The Involvement of Arginase and Nitric Oxide Synthase in...

Research ArticleThe Involvement of Arginase and Nitric Oxide Synthase inBreast Cancer Development Arginase and NO Synthase asTherapeutic Targets in Cancer

Nikolay Avtandilyan12 Hayarpi Javrushyan2

Gayane Petrosyan2 and Armen Trchounian 2

1Laboratory of Biochemistry Research Institute of Biology Faculty of Biology Yerevan State University Yerevan Armenia2Department of Biochemistry Microbiology and Biotechnology Faculty of Biology Yerevan State University Yerevan Armenia

Correspondence should be addressed to Armen Trchounian trchounianysuam

Received 21 December 2017 Accepted 15 March 2018 Published 30 April 2018

Academic Editor Swaran J S Flora

Copyright copy 2018 Nikolay Avtandilyan 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

It is well established that during development of malignancies metabolic changes occur including alterations of enzyme activitiesand isoenzyme expression Arginase and nitric oxide (NO) synthase (NOS) are two of those enzymes considered to be involvedin tumorigenesis The goal of this article was to study the involvement of arginase and NOS in the development of different stagesof breast cancer Our results have shown that human serum arginase activity and NO (resp and NOS activity) and polyaminesquantities increased in parallel with cancer stage progression and decreased after neoadjuvant chemotherapy For breast cancer theonly isoenzyme of arginase expressed in serum before and after chemotherapy was in a cationic form The data of Lineweaver-Burk plot with a 119870119898 value of 2mM was calculated which is characteristic for human liver type isoform of arginase Duringelectrophoresis at pH 89 the enzyme exhibited high electrophoretic mobility and was detected near the anode The presentedresults demonstrated that arginase in human serumwith breast cancer and after chemotherapy is not polymorphicWe suggest thatarginase and NOS inhibition has antitumor effects on cancer development as it can inhibit polyamines and NO levels a precursorof cancer cell proliferation metastasis and tumor angiogenesis

1 Introduction

Breast cancer is one of the most common types of canceraccounting for 19 of all cancer-related mortalities inwomen Therefore it is important to continue improvingcurrent diagnostic and treatment tools and to determine newprognostic variables Research over the last years has con-vincingly demonstrated an important role for arginase andnitric oxide (NO) synthase (NOS) in tumor immunobiology[1 2] Earlier reports focused on the expression of arginaseand NOS in murine or human primary cancer tissue aswell as malignant cell lines and emphasized its potential rolein the promotion of tumor growth via polyamine synthesisor downregulation of NO-mediated tumor cytotoxicity [34] L-Arginine is used as a substrate by both NOS andarginase to produce NO and urea respectively Arginase (EC

3531) is a hydrolase which catalyzes the hydrolysis of L-arginine into L-ornithine and urea [5 6] Due to the genera-tion of L-ornithine arginase is involved in several importantdownstreammetabolic pathways L-Ornithine can be furthermetabolized to polyamines (putrescine spermidine andspermine) via ornithine decarboxylase (ODC) [7]Thehepat-ic arginase arginase I is a cytosolic enzyme that catalyzes thefifth and final step in the urea cycle a series of biochemicalreactions in mammals during which the body disposes ofharmful ammonia Extrahepatic arginase arginase II is amitochondrial isoform and its biological function has beenthe subject of considerable interest It seems that this isoformprovides a supply of ornithine a crucial metabolite in biosyn-thesis of glutamic acid proline and polyamines

Currently there is a rapid growth of polyamines (sper-mine spermidine and putrescine) quantity in blood serum

HindawiBioMed Research InternationalVolume 2018 Article ID 8696923 9 pageshttpsdoiorg10115520188696923

2 BioMed Research International

and urine during malignant tumors in different organs [8]The large quantity of polyamines allows the oncogenes toovercome the immune system and allows the cancer cells tometastasize Since polyamines are vital for cell proliferationit is possible that the increased level of ornithine due to theelevated arginase activity may be linked to the developmentof carcinogenesis Increased polyamine uptake by immunecells results in decreased production of tumoricidal cytokinesand the amount of adhesion molecules and these eventuallyattenuate the cytotoxic activities of immune cells

NO is an unorthodox messenger molecule which hasnumerous molecular targets It is the smallest signalingmolecule known which is produced by three isoforms ofNOS (EC 1141339) [2] Data indicate that NO is the integralpart of human physiological response to hypoxia It hasbeen shown that the concentration of NO biomarkers inblood plasma (nitrite nitrate) increases in high altitudes atacclimatization Various oxides of nitrogen are involved inthe increase of NO which provides tolerance for hypoxia [9]High-altitude residents produce more nitric oxide than lowdwellers which is expressed as a high concentration of NO inthe exhaled air as so by increased level of NO metabolites inblood (nitrites nitrates and nitroso compounds)The impor-tance is increasing taking into account the fact that breastcancer patients with metabolic dysregulation are associatedwith poor response to current chemotherapy [10]

Whereas Armenia is a high-altitude locality it was inter-esting to study the quantity change of total nitrite anionsin blood serum during the diseases and after chemotherapyfor revealing the specific regional phenomena Moreoveraccording to the World Health Organization Armenia hasa high smoking percent stress level and coronary heartdisease which also can be responsible for the triggering of NOproduction (there is no clear data for the increase in nitricoxide concentration) Furthermore NO either facilitatescancer-promoting characters or acts as an anticancer agentThe dilemma in this regard still remains unanswered It isimportant once again to examine and understand that differ-ent actions of NO in breast cancer at the molecular level canhelp in providingNObased diagnostic or prognosticmarkersand also in devising potential strategies for prevention andtreatment

Increased NO generation in cancer cells may contributeto tumor angiogenesis by upregulating vascular endothelialgrowth factor (VEGF) and VEGF-induced neovasculariza-tion may increase the tumorsrsquo metastatic ability Increasedamounts of NO have been observed in the blood of breastcancer patients and higherNOS activitywas found in invasivebreast tumors when compared with benign or normal breasttissue [2 11] NO increases tumor blood flow and promotesangiogenesis which could explain the positive correlationbetweenNObiosynthesis and grade ofmalignancy Althoughseveral reports have addressed the protumoral effects ofNO few have demonstrated the contrasting role of NO inmediating tumor regression [12] Although these tumoricidalroles of NO have been proposed most experiments havebeen performed in vitro and such findings have not beenreported in cancer patients It has been suggested that NOconcentrations found in tumors are insufficient to produce

apoptosis and other tumoricidal effects and are likely tofacilitate angiogenesis and tumor dissemination

The levels of arginase activity and NO and polyaminesquantity in malignant tissues have been reported by severalresearchers to be increased compared with healthy tissuesHowever besides the limited number of these studies noneof the authors traced the relation between arginase isoen-zymes activity and biological behavior of tumors and NOand polyamines quantity Therefore the present study wasdesigned not only to determine arginase activity nitriteanions and polyamine concentrations levels in blood serumof breast cancer patients but also to correlate them withbiological behavior of this tumor

The goal of this article was to study the involvement ofarginase and NOS in breast cancer development by thechange of polyamines and NO quantities Arginase activityand polyamines and NO quantity in different stages of breastcancer and after breast cancer stage II patientsrsquo chemotherapyin blood serum were investigated Serum arginase isoen-zymes activity and protein quantity by gel and ion-exchangechromatographies were also studied

2 Materials and Methods

21 Patients This study was performed with blood serumof patients with breast cancer who were hospitalized at theNational Center of Oncology named after V A Fanarjyan(Yerevan Armenia) It included women with primary breastcancer who were hospitalized between 2014 and 2017 Noneof the patients had received preoperative radiotherapy orchemotherapy (besides patients with breast cancer stage II)None of the patients had other diseases The Tumor-Nodes-Metastasis (TNM) system was used to describe the cancerstages (American Joint Committee on Cancer) (Table 1) [13]The surgical intervention and also the chemotherapy (chemo)after surgery on the patients in different stages of diseasedevelopment were not taken into account For all women inchemotherapy group neoadjuvant chemotherapy is usedThedrugs used for neoadjuvant chemotherapy include doxoru-bicin and epirubicin paclitaxel and docetaxel 5-fluorouracil(5-FU) cyclophosphamide (Cytoxan) and carboplatin (Para-platin)Most often combinations of 2 or 3 of these drugs wereused In our experiments blood serums were used after totalperiod of treatment Neoadjuvant chemotherapy was givenfor a total of 3 to 6 months depending on the drugs usedAll work was conducted in accordance with the Declarationof Helsinki (1964) The study was approved by the BioethicsCommittee of Armenia and informed consent was obtainedfrom all patients

22 Chemicals Chemicals for determination of arginaseactivity proteins and NO quantity electrophoresis and TLCwere obtained from Sigma-Aldrich Co Ltd (TaufkirchenGermany) and Carl Roth GmbH + Co KG (Germany)

23 Separation of Serum Arginase For purification of theenzyme the procedure described by Porembska et al for

BioMed Research International 3

Table 1 Characteristics of patients and arginase activity test in blood serum

Patients number Age Stage TNM Characteristic ofarginase activity test Number of patients

11 49 plusmn 15 Healthy -

15 57 plusmn 8 IT1N0M0T1N1miM0T0-1N1miM0

True positive (TP) 53lowast 706

21 53 plusmn 10 IIT0N1M0

T1-2N0-1M0T2-3N0-1M0

False positive (FP) 2 28

18 55 plusmn 11 III

T0minus2N2M0T3N1-2M0T4N0minus2M0TanyN3M0

True negative (TN) 9 12

13 51 plusmn 12II

Chemo

T0N1M0T1-2N0-1M0T2-3N0-1M0

False negative (FN) 11 146

lowast3 women in chemo group also involved in cancer group

human liver and erythrocytes was used with some modifica-tions [14] Separation of arginase isoenzymes was performedby the method of Kossman with some modifications [15 16]The column (2 times 40 cm) containing Sephadex G-150 wasbalanced with Na-phosphate buffer (pH 72) and 40 fractionseach one of 4mlwere collected 4ml of low-molecular-weightprotein fraction after gel filtration was passed through theCM-cellulose column (CM52 1 times 18 cm) balanced against5mM Tris-HCl buffer pH 72 with elution speed of 24mlhwhere 32 fractions each one of 4ml were collected The peakof arginase activity was adsorbed on the column with a linearKCl gradient (005ndash025M)

24 Kinetic Properties of Serum Arginase The double-recip-rocal plot was used for analyzing119870119898 Plotting the reciprocalsof the Michaelis-Menten data pointed yields a ldquodouble-reciprocalrdquo or Lineweaver-Burk plot To reveal 119870119898 the de-pendence of arginase isoenzymes on different concentrationsof the substrate (01 025 04 05 1 15 and 2mML-arginine)was determined Velocities are expressed as 120583M of ureaproduced in 1 sec per ml fraction (after gel chromatographythe fractions from 100 to 124ml)

25 Polyacrylamide Gel Electrophoresis (PAGE) PAGE wasperformed according to the method of Porembska et al [14]Electrophoresis was at 16ndash20∘C and 100ndash120V for 4 h at pH67 through the stacking gel (14wv polyacrylamide) and atpH 89 through the resolving gel (77wv polyacrylamide)Electrophoresis was performed in Tris-glycine buffer Gelswere stained using 200ml of the fixative solution contain-ing 01 (wv) Coomassie Brilliant Blue R250 for 1 h withshaking Gels were subsequently destained in 10 (vv) aceticacid for approximately 12 h with shaking Arginase activitywas determined in 5mm gel slices eluted with NaOH-glycinebuffer pH 95

26 Determination of Arginase Activity Arginase activity wasdetermined by the colorimetric method of Van Slyke andArchibald with some modifications [14 16] 15ml 02Mglycine buffer (pH 95) 05ml blood serum 02ml 5120583MMnCl2 and 04ml 50120583ML-argininewere added in test tubesIn the supernatant the final product of the catalysis wasdetermined which was urea 1ml supernatant and 025ml3 (wv) diacetylmonoxime (DAMO)were added andboiledin a water bath for 45min The intensity was measuredwith a spectrophotometer at 487 nm Activity of enzyme wasevaluated with the received urea in micromoles in 1 sec (kat)

27 Dansylation andThin Layer Chromatography (TLC) Anal-ysis Themethod of Seiler was usedwith somemodificationsas follows [16 17] Tissueswere extracted in 02McoldHClO4at a ratio of about 100mgml After extraction for 1 h in anice bath samples were centrifuged for 20min at +4∘C and11500 g (119903av = 11 cm) 200120583l of HClO4 extract wasmixed with400 120583l of dansyl chloride (5mgml in acetone) and 200 120583l ofsaturated sodium carbonate was added Dansyl polyamineswere extracted in 05ml benzene and vortexed for 30 sec Upto 50 120583l of dansylated extract was loaded on the preadsorbentzone of silica gel plates and the chromatogram was devel-oped for about 2 h with chloroform-triethylamine (25 2 vv)solvent system The dansyl polyamine bands were scrapedeluted in 2ml ethyl acetate and quantified in 505 nm Thequantity of polyamines is presented in nMpolyamines in 1mlof serum

28 Griess Assay for NO Quantity Nitrite was measured bythe Griess assay as described [16 18] Briefly 100 120583l Griessreagent was added to 100 120583l of each of the above supernatantsThe plates were read at 550 nm against a standard curve ofNaNO2 The values were corrected for the NO2minus + NO3minuscontent of water and the recovery of NO2minus was calculated


29 Protein Quantity Protein quantity was determined ac-cording to Lowry method [19]

210 Data Processing Results were expressed as means plusmn SDand evaluated by Studentrsquos 119905-test (single sample) using Statis-tica software (StatSoft 100)

3 Results and Discussion

In our research work arginase activity and polyamines andNO quantities were determined in blood serum of 11 healthyindividuals (female 34ndash63 years old) patients with breastcancer (54 patients female stages IndashIII 43ndash66 years old)and 13 patients after chemotherapy (stage II 39ndash63 years old)(Table 1)

A low level of arginase is normally present in plasma ofhealthy individuals but can become elevated in certain condi-tions or diseases As plasma arginase activity is not routinelyassayed by clinical chemistry laboratories the full range ofconditions in which it becomes elevated is not yet known [6]

Our studies have shown that in women from the breastcancer group of stage I activity of serum arginase wasincreased by 50 in the group of stage II by 721 and in thegroup of stage III by 1317 compared to the healthy group (11healthy individuals) (Figure 1) This is an interesting findingsince obtained data show statistically significant correlationfor all stages in contrast to other published data where corre-lationswere found only in advanced stages [6 9 14 20]Theseresults approve that arginase activity increase correspondedto breast cancer progression An increase of arginase activitywas found in 706 of patients (true positive) (Table 1)

Special attention was worth giving to the decrease ofarginase activity after chemotherapy In postchemotherapygroup (stage II) arginase activity showed 356 decreasecompared with breast cancer stage I patients (see Figure 1)

Arginase activity after chemotherapy had closer values tostage I however it did not reach the control group valueThisinsinuates that arginase might have some role in promotingtumor growth and development and its activity can be con-sidered as an importantmarker to determine disease progres-sion or regression It is difficult to follow up and control thechanges of biochemical options in blood serum in the samepatients after half andor one year after chemotherapyThere-fore we have scant data processing which is not reliable andcannot be submitted in this work Later with the necessaryamount of data it can serve as a material for a further article

The calculated values of arginase activity test sensitivity[TP(TP + FN)] specificity [TN(TN + FP)] and accuracy[(TP + TN)(TP + FP + FN + TN)] in serum of women withbreast cancer were 83 81 and 826 respectively (seeTable 1)

In blood serum of control group (healthy) gel chro-matography (GC) revealed 1 peak for arginase activity (119881119890 =92ml) and 2 peaks for protein quantity (119881119890 = 48 and 92ml)(Figure 2(a))

In breast cancer group stage II and after chemotherapyarginase activity was distributed in one (119881119890 = 116ml) and

Arg

inas

e act

ivity

M

ure

ase

c (ka

t)

3

25

2

15

1

05

0Norm Stage I Stage II Stage III Chemotherapy

Stage II

lowast

lowastlowast

Figure 1 The change of arginase activity in blood serum duringdifferent stages of breast cancer and after chemotherapy of patientsin stage II 119901 lt 005 119899 is the number of patients for 119899 see Table 1for others see Materials and Methods lowast119901 lt 0001 lowastlowast119901 lt 005

protein quantity was distributed over two peaks (119881119890 = 52 and116ml) (Figures 2(b) and 2(c))

The influence of substrate concentrations varying from01 to 2mM on enzymic activity (fractions after gel chro-matography 92ndash116ml for each group 119899 = 7 119901 lt 005)was studied in 02M glycine buffer (pH 95) and 02ml 5120583MMnCl2 From this data a Lineweaver-Burk plot with a 119870119898value of 2mM was calculated which is characteristic for theliver and erythrocytes type isoform of arginase (arginase I)(Figure 3)

To confirm this fact ion-exchange chromatography (IEC)of the second peak fraction (low molecular fraction 119881119890 =92 and 116ml) was performed In ion-exchange chromatog-raphy arginase activity curve is presented with 1 cationicisoenzyme peak (119881119890 = 80ml) in postchemotherapy group 1peak was also visible (119881119890 = 92ml) (Figures 4(b) and 4(c)) Atthis point very few papers are available regarding isoenzymespectrums in serum For breast cancer it was revealed that theonly isoenzyme expressed in serum before and after chemo-therapy was a cationic form (arginase I) (Figures 3ndash5)

Our studies have shown the increase of arginase activityin fractions after gel and ion-exchange chromatographycompared to the control group and the decrease in gel andion-exchange chromatography fractions after chemotherapycompared to the cancer group fractions (see Figures 2 and4) In women with breast cancer arginase activity (kat) in GCfraction accounted for 0068 plusmn 0007 kat (increased by 97compared with norm stage II 119899 = 7 119901 lt 005 Figure 2(b))and after IEC it is 0052plusmn 0005 (increased by 58 comparedwith norm stage II 119899 = 7 119901 lt 005 Figure 4(b))The activityin serum from healthy women was lower and it accountedfor 0062 plusmn 0006 (119901 lt 005 after IEC it is 0049 plusmn 0006Figures 2(a) and 4(a)) The activity in serum from womenafter chemotherapy was lower than norm and cancer groupwomen and it accounted for 0054plusmn0003 (decreased by 206compared with breast cancer group stage II 119899 = 7 119901 lt 005


mg

prot

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ract

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35

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4 12 20 28 36

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52

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76 84 92

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Arg

inas

e act

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m

oles

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ase

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(a)

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4 12 20 28 36

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(b)

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prot

ein

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ract

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45

3

25

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05

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Elution volume (ml)

4 12 20 28 36

44

52

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76 84 92

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156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

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001

0

(c)

Figure 2 Arginase activity (998771) and protein quantity (◼) curves after gel chromatography of serum proteins in healthy individuals breastcancer patients and patients after chemotherapy (stage II 119899 = 7 119901 lt 005) (a) Healthy (b) breast cancer and (c) chemotherapyThe enzymeobtained after several steps of purification (see Materials and Methods 45mg 51mg and 52mg protein resp for healthy individuals breastcancer patients and patients after chemotherapy) was applied to a Sephadex G-150 column (2 times 40 cm)

1(

M u

rea

sec

ml f

ract

ion)

100

90

80

70

60

50

40

30

20

10

0

minus10

minus20

minus1Km

1[L-arginine (mM)]minus2 minus1minus15 minus05 0 05 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10

Figure 3 Lineweaver-Burk plot of human serum arginase [serumproteins obtained (9 11 and 13mg resp for healthy individuals (998771119881119890 = 92ml) breast cancer patients (◼ 119881119890 = 116ml) and patientsafter chemotherapy (e119881119890 = 116ml)) after gel chromatography wasused]

Figure 2(c)) and after IEC it is 0045 plusmn 0007 (decreased by82 compared with breast cancer group stage II 119899 = 7119901 lt 005 Figure 4(c))

Ion-exchange protein purification is possible becausemost proteins bear nonzero net electrostatic charges at all pHvalues except at pH = p119868 (isoelectric point) At pH gt p119868 ofa given protein that protein becomes negatively charged (ananion) but at the pH lt p119868 of that same protein it becomespositively charged (a cation)Mammalian liver arginases tendto have basic isoelectric point (p119868) values of about 88ndash94[21]

Figure 5 illustrates polyacrylamide gel electrophoresis ofhuman serumarginase after gel chromatography (2ndproteinquantity peak 119881119890 = 116ml) During electrophoresis at pH89 (at pH 89 most proteins become negatively charged)the enzyme exhibited high electrophoretic mobility and wasdetected near the anode Arginase activity for cancer andchemo group was found in the first lane (see Figure 5 serumarginase) This means that p119868 for serum arginase is lt89 Our


mg

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Arg

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0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

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ract

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4 12 20 28 36

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76 84 92

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Arg

inas

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oles

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ase

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005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

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Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

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108

116

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mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

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005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)

Figure 4 Arginase activity (998771) and protein quantity (◼) curves after CM-cellulose chromatography of human serum arginase (stage II 119899 = 7119901 lt 005) (a) Healthy (b) breast cancer and (c) chemotherapyThe enzyme obtained after gel chromatography (9 11 and 13mg protein respfor healthy individuals breast cancer patients and patients after chemotherapy) to a CM-cellulose column (1 times 18 cm) equilibrated with 5mMTris-HCI pH 75 eluted with a KCl concentration gradient Fractions (4ml) were collected at a flow rate of 24mlh

results have shown that human serum arginase is a cationicprotein and its mobility at pH 89 was similar to the mobilityof the arginase form from rat and human liver (see Figures 4and 5) In human liver kidney erythrocytes and mammaryglands multiple molecular forms of arginase were shown tobe present Our presented results (after ion-exchange chro-matography and electrophoresis) demonstrated that arginasein human serum with breast cancer and after chemotherapyis not polymorphic

Increase of arginase activity allows suggesting that itshould have reflection on polyamines quantity To confirmthis assumption in further studies the quantity of polyamineswas determined by thin layer chromatography (TLC) Chro-matography results showed that in every stage of cancer alongwith arginase activity increasing the quantity of polyamineswas also growing The increase of total polyamines quantityin serum compared with standard was 926 1343 1742

and 1041 respectively in stages I II and III of breast cancerand after chemotherapy (Table 2)

This coincides with the increase of arginase activityshowing the correlation between them during the diseaseThe results indicate the acceleration of metabolism as a resultof disease because a large amount of building material isneeded and polyamines are an essential factor for tumorgrowth

The results of our study have shown that in blood serumof patients with breast cancer stage II before chemotherapycompared to the control group the quantity of nitrite anionswas increased by 357 and after chemotherapy it wasincreased by 147 (decreased by 21 compared with cancergroup) (Figure 6) Results show that the quantity of nitriteanions in blood serum is rapidly increased during the diseaseand decreases after treatment This once again indicates theinvolvement of NO in the processes of tumor development


Breastcancer Chemo

Standardproteinmixture

220 kDa Myosin

170 kDa Alcoholdehydrogenase

69 kDa Bovine serumalbumin

45 kDa Ovalbumin

38 kDa Bovine liverArginase+

Gamma globulin

Beta 2 globulinBeta 1 globulin

Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase

Figure 5 Diagram of electrophoretic patterns of human serum pro-teins obtained (20120583g) after gel chromatography was used (stage II)Standard protein mixture (each of 10120583g) lane containing molecularweight standards from Sigma 38 kDa bovine liver arginase 45 kDaovalbumin 69 kDa serum albumin 170 kDa alcohol dehydrogenaseand 220 kDa myosin

M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005

0Norm Breast cancer stage II Chemotherapy

Figure 6 The change of nitrite anions quantity in blood serum ofbreast cancer patients and after breast cancer patientsrsquo chemotherapyof stage II (119899 = 7 for norm 119899 = 11 for cancer of stage II abd 119899 = 7after chemotherapy 119901 lt 005)

Arginase activity and polyamines and NO quantities aresignificantly increased during breast malignant cancer andthis enhancement is in correlation with the cancer stages Inthe current study for the first time it was shown that arginaseand NOS activities decrease after chemotherapy as well asthe suggestion was supported that this enzymes activity waselevated in case of breast malignancies

Table 2The changes of polyamines quantity (SPM spermine SPDspermidine PUT putrescine) in blood serumduring different stagesof breast cancer (119899 = 10 for each stage 119901 lt 0001) and afterchemotherapy in breast cancer patients in stage II (119899 = 7 119901 lt 005)

Stage of cancer Polyamine nM Polyaminesml serumBreast Chemotherapy

NormPUT 72 plusmn 053 -SPD 86 plusmn 074 -SPM 113 plusmn 09 -

IPUT 142 plusmn 11 -SPD 174 plusmn 19 -SPM 206 plusmn 18 -

IIPUT 178 plusmn 17 153 plusmn 15

SPD 213 plusmn 12 164 plusmn 18

SPM 244 plusmn 17 236 plusmn 21

IIIPUT 199 plusmn 11 -SPD 226 plusmn 13 -SPM 318 plusmn 22 -

119877119891 values for serumpolyamines in healthy individuals are119877119891 SPM=029119877119891SPD=045 and119877119891 PUT=072 for serumpolyamines of the group ofwomenwith breast cancer are stage I 119877119891 SPM = 03 119877119891 SPD = 042 and 119877119891 PUT =067 stage II 119877119891 SPM = 033 119877119891 SPD = 042 and 119877119891 PUT = 075 stage III119877119891 SPM = 032 119877119891 SPD = 044 and 119877119891 PUT = 065 and after chemotherapy119877119891 SPM = 031 119877119891 SPD = 041 and 119877119891 PUT = 068

4 Conclusions and Significance

After chemotherapy blood serumarginase activity and nitriteanions quantity are decreased but they still maintain a rela-tively high level The decrease of effectivity of chemotherapyand the less sensitivity of patients to it can be related hence todisbalance in L-arginine metabolism because of the initiallyhigh level ofNO in blood serumThe last assumption requiresfurther research and a future study will be focused on morethorough examination of the mentioned processes We candraw a conclusion that arginase and NOS are the importantpartakers of cancerogenesis and can be considered as animportant diagnostic and treatment tools for cancer develop-mentThe feature of the arginase and NOS family that makesthem attractive as therapeutic targets is that there are smallmolecules inhibiting these enzymes [1 10 20 21]

The study in our laboratory (electrophoresis119870119898 and geland ion-exchange chromatography) show that we have justarginase I in blood serumThese results allowus to narrow thescope of investigation and find noncytotoxic concentrationof natural or synthetic specific inhibitor for arginase I Basedon our investigations we suggest applying direct specificinhibitors for thementioned enzymes whichwill have imme-diate influence on them in contrast to other research workswhere there is nonspecific chemicalrsquos influence on the changeof activity of the mentioned enzymes [22 23] It should benoted that in this case undesirable side effects are possiblewhich can also activate other metabolic pathways during thediseases

We suggest that arginase and NOS inhibition may havesome antitumor effects on cancer development as it inhibits


polyamines and NO levels a precursor of cancer cell proli-feration and tumor angiogenesisThe intermediate ofNO syn-thesis NG-hydroxy-nor-L-arginine (nor-NOHA) is a well-known arginase inhibitor Noncompetitive inhibition of nor-NOHA and high affinity for the anionic isoform of arginase IIcreate a chance to escape from the possible hyperammone-mia due to arginase activity inhibition NG-nitro-L-argininemethyl ester (L-NAME) is one of the most clinically devel-oped NOS inhibitors [10] Collectively these observationssupport the preclinical evaluation of nor-NOHA and L-NAME for the treatment of different types of cancer Sincemodern medicine has no effective cure for the malignantcancers and tumors scientists are interested in finding apotent agent with noncytotoxic properties

Currently we study the anticancer influence of differentconcentrations ofNG-hydroxy-nor-L-arginine andNG-nitro-L-argininemethyl ester (inhibiting arginase andNOS activitycorrespondingly) in an experimental model of rats withbreast cancer induced by 712-dimethylbenz(a)anthracene(DMBA) At this stage (studies are in progress) studies showlong-term anticancer effect of nor-NOHA and L-NAMEThis was mainly manifested by the decrease of tumor sizesand quantity as well as mortality and also the biochemicalparameters for quantity of polyamines NOmalondialdehyde(MDA) and histopathological alterations were close to thehealthy rats The obtained results can serve as a base to useour model for determination of productive and noncytotoxicantitumor concentration of anticancer agents Perspectivesof targeting arginase and NOS in cancer management canground application in clinical medicine It can also be usedto reveal new inhibiting model of enzymes by experimentaland theoretical ways

Data Availability

Thedata supporting the conclusions of this article are includ-ed within this article

Ethical Approval

All experiments were conducted according to the principlesof the National Center of Bioethics (Armenia)

Disclosure

Some results presented were reported as a poster at the 42thFEBS Congress (2017) in Jerusalem Israel [24] All authorsagree to publish the manuscript in its present form

Conflicts of Interest

The authors declare that there are no conflicts of interest re-garding the publication of this article

Authorsrsquo Contributions

Nikolay Avtandilyan Hayarpi Javrushyan and GayanePetrosyan performed the experiments and analyzed the

results Nikolay Avtandilyan wrote the manuscript HayarpiJavrushyan edited the manuscript Armen Trchouniandirected the research and revised and reedited the manu-script All authors read and approved the final version of themanuscript

Acknowledgments

The authors would like to thank the National Center ofOncology named after V A Fanarjyan (Yerevan Armenia)and especially Dr Knarik Aleksanyan for providing cancerserum specimens for this research This work was supportedby the State Committee of ScienceMinistry of Education andScience of Armenia in the frames of the Research Project no16YR-1F023

References

[1] P C Rodrıguez and A C Ochoa ldquoArginine regulation by mye-loid derived suppressor cells and tolerance in cancer mech-anisms and therapeutic perspectivesrdquo Immunological Reviewsvol 222 no 1 pp 180ndash191 2008

[2] B Bonavida Nitric Oxide and Cancer Pathogenesis and Ther-apy Springer International Publishing Cham Switzerland2015

[3] C Engblom C Pfirschke and M J Pittet ldquoThe role of myeloidcells in cancer therapiesrdquo Nature Reviews Cancer vol 16 no 7pp 447ndash462 2016

[4] S K Choudhari M Chaudhary S Bagde A R Gadbail andV Joshi ldquoNitric oxide and cancer a reviewrdquo World Journal ofSurgical Oncology vol 11 article 118 11 pages 2013

[5] R Lucas The Role of Arginase in Endothelial DysfunctionFrontiers in Immunology Frontiers Media SA 2015

[6] S M Morris ldquoArginases and arginine deficiency syndromesrdquoCurrent Opinion in Clinical Nutrition amp Metabolic Care vol 15no 1 pp 64ndash70 2012

[7] M H Park and K Igarashi ldquoPolyamines and their metabolitesas diagnostic markers of human diseasesrdquo Biomolecules ampTherapeutics vol 21 no 1 pp 1ndash9 2013

[8] K Soda ldquoThe mechanisms by which polyamines accelerate tu-mor spreadrdquo Journal of Experimental amp Clinical Cancer Re-search vol 30 no 1 article 95 2011

[9] D Z Levett B O Fernandez H L Riley et al ldquoThe role ofnitrogen oxides in human adaptation to hypoxiardquo Scientific Re-ports vol 1 article 109 2011

[10] J Steppan D Nyhan and D E Berkowitz ldquoDevelopment ofnovel arginase inhibitors for therapy of endothelial dysfunc-tionrdquo Frontiers in Immunology vol 4 article 278 2013

[11] M Munder ldquoArginase an emerging key player in the mam-malian immune systemrdquo British Journal of Pharmacology vol158 no 3 pp 638ndash651 2009

[12] H Vahora M A Khan U Alalami and A Hussain ldquoThe po-tential role of nitric oxide in halting cancer progression throughchemopreventionrdquo Journal of Cancer Prevention vol 21 no 1pp 1ndash12 2016

[13] National Comprehensive Cancer Network (NCCN) PracticeGuidelines in Oncology Version 3 NCCN 2014

[14] Z Porembska G Luboinski A Chrzanowska M MielczarekJ Magnuska and A Baranczyk-Kuzma ldquoArginase in patients


with breast cancerrdquo Clinica Chimica Acta vol 328 no 1-2 pp105ndash111 2003

[15] N Avtandilyan S Karapetyan and K Aleksanyan ldquoThe rela-tionship between arginase activity and change of polyaminesquantity in humanblood serumduring breast cancerrdquoBiologicalJournal of Armenia vol 2 no 68 pp 92ndash99 2016

[16] H JavrushyanNAvtandilyanAMamikonyan andATrchou-nian ldquoThe changes of polyamines and nitric oxide quantities inhuman blood serum of prostate and bladder cancerrdquo BiologicalJournal of Armenia vol 1 no 69 pp 125ndash132 2017

[17] DMorganMethods inMolecular Biology Volume 79 PolyamineProtocols Humana Press 1998

[18] Y Vodovotz ldquoModified microassay for serum nitrite and ni-traterdquo BioTechniques vol 20 no 3 pp 390ndash394 1996

[19] OHLowryN J RosebroughA L Farr andR J Randall ldquoPro-tein measurement with the folin phenol reagentrdquoThe Journal ofBiological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] MV PokrovskiyM V Korokin S A Tsepeleva et al ldquoArginaseinhibitor in the pharmacological correction of endothelialdysfunctionrdquo International Journal of Hypertension vol 2011Article ID 515047 4 pages 2011

[21] C P Jenkinson W W Grody and S D Cederbaum ldquoCom-parative properties of arginasesrdquoComparative Biochemistry andPhysiologymdashPart B Biochemistry amp Molecular Biology vol 114no 1 pp 107ndash132 1996

[22] H ErbasO Bal andE Cakır ldquoEffect of rosuvastatin on arginaseenzyme activity and polyamine production in experimentalbreast cancerrdquo Balkan Medical Journal vol 32 no 1 pp 89ndash952015

[23] A J Espanol A Salem D Rojo and M E Sales ldquoParticipationof non-neuronal muscarinic receptors in the effect of carbacholwith paclitaxel on human breast adenocarcinoma cells Roles ofnitric oxide synthase and arginaserdquo International Immunophar-macology vol 29 no 1 pp 87ndash92 2015

[24] N Avtandilyan and A Trchounian ldquoArginase andNO-synthaseare target enzymes in breast anticancer therapyrdquo The FEBSJournal vol 284 no supplement 1 p 256 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

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ObesityJournal of



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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


and urine during malignant tumors in different organs [8]The large quantity of polyamines allows the oncogenes toovercome the immune system and allows the cancer cells tometastasize Since polyamines are vital for cell proliferationit is possible that the increased level of ornithine due to theelevated arginase activity may be linked to the developmentof carcinogenesis Increased polyamine uptake by immunecells results in decreased production of tumoricidal cytokinesand the amount of adhesion molecules and these eventuallyattenuate the cytotoxic activities of immune cells

NO is an unorthodox messenger molecule which hasnumerous molecular targets It is the smallest signalingmolecule known which is produced by three isoforms ofNOS (EC 1141339) [2] Data indicate that NO is the integralpart of human physiological response to hypoxia It hasbeen shown that the concentration of NO biomarkers inblood plasma (nitrite nitrate) increases in high altitudes atacclimatization Various oxides of nitrogen are involved inthe increase of NO which provides tolerance for hypoxia [9]High-altitude residents produce more nitric oxide than lowdwellers which is expressed as a high concentration of NO inthe exhaled air as so by increased level of NO metabolites inblood (nitrites nitrates and nitroso compounds)The impor-tance is increasing taking into account the fact that breastcancer patients with metabolic dysregulation are associatedwith poor response to current chemotherapy [10]

Whereas Armenia is a high-altitude locality it was inter-esting to study the quantity change of total nitrite anionsin blood serum during the diseases and after chemotherapyfor revealing the specific regional phenomena Moreoveraccording to the World Health Organization Armenia hasa high smoking percent stress level and coronary heartdisease which also can be responsible for the triggering of NOproduction (there is no clear data for the increase in nitricoxide concentration) Furthermore NO either facilitatescancer-promoting characters or acts as an anticancer agentThe dilemma in this regard still remains unanswered It isimportant once again to examine and understand that differ-ent actions of NO in breast cancer at the molecular level canhelp in providingNObased diagnostic or prognosticmarkersand also in devising potential strategies for prevention andtreatment

Increased NO generation in cancer cells may contributeto tumor angiogenesis by upregulating vascular endothelialgrowth factor (VEGF) and VEGF-induced neovasculariza-tion may increase the tumorsrsquo metastatic ability Increasedamounts of NO have been observed in the blood of breastcancer patients and higherNOS activitywas found in invasivebreast tumors when compared with benign or normal breasttissue [2 11] NO increases tumor blood flow and promotesangiogenesis which could explain the positive correlationbetweenNObiosynthesis and grade ofmalignancy Althoughseveral reports have addressed the protumoral effects ofNO few have demonstrated the contrasting role of NO inmediating tumor regression [12] Although these tumoricidalroles of NO have been proposed most experiments havebeen performed in vitro and such findings have not beenreported in cancer patients It has been suggested that NOconcentrations found in tumors are insufficient to produce

apoptosis and other tumoricidal effects and are likely tofacilitate angiogenesis and tumor dissemination

The levels of arginase activity and NO and polyaminesquantity in malignant tissues have been reported by severalresearchers to be increased compared with healthy tissuesHowever besides the limited number of these studies noneof the authors traced the relation between arginase isoen-zymes activity and biological behavior of tumors and NOand polyamines quantity Therefore the present study wasdesigned not only to determine arginase activity nitriteanions and polyamine concentrations levels in blood serumof breast cancer patients but also to correlate them withbiological behavior of this tumor

The goal of this article was to study the involvement ofarginase and NOS in breast cancer development by thechange of polyamines and NO quantities Arginase activityand polyamines and NO quantity in different stages of breastcancer and after breast cancer stage II patientsrsquo chemotherapyin blood serum were investigated Serum arginase isoen-zymes activity and protein quantity by gel and ion-exchangechromatographies were also studied

2 Materials and Methods

21 Patients This study was performed with blood serumof patients with breast cancer who were hospitalized at theNational Center of Oncology named after V A Fanarjyan(Yerevan Armenia) It included women with primary breastcancer who were hospitalized between 2014 and 2017 Noneof the patients had received preoperative radiotherapy orchemotherapy (besides patients with breast cancer stage II)None of the patients had other diseases The Tumor-Nodes-Metastasis (TNM) system was used to describe the cancerstages (American Joint Committee on Cancer) (Table 1) [13]The surgical intervention and also the chemotherapy (chemo)after surgery on the patients in different stages of diseasedevelopment were not taken into account For all women inchemotherapy group neoadjuvant chemotherapy is usedThedrugs used for neoadjuvant chemotherapy include doxoru-bicin and epirubicin paclitaxel and docetaxel 5-fluorouracil(5-FU) cyclophosphamide (Cytoxan) and carboplatin (Para-platin)Most often combinations of 2 or 3 of these drugs wereused In our experiments blood serums were used after totalperiod of treatment Neoadjuvant chemotherapy was givenfor a total of 3 to 6 months depending on the drugs usedAll work was conducted in accordance with the Declarationof Helsinki (1964) The study was approved by the BioethicsCommittee of Armenia and informed consent was obtainedfrom all patients

22 Chemicals Chemicals for determination of arginaseactivity proteins and NO quantity electrophoresis and TLCwere obtained from Sigma-Aldrich Co Ltd (TaufkirchenGermany) and Carl Roth GmbH + Co KG (Germany)

23 Separation of Serum Arginase For purification of theenzyme the procedure described by Porembska et al for








T1-2N0-1M0T2-3N0-1M0


18 55 plusmn 11 III



13 51 plusmn 12II

Chemo

T0N1M0T1-2N0-1M0T2-3N0-1M0





















Arg

inas

e act

ivity

M

ure

ase

c (ka

t)

3

25

2

15

1

05


Stage II

lowast

lowastlowast







mg

prot

ein

ml f

ract

ion

4

35

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

007

006

005

004

003

002

001

0

(a)

mg

prot

ein

ml f

ract

ion

4

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

008

007

006

005

004

003

002

001

0

(b)

mg

prot

ein

ml f

ract

ion

4

5

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

(c)


1(

M u

rea

sec

ml f

ract

ion)

100

90

80

70

60

50

40

30

20

10

0

minus10

minus20

minus1Km







mg

prot

ein

ml f

ract

ion

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

ein

ml f

ract

ion

09

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

06

04

02

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

004

0045

005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)








Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























T1-2N0-1M0T2-3N0-1M0


18 55 plusmn 11 III



13 51 plusmn 12II

Chemo

T0N1M0T1-2N0-1M0T2-3N0-1M0





















Arg

inas

e act

ivity

M

ure

ase

c (ka

t)

3

25

2

15

1

05


Stage II

lowast

lowastlowast







mg

prot

ein

ml f

ract

ion

4

35

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

007

006

005

004

003

002

001

0

(a)

mg

prot

ein

ml f

ract

ion

4

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

008

007

006

005

004

003

002

001

0

(b)

mg

prot

ein

ml f

ract

ion

4

5

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

(c)


1(

M u

rea

sec

ml f

ract

ion)

100

90

80

70

60

50

40

30

20

10

0

minus10

minus20

minus1Km







mg

prot

ein

ml f

ract

ion

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

ein

ml f

ract

ion

09

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

06

04

02

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

004

0045

005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)








Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































Arg

inas

e act

ivity

M

ure

ase

c (ka

t)

3

25

2

15

1

05


Stage II

lowast

lowastlowast







mg

prot

ein

ml f

ract

ion

4

35

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

007

006

005

004

003

002

001

0

(a)

mg

prot

ein

ml f

ract

ion

4

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

008

007

006

005

004

003

002

001

0

(b)

mg

prot

ein

ml f

ract

ion

4

5

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

(c)


1(

M u

rea

sec

ml f

ract

ion)

100

90

80

70

60

50

40

30

20

10

0

minus10

minus20

minus1Km







mg

prot

ein

ml f

ract

ion

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

ein

ml f

ract

ion

09

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

06

04

02

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

004

0045

005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)








Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















mg

prot

ein

ml f

ract

ion

4

35

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

007

006

005

004

003

002

001

0

(a)

mg

prot

ein

ml f

ract

ion

4

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

008

007

006

005

004

003

002

001

0

(b)

mg

prot

ein

ml f

ract

ion

4

5

35

45

3

25

2

15

1

05

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

132

140

148

156

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

(c)


1(

M u

rea

sec

ml f

ract

ion)

100

90

80

70

60

50

40

30

20

10

0

minus10

minus20

minus1Km







mg

prot

ein

ml f

ract

ion

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

ein

ml f

ract

ion

09

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

06

04

02

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

004

0045

005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)








Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















mg

prot

ein

ml f

ract

ion

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0005 M KCl

01M KCl

015M KCl02M KCl

025 M KCl

(a)

mg

prot

ein

ml f

ract

ion

09

08

07

06

05

04

03

02

01

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

006

005

004

003

002

001

0

005 M KCl01M KCl

015M KCl02M KCl

025 M KCl

(b)12

1

08

06

04

02

0

Elution volume (ml)

4 12 20 28 36

44

52

60

68

76 84 92

100

108

116

124

mg

prot

ein

ml f

ract

ion

Arg

inas

e act

ivity

m

oles

ure

ase

c (ka

t)

003

0035

004

0045

005

0025

002

0015

001

0005

0005 M KCl

01M KCl015M KCl

02M KCl

025 M KCl

(c)








Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Breastcancer Chemo


220 kDa Myosin



45 kDa Ovalbumin


Gamma globulin


Alpha 2 globulin

Alpha 1 globulin

Serum albumin

Serum arginase


M

nitr

ite an

ions

ml s

erum

005

0045

004

0035

003

0025

002

0015

001

0005




















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Data Availability


Ethical Approval


Disclosure







Acknowledgments


References































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















The Involvement of Arginase and Nitric Oxide Synthase in...

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