AFM

+ A

snA

FM +

CQ

A

FM +

3M

AA

FM

0h 05h 4h 8h

(b)



CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CM AFM

LC3I

LC3II

ph-p70-S6K

Actin


ph-4E-BP1120572120573120574




4 Discussion




BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BECLIN 1

Actin

Intensity ()


466 plusmn 13 100

(a)

Cel

l gro

wth

()

0

20

40

60

80

100

120

140

160


AFM (96h)

lowast

AFM rarr CM (72h)

(b)


0

20

40

60

80

100

120

140

160

180

6 4 2

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

Days

AFM rarr CM (AFM

72h)

(a)

0

20

40

60

80

100

120

140

160

180

Resto

ratio

n of

cell

grow

th aft

er tr

eatm

ent (

)

6 4 2 Days

lowastlowast


(b)








Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article single amino acid arginine deprivation

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