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Fiziologia - Physiology • 2015.25.3(87)FIZIOLOGIA

physiology
CONTENTS
1. Vitamin D Improves Vascular Function in Experimental Diabetes ........................................................................................................4
Sturza A, Duicu O, Vaduva A, Noveanu L, Danila M, Privistirescu A, Munteanu M, Muntean D
2. In Vitro Assessment of Tumor-Associated Fibroblasts’ Proliferation Ability and Viability ..............................................................10
Anastasiu MD, Cernat L, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Crisnic D, Tatu C, Tanasie G, Panaitescu C,
Paunescu V
3. The Relationship between Vitamin D, Inflammation and the Activity of Systemic Lupus Erythematosus .....................................16
Buleu F, Gurban C, Sarbu E, Serban MC, Tudor A, Ardelean F, Stoichescu-Hogea G, Dragan S
4. Assessment of a Murine Melanoma Model ............................................................................................................................................21
Coricovac D, Berceanu R, Bratu T, Muntean D, Soica C, Ciurlea S, Dehelean C
5. Lupeol a Potent Anti-Inflammtory Agent in Acute Inflammation Mouse Ear Model ..........................................................................25
Minda D, Coricovac D, Pinzaru I, Dehelean C, Borcan F, Muntean D
6. Molecular Markers in Breast Cancer and Their Clinical Significance ................................................................................................29
Margan MM
7. In Vitro Morphological Characterization of Endothelial Cells Derived from Bovine Cornea ............................................................36
Ivanescu R, Toma M, Lazarovicz R, Bojin F, Nistor D, Panaitescu C, Paunescu V, Tanasie G
8. Anthropometric Basis for the Physiological Demand of Anaerobic Power and Agility in Young Indian
National Level Field Hockey Players ......................................................................................................................................................41
Barun Hanjabam, Konthoujam Kosana Meitei

CUPRINS
1. Vitamina D îmbunătățește funcția cardiacă în diabetul zaharat experimental .....................................................................................4
Sturza A, Duicu O, Vaduva A, Noveanu L, Danila M, Privistirescu A, Munteanu M, Muntean D
2. Analiza in vitro a capacității de proliferare și a viabilității fibroblastelor peri-tumorale ...................................................................10
Anastasiu MD, Cean A, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Tatu C, Tanasie G, Panaitescu C, Paunescu V
3. Relația dintre vitamina D, inflamație și indexul de activitate al lupusului eritematos sistemic .......................................................16
Buleu F, Gurban C, Sarbu E, Serban MC, Tudor A, Ardelean F, Stoichescu-Hogea G, Dragan S
4. Realizarea unui model animal de melanom murinic .............................................................................................................................21
Coricovac D, Berceanu R, Bratu T, Muntean D, Soica C, Ciurlea S, Dehelean C
5. Lupeolul – un agent anti-inflamator puternic în inflmația acută pe un model experimental de ureche murină .............................25
Minda D, Coricovac D, Pinzaru I, Dehelean C, Borcan F, Muntean D
6. Markeri moleculari în cancerul mamar și semnificația lor clinică .......................................................................................................29
Margan MM
7. Caracterizarea morfologică in vitro a celulelor endoteliale obținute din corneea bovină ................................................................36
Ivanescu R, Toma M, Lazarovicz R, Bojin F, Panaitescu C, Paunescu V, Tanasie G
8. Bazele antropometrice ale cererii fiziologice de putere anaerobă și agilitate la jucătorii de hochei pe iarbă
din lotul național de juniori din India ......................................................................................................................................................41
Barun Hanjabam, Konthoujam Kosana Meitei

Fiziologia - Physiology • 2015.25.3(87)3VITAMIN D IMPROVES VASCULAR FUNCTION IN
EXPERIMENTAL DIABETESADRIAN STURZA1,4, OANA DUICU1,4, ADRIAN VADUVA2, LAVINIA NOVEANU1,4, MARIADANILA1,4, ANDREEA PRIVISTIRESCU1, MIRCEA MUNTEANU3, DANINA MUNTEAN1,4
1Department of Pathophysiology
2Department of Morphopathology
3Department of Internal Medicine II
4Center for Translational Research and Systems Medicine“Victor Babeș” University of Medicine and Pharmacy, Timișoara, Romania

ABSTRACTControversy exists over the role of the active form of vitamin D, 1,25-dihydroxycholecalciferol, in the cardiovascularsystem in the presence of diabetes mellitus. The present study were purported to investigate the protective effect of
1,25-vitamin D3 on endothelial function in diabetic rats. To this aim, male Sprague-Dawley rats (8 weeks old) were
injected with streptozotocin (STZ, 50 mg/kg, single injection intraperitoneally) to induce diabetes. After one month ofhyperglycemia, thoracic aorta was removed and used for organ bath experiments. Endothelial function was evaluatedin aortic rings isolated from diabetic and control animals, after 24 hours incubation in the absence or presence of
1,25-cholecalciferol (0.1µM). Endothelial-dependent relaxation to acetylcholine was significantly reduced in diabetic ascompared with normal rats. Vitamin D significantly improved vascular function in diabetic rat aortic segments. In
conclusion, in vitro administration of vitamin D alleviated diabetes-induced endothelial dysfunction in experimental
setting.
Keywords: vitamin D, endothelial dysfunction, diabetes mellitus

INTRODUCTION
Cardiovascular disease is the major cause ofmorbidity and mortality in patients with diabetes. During
the pathophysiological process of vascular damage,endothelial dysfunction is an early stage of diseasedevelopment [1]. There is a large body of evidence
suggesting that conditions associated with hyperglycemia
can lead to different perturbations in endothelial function
via activation of multiple oxidative stress pathways [2-5].
In diabetes, the evolution of atherosclerosis starting fromendothelial dysfunction and ending up with thecomplicated plaques (prone to rupture) is accelerated[6-9]. Several cardiovascular risk factors have beenrecognized as contributors to the atherosclerotic process.
Among them, vitamin D deficiency is emerging as a newone, being a highly investigated issue [10-12]. The active
form of vitamin D, 1,25-dihydroxy-vitamin D3 (calcitriol)
which functions via binding to vitamin D receptors. An
increasing body of evidence suggests that vitamin D has
also cardiovascular effects besides the regulation of
calcium-phosphate homeostasis, and calcitriol and its

analogs may play a role in modulating cardiovascularfunction [13-16]. Ecological studies revealed the fact thatthe incidence of cardiovascular disease augments withincreasing distance from the Equator, suggesting an
association with vitamin D insufficiency in regions withless sun exposure [17,18]. In diabetic patients, serum
25-hydroxyvitamin D (25OHD) concentration is inversely
correlated with prevalence of cardiovascular disease [19],but the mechanism of vascular improvement induced byvitamin D is not very well understood. Vitamin D exerts avariety of favorable effects on endothelial dysfunction,
VSMC proliferation [20,21], inflammatory process ofatherosclerosis [22-25], insulin resistance, β-cell dysfunction[26,27], dyslipidemia [28], renin-angiotensin-aldosteronesystem [22,29], therefore suggesting a potential therapeuticrole, but the information regarding the mechanisms are
missing.
Progression of endothelial dysfunction in diabetes
involves the following general mechanism: high glucose
levels increase reactive oxygen species (ROS)
generation in vascular tissues with subsequentdevelopment and progression of endothelial dysfunction

Received 10th of March 2015. Accepted 15th of April 2015. Address for correspondence: Danina Muntean, MD, PhD,
Department of Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st.,300173 Timisoara, RO, Tel/Fax: +40-256-493085; e-mail: daninamuntean@umft.ro4

Fiziologia - Physiology • 2015.25.3(87)[30]. Several experimental and clinical studies revealed
the fact that NADPH oxidase and the uncoupling ofeNOS are the most important enzymatic systems of ROS
generation [31,36]. In this regard it was showed thatvitamin D down-regulates subunits p47 and p22 of
NADPH oxidase [37] with a significant reduction in ROS
production.
The present study was purported to assess theeffect of 1,25-dihydroxy-vitamin D3, the major metaboliteof vitamin D, on vascular reactivity in aortic segments
harvested from diabetic rats.

MATERIAL AND METHODS
Animal model and tissue preparation
Male Wistar male rats were purchased from the
Cantacuzino Institute (Bucharest, Romania) and wereacclimated for 2 weeks prior to the study. At the age of 8
weeks, diabetes was induced by a single injection of
streptozotocin (50 mg/kg STZ, IP). Age-matched control
rats received an equal volume of vehicle (0.01 M citratebuffer, pH 4.5). Two days after the injection, a blood
sample was collected from the tail vein to measure theblood glucose. Rats with blood glucose over 200 mg/dl
were considered diabetic. Animals were housed understandard conditions (constant temperature and humidityof 22.5 ± 2° C and 55 + 5%, 12-h light/dark cycle). The
duration of the diabetes evolution was 8 weeks and blood
glucose and body weight were systematically monitored.
Twenty-four hours prior to the experiment solid food waswithdrawn with no limitation in water supply.
All experimental procedures used in this study wereconducted in accordance with the Directive 2010/63/EU
and the Romanian Law nr. 43/May 2014 concerning theprotection of animals used for scientific purposes. The
experimental protocol was approved by the Committee
for Research Ethics of “Victor Babes” University for
Medicine and Pharmacy of Timişoara, Romania.
All reagents used were purchased from Sigma
Aldrich, Invitrogen, Applichem and Abcam.

force recording. The rings were stretched to an optimal
tension of 2 g and allowed to equilibrate for 60 min. They
were then exposed to 80 mMKCl to obtain a submaximal
contraction. Organ bath experiments were performed in
the presence of diclofenac (10 μmol/L). The
concentration of phenylephrine, used for pre-constriction,
was adjusted to obtain a pre-constriction level of 80% of
the contraction elicited by KCl(80 mmol/L). Subsequently,
was recorded the endothelium-dependent relaxation to
cumulative concentrations of acetylcholine (Ach) andcontractility to endothelial nitric oxide synthase (eNOS)
inhibitor L-NAME (10μM).
Statistics
Data are presented as mean + SEM and wereanalyzed using a one-way ANOVA or student t-test whereappropriate. Post-hoc comparison among the groups was
performed using the Tukey’s test. Data analysis of thedose-effect response curves was performed using the
ANOVA F-test (comparisons of bottom and top values,
EC50 and the Hill slope). Values of p<0.05 wereconsidered statistically significant.

RESULTS
Animals
Body weight and blood glucose from the day ofexperiment are shown in Table I. Four weeks after STZ
administration, body weight was decreased in STZ rats
than in normal rats. Blood glucose levels in STZ rats
were significantly higher vs. controls.
Table I. Body weight and blood glucose at 4 weeks after STZ
administration.

nBody weight (g)Blood glucose(mg/dL)CONTROL8
421.6 ± 24.8

STZ
8
282.5 ± 49.5*

104.4 ± 10.6490 ± 38.4**p < 0.05 vs. CONTROL
Organ culture
Rat aortic segments were dissected under sterile
conditions, cleaned, and incubated for the times
indicated at 37°C in EBM culture medium containing
0.1% BSA, in the presence or absence of
1,25-cholecalciferol(0.1µM,
Sigma-Aldrich).
Subsequently, the tissue was studied in organ chambers.
Organ bath studies
The rat aortic rings were suspended in organchambers that contained 5 ml of Krebs solution (37°C)
aerated with 95% O2-5% CO2 gas mixture (pH 7.4). Eachring was connected to a force transducer for isometricFiziologia - Physiology • 2015.25.3(87)Vitamin D improves vascular contractility indiabetic aortic segments
The role of Vitamin D in improving vascular function
was studied here in rats with experimental diabetes
induced with STZ. After 1 month of constant
hyperglycemia, thoracic aorta were isolated, incubatedfor 24 h with 1,25-cholecalciferol (0.1 µM) and studied inorgan bath setup. The vascular contractility in diabetic
segments was significantly increased in response tocumulative doses of phenylephrine and incubation withvitamin D partially normalized the contractility response(Figure 1).

5Vitamin D improves NO bioavailability in aortas
form diabetes rats
In diseased vessels the contractility to L-NAME(Nω-Nitro-L-arginine methyl ester hydrochloride, 10 μM)
was increased and was significantly attenuated after
incubation with vitamin D overnight, observationsuggestive for the implication of NO in the mechanism ofvitamin D-dependent vascular function improvement.

Fig. 1.The effects of vitamin D3 incubation on vascular
contractility in diabetic aortas. Aortic segments from
STZ-diabetic rats and controls were treated in vitro 24 hwith 1,25-cholecalciferol (organ culture, 0.1 µM) andevaluated in organ bath system. Phenylephrine-induced
contractions, n=10, *p<0.05 with and without diabetes(DIAB), #p<0.05 with and without vitamin D.

Vitamin D restores the endothelial-dependent
relaxation in aortas from diabetic rats
Also, endothelium-dependent relaxation, after 4
weeks of hyperglycemia was significantly attenuated and
24 h incubation; again, vitamin D partially restored therelaxation response (Figure 2).

Fig. 3.The effects of vitamin D incubation on NO
bioavailability in diabetic aortas. Aortic segments from
STZ-diabetic rats and controls were treated in vitro 24 hwith 1,25-cholecalciferol (organ culture, 0.1 µM) andevaluated in organ bath system. Contraction to L-NAME(Nω-Nitro-L-arginine methyl ester hydrochloride, 10μM),
DELTA= Max.– 10% Phe contr. (Max.= maximal
contraction to L-NAME, 10% Phe.contr.= 10% fromcontraction to 80 mMKCl induced by Phe), n=6, *p<0.05
with and without diabetes (DIAB), #p<0.05 with andwithout vitamin D.

DISCUSSION

Fig. 2.The effects of vitamin D3 incubation on
endothelial-dependent relaxation in diabetic aortas.
Aortic segments from STZ-diabetic rats and controls weretreated in vitro 24 h with 1,25-cholecalciferol (organ
culture, 0.1 µM) and evaluated in organ bath system.
Acetylcholine-induced endothelium-dependent relaxation.
n=6, *p<0.05 with and without diabetes (DIAB), #p<0.05
with and without vitamin D.

6

Atherosclerosis represents the most important causeof cardiovascular diseases; its pathogenesis hascomplex mechanisms involving several cells - endothelial,
vascular smooth muscle and immune cells, and also
different mediators of inflammation [18]. The process ofendothelial dysfunction is extremely accelerated in thepresence of diabetes mellitus, the most severe metabolic
disease.
An increasing body of evidence links vitamin D
deficiency with atherosclerosis and cardiovascular disease[18]. Vitamin D, in addition to its role in phosphate andcalcium metabolism, has been recognized to be an
important factor in the cardiovascular system. The active
metaboliteofvitaminD,
calcitriol(1,25-dihydroxicholecalciferol), has been revealed to exert
protective effects by several mechanisms: inhibition ofplatelets' adhesion and aggregation, inhibition of the
Fiziologia - Physiology • 2015.25.3(87)release of pro-inflammatory mediators and adhesion
molecules (intercellular adhesion molecule ICAM-1,
vascular cell adhesion molecule - VCAM-1) [38,39].
The present study demonstrates an importantvascular effect of the main active metabolite of vitamin D.
The major finding of the study is that 1,25-cholecalciferol
modulate vascular tone by reducing the contraction andendothelial-dependent relaxation in vasculature in
conditions associated with hyperglycemia.
In organ bath experiments, we report a significant
reduction in the endothelium-dependent relaxation ofvascular segments in diabetic animals vs. controls.
Incubation with the vitamin D3 partially restored theendothelium-dependent relaxation, suggesting abeneficial effect, at least in this in vitro model. Obviously,
endothelial dysfunction in diabetes is attributable tovascular ROS production. Several clinical andexperimental studies has shown that among the many
enzymatic systems capable of producing ROS, NADPH
oxidase and uncoupled eNOS are the main sources inthe vascular wall in diabetic patients [40,41]. Besides
these classical sources of ROS generation, monoamineoxidase (MAO), with 2 isoforms, at the outermitochondrial membrane has emerged as a novel sourceof oxidative stress in the cardiovascular system [42,43].
Moreover, in a very recent study we observed an
upregulation of MAO-A in the aortic rings and the hearts
harvested from STZ-diabetic rats [44].
To date it has been demonstrated that vitamin D canmodulate the activity and expression of NADPH oxidase[18,37], classically the most important source of ROS.
However, modulation of other sources of oxyradicals has
not been investigated so far.
Endothelial dysfunction in diabetes can be aconsequenceofhyperglycemia,
inflammation,
hypertension [39,45,46] and vitamin D can play animportant role in modulation of each of these pathogenic
mechanisms: future studies are warranted to investigate
these directions.

CONCLUSIONSIn summary, the present study demonstrates that themajor
activemetaboliteofvitaminD,
1,25-dihydroxy-cholecalciferol,
reduces
endothelial
dysfunction by decreasing the vascular contractility and byimproving the endothelial-dependent relaxation. These
results may help to explain the link between the lower
levels of vitamin D and vascular complications in diabetes.
Elucidating the signal transduction of the vascular effectsof vitamin D in pathological conditions, including diabetes,
might bring novel therapeutic alternatives in the
management of diabetes complications.

Fiziologia - Physiology • 2015.25.3(87)ACKNOWLEDGEMENTS
The research was supported by the POSDRU grant no.
159/1.5/S/136893 titled: “Strategic partnership for theincrease of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”.

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22. Mandarino NR, Junior F, Salgado JV, et al. Is vitamin d
deficiency a new risk factor for cardiovascular disease? Open
Cardiovasc Med J. 2015; 9: 40-9.
23. Menezes AR, Lamb MC, Lavie CJ, et al. Vitamin D andatherosclerosis. Curr Opin Cardiol. 2014; 29(6): 571-7.
24. Singh J, Merrill ED, Sandesara PB, et al. Vitamin D,
Low-Grade Inflammation and Cardiovascular Risk in YoungChildren: A Pilot Study. Pediatr Cardiol. 2015.
25. Wobke TK, Sorg BL, Steinhilber D. Vitamin D in
inflammatory diseases. Front Physiol. 2014; 5: 244.
26. Challa AS, Makariou SE, Siomou EC. The relation ofvitamin D status with metabolic syndrome in childhood andadolescence: an update. J Pediatr Endocrinol. 2015
27. Kavadar G, Demircioglu DT, Ozgonenel L, et al. The
relationship between vitamin D status, physical activity andinsulin resistance in overweight and obese subjects. Bosn J
Basic Med Sci. 2015; 15(2): 62-6.
28. Rusconi RE, De Cosmi V, Gianluca G, et al. Vitamin D
insufficiency in obese children and relation with lipid profile.
Int J Food Sci Nutr. 2015; 66(2): 132-4.
29. Vaidya A, Brown JM, Williams JS. The
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30. Giacco F, Brownlee M. Oxidative stress and diabetic
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31. Huynh K, Kiriazis H, Du XJ, et al. Targeting theupregulation of reactive oxygen species subsequent tohyperglycemia prevents type 1 diabetic cardiomyopathy inmice. Free Radic Biol Med. 2013; 60: 307-17.
32. Li H, Horke S, Forstermann U. Vascular oxidative stress,
nitric oxide and atherosclerosis. Atherosclerosis. 2014; 237(1):208-19.

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33. Patel H, Chen J, Das KC, et al. Hyperglycemia induces
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34. Teshima Y, Takahashi N, Nishio S, et al. Production ofreactive oxygen species in the diabetic heart. Roles of
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35. Brandes RP, Schroder K. Differential vascular functions of
Nox family NADPH oxidases. Curr Opin Lipidol. 2008; 19(5):513-8.
36. Ebrahimian TG, Heymes C, You D, et al. NADPH
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impairs postischemic neovascularization in mice with type 1
diabetes. Am J Pathol. 2006; 169(2): 719-28.
37. Kono K, Fujii H, Nakai K, et al. Anti-oxidative effect ofvitamin D analog on incipient vascular lesion in non-obese
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38. Hirata M, Serizawa K, Aizawa K, et al. 22-Oxacalcitriol
prevents progression of endothelial dysfunction throughantioxidative effects in rats with type 2 diabetes andearly-stage nephropathy. Nephrol Dial Transplant. 2013; 28(5):1166-74.
39. Howangyin KY, Silvestre JS. Diabetes mellitus andischemic diseases: molecular mechanisms of vascular repair
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44. Sturza A, Duicu OM, Vaduva A, et al. Monoamineoxidases are novel sources of cardiovascular oxidative
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Diabetes. 2014; 5(5): 601-5.

Fiziologia - Physiology • 2015.25.3(87)VITAMINA D ÎMBUNĂTĂŢEŞTE FUNCŢIA CARDIACĂ
ÎN DIABETUL ZAHARAT EXPERIMENTAL
REZUMAT
Rolul 1,25-colecalciferolului, forma activă a vitaminei D, la nivelul sistemului cardiovascular în prezența diabetului
zaharat este controversat. În orice caz, mecanismele prin care vitamina D exercita aceste efecte nu sunt pe
deplin înțelese. Scopul acestui studiu a fost investigarea rolului protector al vitaminei D asupra funcției
endoteliale la șobolani diabet zaharat. Pentru aceasta, am folosit un model experimental de diabet zaharat cu
streptozotocină la șobolan (administrare intra-peritoneală, 50 mg/kgc, doză unică). Dupa o luna de hiperglicemie
constantă, aorta toracică a fost prelevată și utilizată pentru studii de reactivitate vasculară în baia de organ.
Funcția endotelială a fost evaluată pe segmente aortice, izolate de la animalele din lotul diabetic comparativ cu
cel martor după 24 de ore de stimuare in vitro cu 1,25-colecalciferol (0,1 µM). La animalele diabetice relaxarea
endotelial-dependentă la acetilcolină a fost semnificativ alterată. Vitamina D a îmbunătățit semnificativ funcția
vasculară pe aceste segmente vasculare. În concluzie, administratrea in vitro a vitaminei D a îmbunătățit funcția
vasculară în diabetul zaharat experimental.
Cuvintecheie: vitamină D, disfuncție endotelială, diabet zaharat

Fiziologia - Physiology • 2015.25.3(87)9IN VITRO ASSESSMENT OF TUMOR-ASSOCIATED
FIBROBLASTS’ PROLIFERATION ABILITY AND VIABILITY
ANASTASIU MD1, CERNAT L2, CRISTEA M3, BOJIN F2, GAVRILIUC O2, GLUHOVSCHI A1,
ANASTASIU D1, CRAINA M1, CRISNIC D2, TATU C2, TANASIE G2, PANAITESCU C2,PAUNESCU V2
1Department of Obstetrics and Gynecology, “Victor Babes” University of Medicine and Pharmacy Timisoara
2Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara
3Immunophysiology and Biotechnologies Center, Clinical Emergency County Hospital Timisoara

ABSTRACTThe origin of tumor-associated fibroblasts is not fully understood. There are multiple data according to which
TAFs are a subset of MSCs and are recruited from bone marrow or from tissue level during tumor development
process. Tumor microenvirnment provides the conection between tumor and host, at the level of primary site andat distance site. This communication is possible through the soluble factors which are secreted by tumor cells
and by cellular component of the tumor microenvironment, in which TAFs represent an essential component.
TAFs plasticity and their ability to induce tumor cells development, invasion, migration and metastasis, but also
their involvement in survival of tumor cells and acquirement of drug-resistance, makes exploration ofthese cells apriority for characterization and use as future therapeutic targets.
We investigated the proliferative ability and viability of two types of tmor-associated fibroblasts – breast
cancer-derived TAFs (bTAF) and uterine cancer-derived TAFs (uTAF) – comparative to their physiologicalcounterparts, the bone marrow-derived mesenchymal stem cells (MSCs). We used the in vitro toxicology assay(MTT-based) and flowcytometric evaluation using Annexin V/PI. The results were similar for all cellular types
under investigation, with a difference for bTAFs regarding the late apoptosis stage, which was significantly lower
compared to MSCs and uTAFs.
Although they present many common features with MSCs, there are still some questions to be answered regarding thedistinct characteristics and functions of TAFs, as well as their specific involvement in tumor pathogenesis.
Key words: mesenchymal stem cells (MSCs), tumor-associated fibroblasts (TAFs), viability tests, MTTINTRODUCTION
Tumor-associated fibroblasts (TAFs) represent themain cellular component of tumor stroma. They are
actively involved in tumor development and progression,
providing secretion of pro-tumorigenic factors, enabling
connection with extracellular matrix and de novo formed
vessels, thus contributing to structuring and functioningof tumor in an organ-like fashion. Intercellular interactions
are very complex due to involvement of several cellular
types – tumor cells, endothelial cells, immune systemcells, fibroblasts, pericytes, adipocytes, etc. Secreted
factors are enrolled in establishment of plurivalentsignaling pathways with the role of tumor developing andacquirement of so-called cellular plasticity (acquirementof new features allowing the cells long-term survival,
expansion, and malignant behavior).

Tumor-associated fibroblasts (TAFs) population is nothomogenous; it is endowed with important heterogeneity,cellular plasticity allowing these cells to transform bothmorphologically and functionally, thus contributing totumor pathogenicity. TAFs modulation of tumor
development is both positive, and negative [1].
Heterogeneity of TAFs subpopulations within a tumor
makes difficult to explore and to establish some specificmarkers; there are topographical differences (depending
on originating tissue), and also within the same tumor [2].
Moreover, analysis of gene expression in breast cancer
samples revealed specific molecular markers for multiple
TAF subtypes [3], a certain TAF subtype being able toinduce an inhibitory or stimulatory effect on tumor
development, depending on activation or inhibition ofcertain factors secretion, characteristics which are still
under investigation.

Received 10th of May 2015. Accepted 20th of June 2015. Address for correspondence: Diana Anastasiu, MD, PhD student,“Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-3000041, Timisoara,phone/fax: +40256220479; e-mail: diana_anastasiu@yahoo.com

10

Fiziologia - Physiology • 2015.25.3(87)However, TAFs origin is under debate, numerous
data pleading for mesenchymal origin of these cells,
being considered a subset of mesenchymal stem cells,
which are either migrating from bone marrow [4], or aretransformed locally from stromal cells.
The objective of this study was to investigate theproliferative ability and viability of tumor-associatedfibroblasts (TAFs) isolated from both breast cancer anduterus cancer samples, comparative to bonemarrow-derived mesenchymal stem cells (MSCs).

MATERIALS AND METHODS
1. Isolation and culture of tumor-associatedfibroblasts (TAFs)
TAFs were isolated from 10 breast cancer and 5
uterus cancer surgical pieces using collagenase type
IV-S from Clostridium histolyticum (Sigma-AldrichCompany, Ayrshire, UK) method. Breast cancer surgicalpieces of approximately 5 cm2 were obtained form 10
female patients, with the histopathological diagnosis of
infiltrative ductal mammary carcinoma. Uterus cancer
samples of approximately 10 cm2 were obtained frompatients with the diagnosis of endometroid
adenocarcinoma. Cells isolated from tissues were
washed several times with phosphate buffered saline(PBS, Sigma) solution and passed through 0.70/0.40 μm
strainer filters and then plated as suspension in adherent
plastic culture plates using Dulbecco’s modified Eagle
Medium (DMEM; Sigma), supplemented with 10% fetal
calf serum (FCS; PromoCell, Heidelberg, Germany) and
2% Penicillin/Streptomycin mixture (Pen/Strep, 10,000
IU/ml; PromoCell), and incubated incubation at 37oC in
5% CO2 atmosphere. Every 3 days, medium was
replaced with freshly prepared medium.
All tissue samples were obtained after signing theinformed consent elaborated under an approved protocol,
according to the World Medical Association Declarationof Helsinki.
2. Isolation and culture of bone marrow-derived
mesenchymal stem cells (BM-MSCs)
Human mesenchymal stem cells (MSCs) wereobtained from bone marrow from healthy Orthopedics
patients undergoing bone surgery. Approximately 10 mlof bone marrow were placed in culture plates, and thefibroblastic-like, plastic adherent fraction, was isolated
following multiple passages and used in our experiments.
The MSCs were further cultured and expanded inalpha-minimum essential medium (MEM; Gibco BRL,
Invitrogen), supplemented with 10% fetal calf serum(FCS; PromoCell), 10ng/mL basic fibroblast growth factor(FGF;
Sigma-AldrichCompany)
and2%
Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/ml;
Fiziologia - Physiology • 2015.25.3(87)PromoCell), by incubation at 37ºC in 5% CO2
atmosphere. Medium was replaced every three days andwhen the confluence was 80-90% the cells were passed
using 0.25% Trypsin-EDTA solution (Sigma-AldrichCompany) followed by centrifugation (10 minutes, 300g)
and replated in T75 culture flasks at a density of 10,000
cells/cm2 to ensure optimal proliferation.
3. Analysis of proliferation rate using MTT assays
An MTT-based in vitro toxicology assay kit (Tox-1,
Sigma-Aldrich Company) was used to determine
proliferation rate of BM-MSCs, uterine cancer TAFs
(uTAFs) and breast cancer TAFs (bTAFs) at 24 and 48
hours. Trypan Blue cell counting and viability
assessment were simultaneously performed at thesame time intervals for better correlation of the results.
Cells were passaged at confluence after treatment with
5mM EDTA; the living cells were then assayed by theaddition of 20 μL of 5 mg/mL MTT solution. The intact
mitochondrial reductase converted and precipitated
MTT as blue crystals during a 4h contact period. The
medium was then removed, and the precipitatedcrystals were dissolved in 100 μL of dimethyl sulfoxide(DMSO; Sigma-Aldrich Company, Ayrshire, UK). Cells
were seeded at 2,000 cells/well in 96-well plates inquadruplicate, and the average value of specificmedium extinction was subtracted from the samples
extinction read at 570/655 nm using a benchmark PR
2100 microplate reader from Bio-Rad (Hercules, CA,
USA).
4. Annexin V/PI assay
Annexin V-FITC (Miltenyi Biotec, Gladbach, Germany)
was used in cell death flowcytometric studies (apoptosis)
combined with Propidium Iodide Staining Solution (BDBiosciences, San Jose, CA, USA) following the
manufacturer protocol. Shortly, 106 cells were washed in
1 x Annexin V Binding Buffer (BD Pharmigen) andcentrifuged at 300 x g for 10 minutes, resuspended in thesame solution and incubated with 10 μl of Annexin
V-FITC for 15 minutes in the dark. After washing the cellswith 1 ml specific binding buffer and centrifugation, thecell pellet was resuspended in 500 μl binding buffer and 1
μg/ml of PI solution was added immediately prior to
analysis by flowcytometry. Data acquisition for Annexin
V/PI assay flowcytometric procedure was performed on afour-color capable FACSCalibur (Becton-Dickinson) flow
cytometer, while data analysis employed Flowing
Software 2.5.
5. Statistical analysis
Data were analyzed for statistical relevance using
Excel Microsoft Office 2007 software (Microsoft
Corporation, Redmond, WA, USA). The central

11tendencies of the variables were expressed as a mean(M), and the dispersion ones as a standard deviation (sd).
In order to perform the statistical comparisons, Student's
t test and the variance analysis (ANOVA) were conducted
for continuous variables. Differences were consideredsignificant for p < 0.05.

RESULTSIn vitro toxicology assay (MTT) was performed 24
and 48 hours after plating of cells in appropriate
96-well plates. We comparatively analyzed the meanvalues of 5 TAFs populations obtained from bothbreast cancer and uterine cancer pieces and bonemarrow-derived mesenchymal stem cells (MSCs). As
negative control we used DMSO which was added inamount of 10 µl/well on a layer of breast
cancer-derived TAFs.

Fig. 1. Viability of TAF populations at 24h. Similar values
were obtained for breast cancer-derived TAFs (bTAF),
uterine tumors-derived TAFs (uTAF) comparative to MSCs
viability at 24h.

Fig. 2. Viability of TAF populations at 48h. Similar values
were obtained for breast cancer-derived TAFs (bTAF),
uterine tumors-derived TAFs (uTAF) comparative to MSCs
viability at 48h.

12

Fig. 3. Comparative viability between the three cellular
populations tested at 24 and 48h. Negative control used
DMSO on a layer of breast cancer-derived TAFs.

Analysis of data obtained by MTT assay revealed asimilar proliferation rate and viability for both breast
cancer and uterine cancer-derived TAFs, which wascomparable to bone marrow-derived MSCs proliferationrate.
Annexin V/PI viability assay was performed by
flowcytometric methods, and revealed the 3 stages ofcellular death: early apoptosis (lower right quadrant), late
apoptosis (upper right quadrant) and dead cells (upper
left quadrant). There were no significant differences
between the 3 cellular types analyzed for early apoptosis
and dead cells (p>0.05). Regarding the late apoptosis
stage, analysis revealed differences with statisticalsignificance (p<0.05) between MSCs and breast
cancer-derived TAFs.
Correlations between the MTT and Annexin V/PI
assays showed that bTAFs have a different
proliferative behavior compared to MSCs and uTAFs,
thus demonstrating that breast cancer stromalcompartment has better supportive role in tumor
development. These analyses also demonstrated theheterogeneity between tumor-associated fibroblasts
isolated from different tumor sites, regardless of theiridentical phenotype.

Fig.4. Annexin V/PI viability assay. Late apoptosis of a. MSCs,
b. bTAFs, c. uTAFs.

Fiziologia - Physiology • 2015.25.3(87)CONCLUSION AND DISCUSSION
Proliferation assays performed in this study (MTT
method) revealed a proliferation rate of TAFs similar tothat of mesenchymal stem cells at 24 and 48 hours; theresults were following the same pattern for both breast
cancer and uterine tumors-isolated fibroblasts.
Cellular viability evaluated using the flowcytometrictechnique with Annexin-V/PI showed that bonemarrow-derived mesenchymal stem cells have an increased
apoptotic process compared to tumor-associated fibroblasts
isolated from breast cancer and uterine tumors.
The origin of tumor-associated fibroblasts is not fully
understood. There are multiple data according to which
TAFs are a subset of MSCs and are recruited from bonemarrow or from tissue level during tumor development
process [5]. Tumor microenvirnment provides theconection between tumor and host, at the level of primarysite and at distance site. This communication is possible
through the soluble factors which are secreted by tumor
cells and by cellular component of the tumor
microenvironment, in which TAFs represent an essential
component. TAFs plasticity and their ability to induce
tumor cells development, invasion, migration andmetastasis, but also their involvement in survival of tumor
cells and acquirement of drug-resistance, makes
exploration ofthese cells a priority for characterization
and use as future therapeutic targets.
Apart from genomic alteration which are determinat
for neoplazic potential, recent data revealed theimportance of stromal component of the tumor
microenvironment for initiation, progression, invasion andtumor metastasis [6,7]. The fibroblasts are cells which
are responsible of production and maintenance ofconnective tissues. They are non-vascular, non-epithelial,
and non-inflammatory cells, which are the main
component of the connective tissues [8]. These cells areactively involved in regulation of inflammatory processes,
whound healing, as well as differentiation of epithelial
cells from adjacent areas [9].
Fibroblasts are synthesizing fibrillary proteins of theextracellular matrix (fibronectin, type I, III, and IV
collagen). These cells are a major source of proteases
which are further degrading the matrix (matrix
metalloproteinases), involved in matrix homeostasis.
Under physiological circumstances, proliferation andmetabolic rate of fibroblasts are very low. During thehealing phase of an injury, the fibroblasts will become
activated and will acquire secretory and contractile
qualities, while the proliferation rate increases [10]. In ourstudy we proved that TAFs have an increased
proliferation rate and viability, compared to mesenchymal
stem cells, even under in vitro condition, following
multiple passages, thus demonstrating that TAFs areable to provide a good functional support for long-term
growing of a tumor.
Fiziologia - Physiology • 2015.25.3(87)Moreover, activated fibroblasts are secreting
chemokines which are initiating the recruitment of blood
components at the site of an injury. The activatedfibroblasts will regain their quiescent status after
performing their task, at the end of the injury healing
process [11]. Regarding their phenotype, fibroblastspresent several differences, depending on the site ofharvesting (joints, skin, etc.) [12].
However, the fibroblasts possess a wide variety in
topographic expression of genes involved in synthesis ofextracellular matrix, cellular growth and differentiation,and cellular migration. Some authors [13] noticed thedifferences in size and expression of some receptors(TGF- β1 receptor II) by fibroblasts isolated from different
body sites. They suggested the idea that this regional
diversity could be accounted for susceptibility anddifferent behavior of different anatomic sites during thehealing process and cheloid formation. Other studies
revealed an increased capacity of oral mucosa-derived
fibroblasts to reorganize the collagen fibers due to highexpression of MMP-2, comparative to skin-isolated
fibroblasts [14]. Fibroblasts heterogeneity was also
shown in studies regarding the mechanisms of tissue
injury and wound healing. This last process develops
along 3 phases, all of them involving the stromalcomponent: inflammation, proliferation, maturation [15].
Tumorigenesis is always accompanied bystromagenesis. The stromal compartment functions astumorigenic barrier. In the first stages, the tumor cells are
inducing stromal development and install a feed-backprocess between tumor ad stroma, which will further
become bidirectional, maintaining both processes [16].
Consequent alterations of initially normal stroma lead totransient changes, which further will trigger phenotypicalchanges with the characteristics of activated stroma[17,18]. In advanced phases, the tumor and stroma
become irreversible transformed from the phenotypic
point of view, presenting different degrees of reciprocal
independence. An interesting aspect to be elucidated
would be the identification of “point of no return”, in which
there is the switch between classical stroma phenotype
and activated one [19].
Tumor-associated fibroblasts can be found in variable
proportions at the level of the entire spectrum ofcarcinomas, constituting the most abundant cellularelement within the tumor stroma. The term
tumor/cancer-associated fibroblasts comprises at least 2
cellular types: (1) fibroblast-like cells, which are thesupportive element for epithelial cells; (2) myofibroblasts
with different roles and biological characteristics
compared to tissue fibroblasts. Tumor myofibroblasts andtissue fibroblasts which suffered phenotypical andfunctional changes have multiple roles during cancer
progression: proliferation, angiogenesis, invasion,
metastasis; these actions were demonstrated in animal
experimental models using mice predisposed to13

tumorigenesis, by injecting TAFs combined with tumor
cells, and by studying genetic and pharmacologicalalteration of cellular functionality [20-22].
Due to secretion of various components ofextracellular matrix, tumor-associated fibroblasts are
involved in formation of desmoplastic stroma, whichcharacterizes advanced forms of carcinomas. There arestill some questions to be answered regarding the distinct
functions of the two cellular groups of TAF and their
specific involvement in tumor pathogenesis.
To summarize, several studies have confirmed thattumor-associated fibroblasts action to favor tumor
progression could be based on somatic mutations at thelevel of tumor suppression genes. Also, somatic
alterations were constantly observed with significant
frequency (more than 30%) even at the level oftumor-associated fibroblasts [23]. Despite these data,
other studies showed that genetic alterations are induced
only at the level of epithelial tumor cells, and not at thestromal level [24], while heterozygots analysis andgenetic copies performed on TAFs isolated from breast
cancer and ovarian cancer samples revealed that genetic
alterations are extremely rare at TAFs level. Thus, at thismoment, the researchers were not able to postulate theidea that cancer progression is based ontumor-associated fibroblasts [25].
ACKNOWLEDGEMENT
This work was supported by CNCS-UEFISCSU,PNII-Parteneriate 120/2012 and PNII-IDEI 318/2011.

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Fiziologia - Physiology • 2015.25.3(87)ANALIZA IN VITRO A CAPACITĂŢII DE PROLIFERARE ŞI A VIABILITĂȚIIFIBROBLASTELOR PERI-TUMORALEREZUMAT
Originea fibroblastelor peri-tumorale nu este pe deplin clarificată. Există date multiple conform cărora TAF sunt
un subset al MSC şi sunt recrutate din măduva osoasă sau de la nivel tisular în momentul dezvoltării tumorii.
Microclimatul tumoral asigură conexiunea tumoră-gazdă atât la nivelul site-ului primar, cât şi la distanţă. Această
comunicare este posibilă prin intermediul factorilor solubili secretaţi de celulele tumorale şi de către componenta
celulară a microclimatului tumoral, TAF reprezentând o componentă esenţială a acesteia. Plasticitatea TAF şi
capacitatea acestora de a induce dezvoltarea celulelor tumorale, invazia, migrarea şi metastazarea, dar şi
implicarea acestora în supravieţuirea celulelor tumorale şi dobândirea rezistenţei la chimioterapice, sunt motive
bine întemeiate în a explora aceste celule în vederea caracterizării lor şi utilizării drept ţinte terapeutice.
În acest studiu am investigat capacitatea de proliferare și viabilitatea a două tipuri de fibroblaste peri-tumorale –TAF izolate din cancere mamare (bTAF) și TAF izolate din tumori uterine (uTAF) – comparativ cu celulele stem
mezenchimale derivate din măduva osoasă (MSC). Am utilizat testul de viabilitate MTT și evaluarea
flowcitometrică folosind Anexina V/iodura de propidiu. Rezultatele au fost similare pentru toate tipurile de celule
investigate, diferențe semnificative statistic obținându-se pentru bTAF aflate în faza de apoptoză tardivă, care auprezentat valori scăzute comparativ cu MSCs și uTAF.
Cu toate că prezintă numeroase caracteristici similare cu MSCs, există încă multe necunoscute în ceea ceprivește caracteristicile și funcțiile distincte ale TAF, precum și în ceea ce privește implicarea specifică a acestora
în patogeneza tumorală.
Cuvinte cheie: celule stem mezenchimale (MSCs), fibroblaste peri-tumorale (TAFs), teste de viabilitate, MTT

Fiziologia - Physiology • 2015.25.3(87)15THE RELATIONSHIP BETWEEN VITAMIN D, INFLAMMATION
AND THE ACTIVITY OF SYSTEMIC LUPUSERYTHEMATOSUSFLORINA BULEU1, CAMELIA GURBAN1, ELENA SARBU2, MARIA-CORINA SERBAN1,
ANCA TUDOR1, FLORINA ARDELEAN1, GHEORGHE STOICHESCU-HOGEA1, SIMONADRAGAN1
1University of Medicine and Pharmacy „Victor Babes” Timisoara
2West University of Timişoara, Romania

ABSTRACT
Objective: Systemic lupus erythematosus (SLE) is considered a chronic inflammatory autoimune disease. The
role of vitamin D in the prevention or therapy of LES is still debated. The objective of our study was to determine
the serum levels of vitamin D in patients with SLE and its relationship with inflammatory markers and Systemic
Lupus Erythematosus Disease Activity Index (SLEDAI).
Method: A prospective study was realised on 20 women diagnosed with SLE according to American College of
Rheumatology (ACR) criteria and 12 aged and gender-matched healthy controls. We determined the serum
levels of high sensitivity C reactive protein (hsCRP), erythrocyte sedimentation rate (ESR), 25-hydroxy vitamin
D, total calcium, phosphorus and parathyroid hormone in all patients. The activity of SLE was evaluated through
SLEDAI. The levels below 30 ng/mL of 25(OH) D were considered abnormal. The serum creatinine levels andglomerular filtration rate (GFR) were measured to assess the renal affection in these patients.
Results: The mean age of SLE patients included in this study was 44.1±11.65 years. It was observed that thelevels of vitamin D were significantly lower in SLE patients then in healthy controls. The SLE patients withhypovitaminosis D had higher levels of serum creatinine, GFR, ESR and PCR. SLEDAI was significantly
increased in patients with hypovitaminosis D compared to patients with normal vitamin D levels.
Conclusion: In our study, the deficiency of vitamin D was more prevalent in SLE patients. The patients with SLE
and hypovitaminosis D have significantly higher values of SLEDAI than the patients with normal Vitamin D levels.
Thise results support the importance of maintaining the normal levels of vitamin D to combat lupus diseaseactivity.
Keywords: vitamin D, SLE, inflammation, SLEDAIINTRODUCTION
Systemic lupus erythematosus (SLE) is a chronic
autoimmune disease characterized by systemic
inflammation and the immune system attacks various"self-tissues" throughout the body [1]. SLE predominantly
affects women mainly at childbearing age, with a ratio ofnine women for every man [2]. The reported prevalenceof SLE is between 20 and 150 cases per 100,000
individuals [3]. The etiology of SLE is still unknown andits progression has been linked with the interaction ofgenetic, immunological, hormonal, and environmental
factors [4]. A low vitamin D status might have an
important role in SLE pathology, since hypovitaminosis D

is more prevalent in SLE patients than in the general
population [5]. It has been shown that vitamin D has animportant role in regulating immune response especiallyrelated with B-cells and T cell homeostasis, and intissues “self -tolerance" [6]. In SLE the balance between
the "effector" / "regulatory" cells is directed in the favor of
the first [9]. Moreover, the “regulatory” cells in thesepatients are dysfunctional [9]. Vitamin D exerts several
actions at the cellular level to modify this balance [10].
It has been demonstrated that SLE patients may
have various risk factors which influence the appearanceof vitamin D deficiency. The patients with SLE also havea typical photosensitivity which might determine the useof sunscreen and a lower sun exposure, which blocks

Received 10th of June 2015. Accepted 5th of July 2015. Address for correspondence: Florina Buleu, MD, PhD student,
University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania;
phone/fax: +40256220479; e-mail: florina_28nick@yahoo.com

16

Fiziologia - Physiology • 2015.25.3(87)UVB radiation, and makes this population at risk fordeveloping vitamin D deficiency. The drugs frequently
used in the treatment of patients with SLE, likecorticosteroids could change the metabolism of thevitamin D in the body [11]. In addition, the renal
involvement, one of the determining factors in morbidity
and mortality of SLE patients, can change the stage ofhydroxylation of vitamin D [2,11]. In fact, deficiency ofvitamin D was demonstrated to be associated with higher
lupus disease activity [12,13]. Additionally, low levels ofvitamin D were associated with severity of disease andnephritis in patients with SLE [14].
The purpose of this study was to evaluate the levelsof vitamin D in SLE patients and to determine therelationship with inflammatory markers and the activity of
disease evaluated thorough Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI).

METHODS
The study was performed on 20 women with SLE
attending the outpatient clinic of Department ofRheumatology, Hospital Municipal Timisoara andDepartment of Nephrology, County Emergency Hospital,
Timisoara. The women with SLE patients was diagnosed
based on the criteria of the American College of
Rheumatology (ACR) 1997 [15]. The exclusion criteria:
pregnant women, individuals with systemic disease suchas vasculitis, acute infectious diseases. In addition, 12
healthy people matched age and sex were recruited ascontrols.
The season in which the patients were included wasconsidered for this study. The patients treated withvitamin D and calcium supplements renounced at thisthearpy for 2 months (considered three times the half-lifeof supplements) before joining the study.
The inflammation status was assessed bydetermining the serum levels of C reactive protein (CRP)
and erythrocyte sedimentation rate (ESR). The activity oflupus disease was evaluated using the Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI). The
values of vitamin D ≥ 30 ng/ml were considered normal,
while the vitamin D insufficiency was defined as a level
between 15 to 29 ng/ml and vitamin deficiency at levels<15 ng/ml (16). The serum creatinine levels andglomerular filtration rate were measured to assess therenal involvement in these patients (GFR).
The statistical processing was carried out using Excel
software packages, Epi Info 7 and SPSSv.17. In all cases
we used the significance threshold of 0.05 (5%),
corresponding to a 95% confidence level.
The local ethics committee approved the protocol,
and written informed consent was obtained from all
patients in accordance with the Helsinki Declaration.

Fiziologia - Physiology • 2015.25.3(87)RESULTSThis study included 32 individuals, 20 SLE patients(SLE group) and 12 healthy patients (Control group),
all female. The characteristics of the study groups
showed that there were no significant age differencesamong groups. The age of SLE patients was between 19
and 67 years, with a mean of 44.1±11.65 years, and theage of heathy controls was between 23 and 68 years,
with a mean of 42.2±12.73. The disease duration ranged
from 1 to 7 years with a mean of 2.7±1.69 years. The
analysis of SLEDAI score has revealed a mean of
9.5±7.34. (Table I)
The SLE patients were diagnosed according toAmerican College of Rheumatology (ACR) criteria
published in 1982 and revised in 1997 [15]. The
analysis of the types of treatment applied to the study
group found that chloroquine was used in a proportionof 70 % (n = 14), azathioprine in 40 % (n = 8),
prednisone in 65% (n=13) and methylprednisolone at a
rate of 15% (n = 3). All of SLE patients had received
therapy with steroids (Table I)
The mean levels of serum creatinine in SLE patientswas 1.1±0.44 (0.62-2.53) mg/dL and the mean
glomerular filtration rate was 81.7±34.51(19-139)
mL/min/1.73 m2. In heathly controls, the serum levelsof creatinine were 0.8±0.21 (0.41-1.13) and the mean
glomerular filtration rate was 94.3±26.84 (53-146)
mL/min/1.73 m2 (Table I).
The results of inflammatory markers showed that themean values of ESR were 44.3±23.04 mm/h in SLE
patients and 7.4±3.26 in healthy control. The mean levelsof CRP were 7.7±8.52 mg/dL in SLE patients and
0.7±1.78 mg/dL in healthy controls (Table I).
Table I. Characteristics of SLE patients (n=20) and healthy
controls (n=12)17

The analysis of vitamin D levels in the studiedgroupsThe mean levels of vitamin D in SLE patients were
25.5±7.95 ng/mL and in healthy control were 33.3±6.01
ng/mL. All patients were considered to have deficiency ofvitamin D when the levels were under 15 ng/mL, vitamin D
insufficiency when the levels were between 15 and 29
ng/mL, and normal levels of vitamin D ≥ 30 ng/mL (Table I)
Table II. The analysis of 25 (OH) D levels in SLE group (n = 20 )The percentage distribution of vitamin D insufficiency in
SLE patients was found in 9 patients in a proportion of 45 %and deficiency in 3 patients in a proportion of 15 %. Only 8
patients with SLE had normal levels of vitamin D (Table II).
In healthy controls, the vitamin D insufficiency was
found in 3 patients (25 %) and the rest had normal levels(n=9). The levels of vitamin D were significantly lower in
SLE patients compared to healthy controls(Mann-Whitney test, p = 0.006).

The correlation between vitamin D levels, SLEDAI
scores, ESR, PCR and renal involvement
The correlative study in SLE patient with vitamin Dlevels less than 30 ng/mL (n=12) and SLE patients withnormal levels (n=8) with respect to vitamin D levels and
SLEDAI scores, ESR, PCR and renal involvement
showed that:
SLEDAI scores were significantly higher in patientswith hypovitaminosis D (12.8 ± 6.82) than inpatients with normal levels (3.4 ± 1.4) Mann-Whitney test, p = 0.003, α = 0.01.
ESR values were significantly higher for patientswith hypovitaminosis D (51.3 ± 24.59) than inpatients with normal levels (31.1 ± 12.78) Mann-Whitney test, p = 0.046, α = 0.05.
PCR values ere not significantly increased inpatients with hypovitaminosis D (9.9 ± 8.43) than inpatients with normal vitamin D evels (3.7 ± 2.82) Mann-Whitney test, p = 0.115, α = 0.05.
The renal impairment, quantified by serum
creatinine and glomerular filtration rate (GFR) was
significantly increased (Z test, p = 0.04, α = 0.05) inpatients with hypovitaminosis D.

DISCUSSION

Fig.1. The graphical representation of vitamin D levels between
SLE patients (n=20) and healthy control (n=12)

The correlation between vitamin D and serum
creatinine is significant (p <0.001, α = 0.001), reverseand strong (Pearson coefficient r = -0.735), in SLE
patients with vitamin D levels under 30 ng/mL (Figure 2).

Fig. 2. Correlation of levels of vitamin D and serum
creatinine in SLE patients (n=12) with hypovitaminosis D
18This study showed that low vitamin D levels arecommon in patients with LES - 60% had levels <30 ng /ml and 10% had <15 ng/ml, despite that this population
live in a country with many sunny days. These results are
related to other studies from similar geographical areas[11]. This means that vitamin D deficiency is moreprevalent in patients with SLE than in healthy controls;
also in healthy control vitamin D insufficiency was foundin 3 patients (25 %), explained by Fragoso et al. [17] tobe a result from the modern life activities, which makepeople to avoid sun exposure, and consequently reduce
vitamin D synthesis.
It is noteworthy that therapy with calcium and vitamin
D did not protect completely against the deficit of25(OH)D; from 20 patients with SLE included, seven wasreceiving treatment with calcium and vitamin D pills, and
4 of them (57%) had levels <30 ng/mL of 25(OH)D.
In this study, the mean serum creatinine issignificantly increased for the group with SLE comparedwith healthy control (p <0.012). Higher values ofcreatinine have been observed in this study in patientswith SLE and hypovitaminosis D ((1.3 ± 0.47) mg / dL) )than patients with normal levels of vitamin D levels (0.9 ±
0.16mg / dl)(p = 0.06, α = 0.01). The value of GFR was
slightly lower in SLE patients compared with healthy
controls (p = 0.288). Most of the patients with SLE
patients included in this study are check their kidney
involvement, which may also influence the value of theresults. Lupus nephritis is a major cause of morbidity and
Fiziologia - Physiology • 2015.25.3(87)mortality in SLE. The association between vitamin D
deficiency and lupus nephritis was assessed in manystudies, Kamen et al. [18] published a study in which was
described an association between them.
SLE - by definition an autoimmune inflammatory
disease – is correlate with high inflammation in patients
suffering from this condition. In this study, a statisticallysignificant increase in the values of ESR and C-reactive
protein was observed in SLE patients compared with thehealthy control (p <0.001, α = 0.001 for ESR and p<0.001, α = 0.001 PCR). There was an inverse
association between low levels of 25 (OH) D and lupus
disease activity index in this study (p = 0.003, α = 0.01).
These findings are in agree with many otherobservational studies which shown that vitamin D levels
are correlate inversely with disease activity index in SLE[13,19,20]. This study has shown, similar with the studyof Amezcua-Guerra et al. [21], a positive association
between SLEDAI scores and ESR/PCR in patients with
SLE. However, Firooz et al. [22] could not demonstrate
an association between these inflammation markers anddisease activity index.
The association between vitamin D levels anddisease activity index in SLE patients is extremely
important. Many clinical trials [23] reported an
association between decreased values of vitamin D andincreased activity of SLE, evaluated through SLEDAI.
However, no study which examined the relationshipbetween vitamin D and the of activity in SLE did notinclude all the main factors influencing the status ofvitamin D. Recent studies suggested a role of vitamin D
in the appearance and severity of fatigue found in SLE,
but various descriptions of the symptoms from patient topatient make it difficult to determine.
This study has few limitations. First of all, this trial
can not suggest any causality between the associations
described and vitamin D deficiency in patients with SLE.
The small number of patients included (n = 20) can
influence the results, and also the low level of SLEDAI
scores due to patients enrollment in remission.
Future studies with larger numbers of participants
and a higher activity of the disease could provide more
concrete results regarding the association between
vitamin D deficiency, lupus nephritis, inflammation
markers and disease activity index.

CONCLUSION
We concluded that low vitamin D levels are moreprevalent in patients with SLE than in healthy controls.
The SLEDAI score, serum creatinine, GFR, ESR andCPR values are significantly higher in patients withhypovitaminosis D than in patients with normal levels ofvitamin D. These results suggest the possible
involvement of low vitamin D levels in the disease
Fiziologia - Physiology • 2015.25.3(87)severity in SLE patients and the importance of
maintaining the normal levels of vitamin D to combat theactivity of lupus.
Competing interests
The authors declare that they have no competinginterests.
ACKNOWLEDGEMENT
This paper was published under the frame of European
Social Found, Human Resources Development
Operational Programme 2007-2013, project no.
POSDRU 159/1.5/S/136893: “Strategic partnership forthe increase of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”.

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RELAŢIA DINTRE VITAMINA D, INFLAMAŢIE ŞI INDEXUL DE ACTIVITATE AL
LUPUSULUI ERITEMATOS SISTEMICREZUMAT
Obiectiv: Lupusul eritematos sistemic (LES) este o boală inflamatorie cronică autoimună. Rolul vitaminei D in
preventia sau tratamentul LES este inca dezbatut. Obiectivul acestui studiu a fost evaluarea nivelul seric alvitaminei D la pacienții cu LES, relatia vitaminei D cu markerii inflamatori si cu indexul de activitate a lupusului
eritematos sistemic (SLEDAI).
Metoda: Acest studiu prospectiv a cuprins 20 de femei diagnosticate cu LES, conform criteriilor Colegiului
American de Reumatologie (ACR) și 12 pacienti sanatosi, de control. Am determinat nivelurile serice ale proteinei
C reactive (CRP), vitezei de sedimentare a hematiilor (VSH), 25-hidroxi vitaminei D, calciului total, fosfatului seric
si hormonului paratiroidian. Activitatea LES a fost evaluată cu ajutorul SLEDAI. Nivelurile sub 30 ng/mL ale
vitaminei D au fost considerate anormale. Au fost măsurate nivelurile creatininei serice și rata de filtrare
glomerulara (RFG) pentru a evalua afectarea renala la acesti pacienti.
Rezultate: Vârsta medie a pacientilor cu LES inclusi în acest studiu a fost 44,1 ± 11.65 ani. S-a observat ca
nivelurile serice ale vitaminei D au fost semnificativ mai scăzute la pacientii cu LES comparativ cu lotul de control.
Pacientii cu LES și cu niveluri scazute ale vitaminei D au avut valori mai crescute ale creatininei serice, RFG,
VSH și PCR. Scorul SLEDAI a fost semnificativ mai mare pentru pacientii cu hipovitaminoza D, comparativ cu
cei cu nivel normal al vitaminei D.
Concluzii: În acest studiu, deficitul vitaminei D a fost mai frecvent observat la pacientii cu LES. Parametrii
inflamatori si scorul SLEDAI au fost semnificativ mai mari la pacientii cu hipovitaminoza D comparativ cu cei cu
valori normale ale vitaminei D. Aceste rezultate sustin importanța menținerii nivelurilor normale ale vitaminei D
pentru a combate activitatea LES.
Cuvinte cheie: vitamina D, LES, inflamație, scorul SLEDAI

20

Fiziologia - Physiology • 2015.25.3(87)ASSESSMENT OF A MURINE MELANOMA MODELDORINA CORICOVAC1,#, RADU BERCEANU1,#, TIBERIU BRATU1, DANINA MUNTEAN1*,CODRUTA SOICA1, SORINA CIURLEA2, CRISTINA DEHELEAN1
1“Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
2“Vasile Goldis” Western University, Arad, Romania# Equal contributions

ABSTRACT
Malignant melanoma, the most aggressive type of skin cancer, still represents a hot topic for the researchers dueto the lack of an effective treatment and finding new drugs with improved properties is a goal to achieve. The useof animal models in melanoma research makes this goal seem closer.
The aim of the present study was to develop a murine melanoma model using C57BL/6J mice by inoculation
subcutaneously of B164A5 cell suspension.
In order accomplish the experiment, we used both female and male C57BL/6J mice (n=7 mice/group) with theage of 10-12 weeks. The day prior the experiment the mice were shaved on the back with a razor and B164A5cell suspension (1X106 cells/100 µl PBS/mice) was subcutaneously inoculated to both females and male mice.
The mice were examined daily.
The initiation of tumor had an early onset in the female group, but all the mice included in the study developed
the tumor. These data sustain the fact that the model that we proposed is a reliable and a reproducible one.
Key words: melanoma, B164A5, C57Bl/6J.

INTRODUCTION
Malignant melanoma is considered the most deadly
skin cancer type and is known to possess an increased
metastatic potential and, in addition, is resistant to theconventional anticancer therapy [1].
It is not fully elucidated the mechanism involved inmelanoma initiation and development, but are wellestablished the histopathological stages of this disease:the presence of common and dysplastic nevi, radial
growth phase melanoma (RGP), vertical growth phasemelanoma (VGP) and the last stage and the most criticalone, is metastatic malignant melanoma [1].
One of the reasons why melanoma still attracts a lotof interest among the researchers consists in the lack ofan effective treatment, especially for the metastatic
patients, and considerable efforts are made in thisdirection in order to find novel and efficient compounds or
to find a better formulation with improved efficacy for theconventional therapy.
The animal models represent an useful tool ingathering information regarding the insights of cellularand molecular mechanisms involved in melanoma
initiation and progression (cellular transformation,
chromosomal mutations) [2-4]. Furthermore, the in vivo

models play key roles in the testing process of new drugs[1, 5].
The melanoma studies regarding the genetic
modifications that may occur during the progression of
the disease or the evaluation of new drugs effectivenessare using the mouse model as a standard [1].
One of the most frequently used melanoma mousemodel is the one developed on black mice, C57BL/6J by
inoculation of different murine melanoma cells (B16F10,
B16BL6, B164A5), this strain of mice being the most
receptive host for this type of tumor cells [5 -7].
The objectives of this study were: (i) to develop a
reproducible murine melanoma mouse model by
inoculation subcutaneously of a B164A5 murine
melanoma cell suspension and (ii) to verify if there areany differences related to mice gender in initiation oftumors.

MATERIALS AND METHODSIn this study we used B164A5 murine melanoma
cells. The cell line was purchased from ECACC(European Collection of Cell Cultures) at passage 3.
B164A5 cells were kept in liquid nitrogen and one week

Received 9th of March 2015. Accepted 27th of April 2015. Address for correspondence: Danina Muntean, MD, PhD, Departmentof Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st., 300173
Timisoara, RO, Tel/Fax: +40-256-493085; e-mail: daninamuntean@umft.roFiziologia - Physiology • 2015.25.3(87)21

before the experiment started, we cultured in specificmedium culture.
The cells (figure 1) were cultured in high glucose (4.5g/l) Dulbecco’s modified Eagle Medium (DMEM – Sigma
Aldrich, Germany) supplemented with 15 mM Hepes, 2
mM L-glutamine, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 10% fetal bovine serum (FBS). Cells
were kept in a humidified atmosphere with 5% CO2 at
37°C and were passaged every two days. Cells number
was assessed by using Neubauer chamber in thepresence of Trypan Blue.

RESULTS AND DISCUSSIONS
The aims of this present study were: (i) to obtain areproducible murine melanoma model using B164A5
xenografts and (ii) to compare the moment of emergenceof melanoma tumor in female vs. male mice.
In order to obtain the animal model of murine
melanoma, we used B164A5 murine melanoma cells,
which were counted and prepared as a suspension of
1X106 cells and a volume of 100 µl PBS/mice wasinjected to both groups of mice, females (n=7 mice/group)and males (n=7 mice/group).
The inoculation/ injection of the cells is a very
important step in the initiation and development of the
subcutaneous tumor, because if this step is done wrong
then the tumor won’t appear.
The suspension of cells must be carefully injectedsubcutaneously and a bubble must be observed at theplace of inoculum, bubble that will be resorbed until thenext day (Figure 2).

Fig. 1. B164A5 murine melanoma cells in culture

All the reagents used in the study (DMEM medium,
phosphate saline buffer – PBS, penicillin, streptomycin,
foetal bovine serum, Trypan Blue) were of analyticalpurity and were purchased from Sigma Aldrich, Germany.
The animals used in the present study were female
and male C57BL/6J mice (10-12 weeks-old) which wereobtained from Charles River Laboratories, Budapest,
Hungary. All experimental procedures were conducted inaccordance with the Directive 2010/63/EU on theprotection of animals used for scientific purposes. The
experimental protocol was approved by the Committee
for Ethics Research of the University for Medicine andPharmacy of Timisoara, Romania. Animals were fed ad
libitum and kept under standard conditions: constanttemperature of 22.5 ± 2° C, humidity 55 ± 5% and a 12-h
light/dark cycle.
The murine melanoma model was obtained
according to the following protocol: B164A5 cellsuspension (1X106 cells/100 µl PBS/mice) was preparedand subcutaneously inoculated to both females and malemice which were shaved on their back a day prior theexperiment. During the inoculation the mice wereanesthetized with Isoflurane.
The evolution of the mice was followed daily and thetumors were measured using a caliper. The width (a) andlength (b) of the tumors were measured by calipers forthe evaluation of tumor volume according to the formulaV = ba2/2 [8].
22Fig. 2. Development of murine melanoma mouse modelusing C57BL/6J mice: a) inoculation of the cells suspension
subcutaneously and b) the bubble aspect that should beseen at the inoculum site

The mice were examined daily and the first signs oftumor initiation (a small, round bubble at skin level in thesame area were the cells were inoculated) wereobserved in the females group starting with the fourth
day post-inoculation whereas in the males group no
signs of tumor were detected at that time point.
At day 6 there were observed the first signs of tumor
initiation in the group of male mice, too, since in thefemales group the tumor was small but well-defined(Figure 3).

Fig. 3. Macroscopic aspect of the tumors at day 6
post-inoculation: a) C57BL/6J female and b) C57BL/6J maleFiziologia - Physiology • 2015.25.3(87)At day 14 the tumors were well-defined in bothgroups and the mice were sacrificed between days 25
and 30 when the tumors reached the volume approvedby the ethics protocol (1000 -1500 mm2) and theircondition became poor.
Our results indicated that we obtained a
reproducible murine melanoma model since all the micefrom the both group developed tumors post-inoculationof the cell suspension. Furthermore, we observed thatthe females were more susceptible to develop tumors,
the time of appearance being more reduced ascompared to the males group.
This model is a valuable source of information
concerning the mechanisms involved in thepathogenesis of this mortal disease, but also offers
opportunities for the test of new drugs that may curemelanoma.
B164A5 murine melanoma cells present
fibroblast-like features and were described to have thecapacity to produce melanin. These cells were derived
from a melanoma tumor that developed in the skin of a
C57BL/6J mouse [9].
The use of animal models offers multiple
advantages, such as: to study and to understand betterthe insights of the pathogenesis of different diseases, toevaluate directly the toxicity and efficiency of new
therapies, it can also be obtained mouse models withhuman tumor allografts this leading to the elucidation of
the encoding genes involved in the initiation andprogression of melanoma and other pathologies [1].
The C57BL/6J murine melanoma mouse modelobtained by inoculation of B16 xenografts are known assyngeneic models and one of their advantages is thefact that they possess a functional immune system [9].
C57BL/6J mouse inbred line is frequently used instudies regarding mouse genetics and its genome has
been incorporated into many genetic reference
populations [10].
The novelty of the murine melanoma mouse modelthat we proposed in this study is represented by the
number of the cells B164A5 (1X10 6 cells/100 µl PBS/
mouse). We chose this number of tumor cells after
several attempts with a smaller number (10 5cells/mouse or 0.5X10 5 cells/mouse) [7, 11], butaccording to our last results this number induced
tumors to all mice involved in the study.
C57BL/6 is the strain of mice that is used in all thestudies concerning identification of biomarkers
characteristic for murine melanoma or the developmentof new therapies with anticancer properties [5-7, 12-14].
Another advantage of this model was that themetastases did not occur so rapidly as in the modelobtained by intraperitoneal injection of the B164A5 cells[15], and this offers the possibility to observe theevolution of tumor after the administration of a novel
and improved treatment.
Fiziologia - Physiology • 2015.25.3(87)CONCLUSION
The animal model of murine melanoma that wedeveloped in the present study proved to be a reliable
and reproducible model and it represents the backgroundfor further studies focused on verifying the effect of somemelanoma-targeted nanoparticles drugs.
AKNOWLEDGEMENTS
This work was supported by the POSDRU grant no.
159/1.5/S/136893 titled “Strategic partnership for theincrease of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”, awarded toDorina Coricovac.

REFERENCES
1. Conde-Perez A, Larue L. Human relevance of NRAS/BRAF
mouse melanoma models. Eur J Cell Biol. 2014; 93(1-2): 82-6.
2. Krauthammer M, Kong Y, Ha BH, Evans P, et al. Exome
sequencing identifies recurrent somatic RAC1 mutations inmelanoma. Nature Genetics 2012; 44: 1006-1014.
3. Kunz M, Dannemann M, Kelso J. High-throughput
sequencing of themelanoma genome. Experimental
Dermatology 2013; 22: 10-17.
4. Nikolaev SI, Rimoldi D, Iseli C, Valsesia A, et al. Exome
sequencing identifies recurrent somatic MAP2K1and MAP2K2
mutations in melanoma. Nature Genetics 2015; 44: 133-139.
5. Zhang X, Teodoro JG, Nadeau JL. Intratumoral
gold-doxorubicin is effective in treating melanoma in mice.
Nanomedicine. 2015; 11(6): 1365-75.
6. Taylor P, Noriega R, Farah C, Abad MJ, Arsenak M, Apitz R.
Ajoene inhibits both primary tumor growth and metastasis of
B16/BL6 melanoma cells in C57BL/6 mice. Cancer Lett. 2006;
239(2): 298-304.
7. Soica C, Danciu C, Savoiu-Balint G, Borcan F, Ambrus R,
Zupko I, Bojin F, Coricovac D, Ciurlea S, Avram S, Dehelean
CA, Olariu T, Matusz P. Betulinic acid in complex with agamma-cyclodextrin derivative decreases proliferation and invivo tumor development of non-metastatic and metastatic
B164A5 cells. Int J Mol Sci. 2014; 15(5): 8235-55.
8. Conway TF Jr, Sabel MS, Sugano M, Frelinger JG, Egilmez
NK, Chen F and Bankert RB. Growth of human tumor
xenografts in SCID mice quantified using an immunoassay fot
tumor marker protein in serum. J Immunol Methods 2000; 233:
57-65.
9. McKinney AJ, Holmen SL. Animal models of melanoma: asomatic cell gene delivery mouse model allows rapid evaluationof genes implicated in human melanoma. Chin J Cancer. 2011;30(3): 153-62.
10. Simon MM, Greenaway S, White JK, Fuchs H,
Gailus-Durner V, Wells S, et al. A comparative phenotypic andgenomic analysis of C57BL/6J and C57BL/6N mouse strains.
Genome Biol. 2013; 14(7): R82.
11. Duicu O, Ciurlea S, Dehelean C, Ardelean S, Andrica F,
Muntean D, Soica C, Antal D, Coricovac D, Pinzaru I: Analysis
of a Betulinic Acid Formulation on Liver Mitochondria Isolated

23

from Mice with Induced Murine Melanoma. Rev. Chim. 2014,
65(8): 956-959.
12. Yu Z, Zhang T, Zhou F, Xiao X, Ding X, et al. Anticancer
Activity of Saponins from Allium chinense against the B16
Melanoma and 4T1 Breast Carcinoma Cell. Evid BasedComplement Alternat Med. 2015; 2015: 725023.
13. Dittz D, Figueiredo C, Lemos FO, Viana CT, Andrade SP, etal. Antiangiogenesis, loss of cell adhesion and apoptosis are
involved in the antitumoral activity of Proteases from V.

cundinamarcensis (C. candamarcensis) in murine melanoma
B16F1. Int J Mol Sci. 2015; 16(4): 7027-44.
14. Li X, Sun J, Hu S, Liu J. Icariin Induced B16 Melanoma
Tumor Cells Apoptosis, Suppressed Tumor Growth andMetastasis. Iran J Public Health. 2014; 43(6): 847-8.
15. Gheorgheosu D, Dehelean C, Cristea M, Muntean D.
Development of the B16 Murine Melanoma Model. Annals of
the Romanian Society for Cell Biology, 2011; XVI (2): 148-156.

REALIZAREA UNUI MODEL ANIMAL DE MELANOM MURINICREZUMAT
Melanomul, cel mai agresiv tip de cancer de piele, reprezintă încă un subiect de interes pentru cercetători
datorită lipsei, la momentul actual al unui tratament curativ, iar găsirea unor noi medicamente cu eficacitate
sporită este un obiectiv ce trebuie îndeplinit. Utilizarea modelelor animale în cercetarea referitoare la melanom
face ca acest obiectiv să pară mai ușor de îndeplinit.
Scopul acestui studiu a fost acela de a realiza un model animal de melanom murinic la șoarecii C57Bl/6J prin
injectarea subcutanată a unei suspensii de celule de melanom murinic B164A5.
Pentru realizarea experimentului s-au folosit șoareci C57BL/6J de sex feminin și masculin (n=7 șoareci/lot) cu
vârsta cuprinsă între 10-12 săptămâni. Cu o zi înainte de injectare, șoarecii au fost epilați pe spate și apoi le-afost injectată suspensia de celule 1x106 celule/100 µl PBS/ per șoarece. Șoarecii au fost examinați zilnic
post-injectare.
Primele semne de apariție a tumorii au fost observate mai întâi la lotul de femele, dar pânâ la finalul
experimentului toți șoarecii incluși în studiu au dezvoltat tumora. Aceste rezultate indică faptul că modelul animal
propus este un model reproductibil.
Cuvinte cheie : melanom, celule B164A5, șoareci C57BL/6J.

24

Fiziologia - Physiology • 2015.25.3(87)LUPEOL A POTENT ANTI-INFLAMMTORY AGENT IN
ACUTE INFLAMMATION MOUSE EAR MODELMINDA DALIANA1, CORICOVAC DORINA2, PINZARU IULIA2,DEHELEAN CRISTINA2, BORCAN FLORIN3, MUNTEAN DANINA1‡
1Department of Pathophysiology
2Department of Toxicology
3Department of Analytical Chemistry‡Center for Translational Research and Systems Medicine“Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACT
Lupeol is a natural compound, member of the pentacyclic triterpene family, family known for its multiple
pharmacological effects. This compound was described as a potent anti-inflammatory agent both in vitro and invivo, and, also possess antitumoral effects. The main objectives of this study were to obtain an ear inflammation
mouse model by topical application of 12-0-tetradecanoylphorbol acetate (TPA) in SKH1 hairless mice and toevaluate the effects of lupeol in this condition.
The mice were divided in 3 groups: control group – TPA treated, group treated with lupeol and group treated withindomethacin. There were evaluated parameters such as: transepidermal water loss (TEWL) by means of a
non-invasive technique and ear oedema using Draize scoring.
Our results showed that lupeol treatment had a protective and anti-inflammatory effect, effect comparable withthe one observed in the case of indomethacin. Moreover, the anti-inflammatory activity of lupeol becomesstronger in time (after 24 h).
Key words: inflammation, lupeol, indomethacin, TPA

INTRODUCTION
Lupeol is a compound of natural origin, member ofthe pentacyclic triterpenes family and is widely distributedin vegetables oils, cereals and fruits [1]. This
plant-derived compound has a lupan-skeleton with four
cyclohexane rings and one cyclopentane ring arranged intrans conformation what confers it a high liposolubility [2].
Lupeol is known to exhibit multiple biologicalproperties, including: anti-inflammatory [3, 4], antitumoral[5], antimicrobial, cytoprotective [6], antidiabetic [7].
The pharmacological effects of lupeol were proved
both in vitro and in vivo and another advantage
associated to this product is that it has no toxicity on
normal cells.
The ear inflammation mouse model obtained by
topical application of 12-0-tetradecanoylphorbol acetate(TPA) is an easy model to develop and it might beconsidered a reliable source of information regarding thebeneficial or toxic effects of some new compounds withpotential anti-inflammatory effects.

The aims of the present study were to obtain an ear
inflammation mouse model and to check theanti-inflammatory effect of lupeol after local
administration in comparison with indomethacin.

MATERIALS AND METHODSAll the reagents used in the study (lupeol, acetone,
TPA) were of analytical purity and were purchased from
Sigma Aldrich, Germany and Chimopar, Bucharest.
The animals used in the present study were SKH1
hairless male mice (10-12 weeks-old) and werepurchased from Charles River Laboratories, Budapest,
Hungary. All experimental procedures were conducted inaccordance with the Directive 2010/63/EU on theprotection of animals used for scientific purposes. The
experimental protocol was approved by the Committee
for Ethics Research of the University for Medicine andPharmacy of Timisoara, Romania. Animals were fed ad
libitum and kept under standard conditions: constantReceived 18th of April 2015. Accepted 14th of May 2015. Address for correspondence: Danina Muntean, MD, PhD, Departmentof Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st., 300173
Timisoara, RO, Tel/Fax: +40-256-493085; e-mail: daninamuntean@umft.roFiziologia - Physiology • 2015.25.3(87)25temperature of 22.5 ± 2° C, humidity 55 ± 5% and a 12-h
light/dark cycle.
The ear inflammation mouse model was obtained
according to the following protocol: TPA acetone solution(2 µg/20µl acetone) was applied on the mice external
ear and after 30 minutes, the test solutions wereadministered. After 24h post-administration of thesolutions, the mice were sacrificed and the ears were cut
and weighed.
The mice were divided in 3 groups: group 1 – control
group that received only the TPA solution, group 2 – thegroup treated with lupeol solution and group 3 – thegroup that was treated with indomethacin.
A non-invasive technique was used in order to measuretransepidermal waterloss (TEWL), a physiologic cutaneous
parameter with important role in the diagnostic of different
skin diseases that affect skin integrity. The measurements
were executed by the means of Multiprobe Adapter System(MPA 5), Courage Khazaka, Germany.
All the measurements from this research were donein triplicate for each sample; the results were expressed
as mean ± standard error. Paired Student’s t tests or
One-way Anova followed by Bonferroni’s post-tests wereused to determine the statistical difference between
different experimental and control group. p < 0.05 wasconsidered statistically significant; *, ** and *** indicate
p<0.05, p<0.01 and <0.001

RESULTS AND DISCUSSIONSThe ear inflammation mouse model represents a
useful tool in the evaluation of the anti-inflammatory
potential of new compounds.
In the present study, we developed this model byusing SKH1 hairless mice and acetone solution of
12-0-tetradecanoylphorbol acetate (TPA).
Another objective was to verify the anti-inflammatory
effects of lupeol acetone solution after topicaladministration as compared to the group that was treatedwith indomethacin.
Regarding the macroscopic aspect of the mice ears,
there were detected significant differences between thework groups (Figure 1).

As it can be seen from the pictures, lupeol was able
to reduce the ear oedema and redness as compared tocontrol group.
More than half a century has passed since theimportance of transepidermal water loss (known as
TEWA or TEWL) was recognized as a procedure used forthe evaluation of skin barrier function. G.E. Nilsson
described for the first time this procedure which is basedon the estimation of the vapour pressure gradient [8, 9].
This technique represents a well-established method
in dermatology to assess the integrity of the skin barrierin vivo. When skin is damaged, its barrier function is
unaccomplished resulting in higher water loss [10].
E. Ahaghotu et al. described ten years ago acorelation between different chemical compounds andthe evolution of TEWA at hairless rats; they used acontrol group with TEWA around 5 g/m2 h, and obtained
an increase to 10-15 g/m2 h for a 100 hours treatment
with xylene or benzene and more than 15 units/100
hours in the case of tetramethyl-benzene isomers [11].
Moreover, the dependence between TEWA values andskin treated with cancer promoters such as
7,12-dimethylbenz[a]anthracene (DMBA) solution followed by
UVB exposure was already proved by our research team [12].
In this study, there was observed that skin of micefrom the control group (C) suffered of a continous andimportant degradation during the 24 hours. The mostimportant degradation was recorded in the first 4 hours of
exposure to TPA (around 2 units/hour). The protective
effect of indomethacin, a well-known non-steroidal
anti-inflammatory drug, leads to an aproximative constanttrend for TEWA values during the experiment (between
3.8 and 5.5 g/m2 h); once again the most important
increase was recorded in the first four hours of evaluationof mice from group I.

Fig. 2. Evolution of TEWA-meter values

Fig. 1. Ear inflammation mouse model induced by topic
application of TPA: a) control group - C, b) ears exposed to
TPA solution and lupeol treatment (group L) and c) ears with
TPA treated with Indomethacin (group I)

26

In the case of mice trated with TPA and lupeol (groupL) the difference between the first 4 hours and the others
is not so important. The increase of average
TEWA-values is more linear between 4.5 and 6.4 g/m2 h.
It is important to observe that lupeol presents a protective
effect similar to indomethacin. A maximum value ofFiziologia - Physiology • 2015.25.3(87)TEWA below 10 units, after 24 hours of treatment with
TPA, an important inducer for endogenous superoxide
production, used as a potent tumor promoter, evidenced
one of the beneficial effects of this phytocompound.

CONCLUSION
These results allow drawing the conclusion that
lupeol has an anti-inflammatory activity which becomesstronger after a longer period, but the values are smaller
than in case of mice treated with indomethacin.
AKNOWLEDGEMENTS
This work was supported by the POSDRU grant no.
159/1.5/S/136893 titled “Strategic partnership for theincrease of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”, awarded toDorina Coricovac.

Fig. 3. Values for ear oedema after 4 and 12 hours, respectively

REFERENCES

Dermal irritation has been assessed using the Draize
test since 1944 [13]. Takashi Nishikawa et al. used the
Draize scoring system to assess the effects of 10
polycyclic aromatic hydrocarbons on the dorsal skin ofhairless mice; using this method, they concluded thatthese compounds caused the in vivo production of
micronucleus which is correlated with the reported
carcinogenicity of these chemical compounds [13].
In our study, increased values of Draize scoring systemwere obtained in the case of group C. The beneficial andsimilar effects of indomethacin and lupeol can be assumed
to the results which indicate half scores for values of group I
and group L than the values of control group.

1. Siddique HR, Saleem M. Beneficial health effects of lupeol
triterpene: A review of preclinical studies. Life Sci. 2011; 88:
285-293
2. Broniatowski M, Flasiński M, Zięba K, Miśkowiec P. Langmuirmonolayer studies of the interaction of monoamphiphilic
pentacyclic triterpenes with anionic mithcondrial and bacterial
membrane phospholipids – Searching for the most active terpene.
Biochim Biophys Acta 2014; 1838: 2460-2472
3. Chaturvedi PK, Bhui K, Shukla Y. Lupeol: Connotations forchemoprevention. Cancer Lett. 2008; 263: 1-13
4. Chatterjee I, Chakravarty AK, Gomes A. Daboia russellii andNaja kaouthia venom neutralization by lupeol acetate isolated
from the root extract of Indian sarsaparilla Hemidesmus indicus R.
J Ethnopharmacol. 2006; 106: 38-43
5. Saleem M. Lupeol, a novel anti-inflammatory and anti-cancer
dietary triterpene. Cancer Lett. 2009; 285: 109-115
6. Sudhahar V, Kumar SA, Mythili Y, Varalakshmi P. Remedial
effect of lupeol and its ester derivative on
hypercholesterolemia-induced oxidative and inflammatorystresses. Nutrition Res. 2007; 27: 778-787.
7. Ramu R, Shirahati PS, Zameer F, Ranganatha LV, Nagendra
Prasad MN. Inhibitor Ry effect of banana (Musa sp. Var.
Nanjangud rasa bale) flower extract and its constituents
Umbelliferone and Lupeol on α-glucosidase, aldose reductase
and glycation at multiple stages. South African Journal of Botany
2014; 95: 54-63.
8. Wilson DR, Maibach HI. A review of transepidermal water
loss. In: Neonatal skin - structure and function. Maibach HI and
Boisits EK Eds., Marcel Dekker Inc, New York 1982.
9. Nilsson GE. Measurement of Water Exchange through Skin.
Med Biol Comput. 1977; 15: 209-18.
10. Pinnagoda J, Tupker RA, Agner T, Serup J. Occupational,
guidelines for transepidermal water loss (TEWL) measurement. A
report from the standardization group of the European society ofcontact dermatitis. Contact Dermatitis 1990; 22(3): 164–178.
11. Ahaghotu E, Babu RJ, Chatterjee A, Singh M. Effect of
methyl substitution of benzene on the percutaneous absorption
and skin irritation in hairless rats. Toxicol Lett. 2005; 159:
261–271.
12. Coricovac D, Danciu C, Ionescu D, Trandafirescu C,

Fig. 4. Inhibition ratio after 4, and respectively 12 hours

The average value for oedema induced by TPA alone
after 12 hours was 4.0 ± 0.3 with an inhibition ratio, IR =0%, while the average value for oedema in case oftreatment with indomethacin was 2.0 ± 0.15 and IR =65.11%; for the lupeol, the Draize scoring system value
was 2.0 ± 0.2 and IR = 49.83%. There were not obtained
important differences between 4 and 12 hours in theevaluation of oedema and inhibition rate, but the results
indicate a better effect of two tested compounds after alonger period.
Fiziologia - Physiology • 2015.25.3(87)27Simu G, Soica C, Kemeny L, Ardelean S, Dehelean C. The
influence of betulinic acid formulated as nanoemulsion on UVB
activity as a tumor promoter in a mouse model of skin carcinoma.
Toxicol Lett. 2014; 229: S50-S51

13. Miles A, Berthet A, Hopf NB, Gilliet M, Raffoul W, Vernez D,
Spring P. A new alternative method for testing skin irritation using
a human skin model: A pilot study. Toxicol In Vitro. 2014; 28(2):240-247.

LUPEOLUL – UN AGENT ANTI-INFLAMATOR PUTERNIC ÎN INFLMAȚIA
ACUTĂ PE UN MODEL EXPERIMENTAL DE URECHE MURINĂREZUMAT
Lupeolul este un compus natural, membru al familiei triterpenelor pentaciclice, familie recunoscută de-alungul
timpului pentru multiplele sale efecte farmacologice. Acest compus a fost descris drept un potent agent
antiinflamator atât in vivo, cât și in vitro, și mai posedă și activitate antitumorală. Obiectivele acestui studiu auconstat în obținerea modelului de inflamație a urechii la șoarece după aplicarea locală a forbolului 12-Otetradecanoil acetat (TPA) la șoarecii fără păr SKH1 și verificarea efectului lupeolului asupra inflamației induse.
Șoarecii au fost împărțiți în 3 loturi: lotul control – TPA, lotul tratat cu lupeol și lotul tratat cu indometacin. Au fostevaluați parametrii precum pierderea transepisermică a apei (TEWL) printr-o tehnică non-invazivă și gradul
edemului urechii cu ajutorul scorului Draize.
Rezultatele obținute au indicat faptul că tratamentul cu lupeol a avut un efect protector și antiinflamator, efectcomparabil cu cel al indometacinului. Mai mult decât atât s-a observat că tratamentul cu lupeol este mai eficace
în timp (după 24 ore de la aplicare).
Cuvinte cheie: inflamație, lupeol, indometacin, TPA.

28

Fiziologia - Physiology • 2015.25.3(87)MOLECULAR MARKERS IN BREAST CANCER
AND THEIR CLINICAL SIGNIFICANCEMARGAN MĂDĂLIN-MARIUS
Department of Obstetrics and Gynecology, “Victor Babeș”

University of Medicine and Pharmacy, Timișoara

ABSTRACT
Breast cancer management has experienced incremental improvements in the past decades due to the discovery of
new biomarkers very useful for early detection, prognosis and prediction of treatment responses. This review aims tohighlight all the biomarkers that can be used in order to obtain individualized therapies in breast cancer treatment.
Established molecular markers such as estrogen receptor and progesterone receptor have played a significant rolein the selection of patients benefiting from endocrine therapy for many years. More recently, a new panel ofbiomarkers has emerged, but additional studies on large patient cohorts and using consistent methodologies areneeded to define their precise value for the diagnosis and prognosis of breast cancer and for their predictive
usefulness. Taking into account the quick development of this molecular biomarkers and the effectiveness oftargeted molecular therapies, there is a continuous need for searching and identifying new markers.

INTRODUCTION
Cancer is the second cause of death worldwide.
More conclusively, breast cancer is one of the mostprevalent forms of cancer in women, and among theleading causes of cancer mortalities [1]. However, due torecent advances in the field, there has been a sustained
decline in the mortality rates over the last decades. For
example, the recurrence rate after surgery in patients
detected with early breast cancer is nowadays relatively
low. Improved imaging methods and screening programs
assure in many cases an early diagnosis, emphasizing
the need for new factors and sets of biomarkers toassess the individual risk of patients and to pinpoint theinherent value of additional treatment methods. Thus, thefocus has shifted to individualized therapeutic
approaches and personalized medicine. The estimationof the net benefit of systemic chemotherapy has to betaken in consideration when making therapeutic
decisions for each patient individually. It becomes
important to avoid unnecessary overtreatment in patients
who only receive a modest benefit in the prolonging andquality of life, while at the same time suffering fromsignificant side effects. On the other hand, physiciansmust also avoid decisions leading to incomplete, orincorrect treatments. It is therefore necessary to define
the specific characteristics, molecular or clinical, which
provide the future option for individual, patient-by-patient,treatment optimization [2].

The morphopathological development of breast
cancer involves a progression through series of stages,
beginning with ductal hyperproliferation, followed bysubsequent evolution to carcinoma in situ, invasive
carcinoma, and finally towards metastatic disease [1].
Given the variability in clinical progression of breast
cancer, the identification of markers that could predicttumor behavior has become significantly important.
Furthermore, the determination of tumor markers fromtests is a useful tool for the clinical management ofcancer patients, assisting in overall diagnostic
procedures, tumor staging, evaluation of response totreatment, detection of relapses and prognosis. Not
least important, studying the emerging field of breast
cancer molecular markers aids in the development of
new personalized treatment courses. Classicalclinicopathological features of breast cancer, thatindicate patient prognosis, include tumor size,
histological subtype and grade, lymph node metastases,
and lymphovascular invasion are all derived fromcareful histological analysis of primary breast cancer
samples. The TNM (tumor size, nodes, and metastasis)
system integrates these into tumor stages that areimportant when establishing the prognosis of eachpatient. However, with recent advancements inhigh-throughput methods, an array of novel bio-markers
have been reported with prognostic and predictive
purposes. Out of these, only a few have made their way
into clinical routine due to the lack of sufficient

Received 11th of July 2015. Accepted 12th of August 2015. Address for correspondence: Margan Mădălin-Marius, MD, PhD
student, “Victor Babeș” University of Medicine and Pharmacy Timișoara, University Clinic of Obstetrics and Gynecology “Bega”,
Victor Babeș Blvd. No. 12, RO-300226, Timișoara, Romania; phone: +40256207355; e-mail: marganmm@gmail.com

Fiziologia - Physiology • 2015.25.3(87)29validation to reach a Level of Evidence I or II according
to the American Society of Clinical Oncology’s Tumor
Marker Utility Grading System [3]. Using this system,
only a few biomarkers, including hormone receptors
such as the estrogen receptor (ER) and progesterone
receptor (PR), as well as the human epidermal growth
factor receptor 2 (HER2), have been established andare assessed routinely in every breast cancer.
Nevertheless, the discovery of new markers has led tomore in depth insight into tumor biology and has
underlined the importance of the existing biomarkers.
The purpose of this review is to aggregate all
established diagnostic and predictive factors, as well assome emerging biomarkers that are currently undergoing
testing for technical validity and clinical utility, in one single
resource (Table I). This will help pathologists and clinicians
have a clear glance at the actual state of research so toprovide more efficient, personalized medicine.

PROGNOSIS AND PREDICTION
IN BREAST CANCER
Prognostic and predictive markers have distinct
roles in the establishment of personalized therapies, butboth are of high relevance for therapeutic decisions.
These factors are derived from either the individual
characteristics of the patient or the tumor type.

Prognostic factors intend to objectively predict patient
clinical outcome independent of treatment, while
predictive factors aim to foretell the response of apatient to a specific therapeutic intervention and areassociated with tumor sensitivity or resistance to a
specific therapy. For example, predictive markers canbe targeted by specific therapies, in of themselves. The
monoclonal antibody trastuzumab targets the oncogene
HER2, giving HER2 positive status mixed
prognostic/predictive significance. In the same manner,
Ki67, has an important prognostic value as a marker oftumor proliferation, while also predicting a good
response to systemic chemotherapy. In general,prognostic markers help determine whether a patient issuitable for treatment, while predictive factors areuseful in deciding which therapeutic course will be themost efficient and have the best outcome. Nowadays,
there has been a surge in the application of multiple
marker sets to establish treatment-specific prognosis.
This is particularly important when defining the residual
recurrence risk of individualized treatments, and toevaluate the potential benefits of further therapeutic
options. Significant efforts have been made recently todiscern which ER-positive early breast cancer patientswould actually benefit from additional chemotherapy
and which ones would not, thus being able to avoid theside effects [4].

Table I. Synthesis of established and emerging biomarkers used in breast cancer, their area of research interest,a score of clinical significance and most relevant references to support the findings
Biomarker

Field of importanceER

Diagnosis/Prognosis/
Prediction of drug
response
Diagnosis/Prognosis/
Prediction of drug
response
Diagnosis/Prognosis/
Prediction of drug
response
Prognosis/
Prediction of drug
response
Prognosis
Diagnosis/Prognosis
Diagnosis/Prognosis
Prognosis/
Prediction of drug
response
Prognosis/
Prediction of drug
response
Prognosis/
Prediction of drug
response

PR
HER-2
Ki67
p53
CA 15-3, CEA
BRCA1, BRCA2
Cyclin D1
Cyclin E
ERβ30

Level of clinicalsignificance++++

References*+++[6], [7]++++[8], [9]

++

[10]

+
+++
+++
+

[11], [12][13], [14][15], [16][17], [18]+[19], [20]+[21], [22][5]Fiziologia - Physiology • 2015.25.3(87)PCNA
Caveolin
CXCR4
CCL2, CCL5
Growth Factors**
FOXP3
MYC
microRNAs

+
+
+
+
+
+
#
+

[23]
[24]
[25], [26][27], [28][31][32], [33][34][35], [36],[37]

uPA+PAI+TF
h-MAM

Prognosis
Prognosis
Prognosis
Prognosis
Prognosis
Prognosis
Prognosis
Prognosis/
Prediction of drug
response
Diagnosis
Diagnosis/ Prognosis++

Osteopontin

Prognosis

+

PTEN
FGFR2
Snail 1
Sirtuins
Twist
Zeb-1
CYP2D6

Prognosis
Prognosis
Prognosis
Prognosis
Prognosis
Prognosis
Prediction of drug
response
Prediction of drug
response
Prediction of drug
response
Prediction of drug
response
Prediction of drug
response
Prediction of drug
response
Diagnosis
Prognosis
Prognosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis

+
+
+
+
+
+
+

[38], [39][40], [41],[42][43], [44],
[45]
[46]
[47], [48][49], [50][51], [52]
[50]
[53]
[54]

+

[55], [53]

+

[54]

+

[55]

+

[59], [60]

+

[61]

3PIK3CARARA
STAT3
TIMP-1
Lin28
RS/DJ-1
HSP60, HSP90
Mucin-related
α-2-HS-glycoprotein
Lipophilin B
Beta-globin
Hemopexin
Vitamin D-binding
protein

+
+
+
^
^
^
^
^

[62]
[63]
[64], [65]
[66]
[66]
[66]
[66]
[66]

* most relevant** EGF, HGF, IGF, VEGF, and TGF-β
Legend:+ + + + = established therapeutic target+ + + = diagnosis, prognosis and predictive factors used in everyday clinical practice+ + = could be considered as a prognostic factor for therapeutic decision; however, standardization of techniques and scoring methods areneeded for integration in everyday practice+ = different level of evidence for clinical significance, bur further investigations are needed before any kind of implementation# = lack of evidence for the prognostic significance; could represent a clinically useful predictive parameter in metastatic breast cancer^ = authors only identified abundant proteins in the nipple aspirate fluid whose over- or underexpression was somewhat modest; because,
there is no analyze from patients with inflammatory breast disease, conclusions cannot be drawn regarding breast cancer specificity ofprotein expression.

MULTIGENE TESTS IN BREAST CANCER
Gene expression profiling of tumors measures thousandsof mRNA transcripts in one single test using DNA
microarrays. These signatures (Table II) are composed ofdifferent gene sets with few overlaying genes, mostly
Fiziologia - Physiology • 2015.25.3(87)those associated with proliferation. Clinical context (e.gage, pure prognosis, endocrine treatment) and available
biopsy material (e.g. MammaPrint and RS need fresh
tissue, the others can use FFPE) should dictate theoncologist choice.

31

Table II. Multigene parameters in breast cancer, the number of genes used in each test,
their area of clinical importance and most relevant references that support
Test
MammaPrint

Numberof genes
70

Oncotype DX

21

Genomic-grade
index

97

Molecular grade
index

5

Rotterdam
signature76

Field of importance
Prognostic: 5-years prognosis ofrecurrence for all node-negativeand node-positive patients
Predictive: Residual risk of DR in
ER+ patients treated withtamoxifen or AIs; predictive of
chemotherapy benefit innode-negative ER+ patients
Prognostic/Predictive: Relapse inendocrine- treated ER+ breast
cancerPredictive: Poor outcome despite
endocrine therapy in ER+ breast
cancerPrognostic: Development of
distant metastases within 5 years*most relevant

Routine use of multigene assays such Oncotype Dx
and MammaPrint to guide adjuvant therapy decisions inbreast cancer has been shown to provide goodcost-effectiveness, since it allows a correct choice ofchemotherapy and reduction in chemotherapy utilization[75, 76]. The St Gallen consensus clearly states that theuse of a validated multigene profiling assay is justified as asupplement to high-quality phenotyping of breast cancer incases in which the indication for adjuvant chemotherapy
remains unclear [77], but this tests are far from being
widespread regarding clinical use.

CONCLUSION
There is an intense interest in breast biomarkers, as
they can offer precious information about diseaseprogression, patient’s prognostic and response to specifictreatment. Knowing and dealing with this complicatedtherapeutic schemes, where each patient’s management
should be individualized, is definitely a challenge for theclinicians. Some of the markers discussed in this review
provide strong evidence about their effectiveness andclinical applicability, whereas others are still under study,
with clear signs they will play a significant role in future
therapies. Unfortunately, at present, there have not been
identified any biomarkers that are capable of an early
detection of breast cancer, and many women are still
diagnosed at advanced stages with poor outcome. Veryfew of this biomarkers are capable on their own to bepredictive and have a veritable clinical use, so a lot of
randomized prospective trials are conducted in order tohave some relevant results. Taking into account all thedevelopment done in this field, there still is a need for
future molecular discoveries, which eventually will lead to32

References*[67][68], [69][70], [71][72][73], [74]new markers and of course to better targeted therapies.
The future will provide us with unique case-specificpatterns of biomarkers that will be able to predict specificresponses.

ABBREVIATIONSBRCA1 and BRCA2: Breast Cancer Susceptibility Genes;
CA 15-3: Carbohydrate 15-3; CEA: Carcinoembryonic
Antigens; CCL2 and CCL5: Chemokine (C-C Motif) Ligands
2 and 5; CXCR4: C-X-C Chemokine Receptor Type
4CYP2D6: Cytochrome P450 2D6; ER: Estrogen Receptor;
EGF: Epidermal Growth Factor; FFPE: Formalin-Fixed
Paraffin-Embedded; FGFR2: Fibroblast Growth Factor
Receptors 2; FOXP3: Forkhead Box Protein 3; GCDFP-15:
Gross Cystic Disease Fluid Protein-15; h-MAM: Human
Mammaglobin; HER2: Human Epidermal Receptor 2; HGF:
Hepatocyte Growth Factor, HSP: Heat Shock Protein; IGF:
Insulin-like Growth Factor, ILC: Infiltrating Lobular
Carcinomas; MGBA: Mammaglobin A; MMPs: Matrix
Metalloproteinases; MYC: V-Myc Myelocytomatosis Viral
Oncogene Homolog (Avian); PAI: Plasminogen Activator
Inhibitor; PARP: Poly ADP Ribose Polymerase; PCNA:
Proliferating
Cell
NuclearAntigen;
PIK3CA:
Phosphatidylinositol-4,5-bipho-3-kinase; PR: Progeteron
Receptor; PTEN: Phosphatase and Tensin Homolog;
RARA: Retinoic Acid Receptor Alpha; SERM: Selective
Estrogen Receptor Modulator; SIRT: Sirtuins; SNPs: Single
Nucleotide Polymorphisms; STAT3: Signal Transducer andActivator of Transcription 3; TF: Thomsen-Friedenreich;
TGFβ: Transforming Growth Factor Beta; TICs:
Tumor-Initiating Cells; TIMP-1: Tisue Inhibitor of
Metalloproteinase 1; TGF-β: Transforming Growth Factor-β;
uPA: Urokinase-Dependent Plasminogen Activator System;
VEGF: Vascular Endothelial Growth Factor.
Fiziologia - Physiology • 2015.25.3(87)ACKNOWLEDGMENT

This work was supported by the POSDRU grant no.
159/1.5/S/136893 titled “Strategic partnership for theincrease of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”, awarded toMargan Madalin-Marius

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MARKERI MOLECULARI ÎN CANCERUL MAMAR ŞI SEMNIFICAŢIA LOR CLINICĂREZUMATManagementul cancerului mamar a cunoscut îmbunătățiri incrementale în ultimele decenii, ca urmare adescoperirii de noi biomarkeri foarte utili în depistarea precoce, prognosticul și predicția răspunsului la terapie.
Acest review își propune să evidențieze toți biomarkerii ce pot fi utilizați pentru a obține terapii individualizate întratamentul cancerului de sân. Markeri moleculari consacrați, cum ar fi receptorul de estrogen și receptorul deprogesteron, au jucat de mai mulți ani un rol important în selectarea pacienților ce pot beneficia de terapie
endocrină. Mai recent, s-a conturat un nou panou de biomarkeri, dar studii suplimentare pe loturi mari de paciente
și folosind metodologii consistente sunt necesare pentru a defini valoarea lor exactă în diagnosticul și prognosticul
cancerului de sân și capacitatea lor predictivă. Luând în considerare dezvoltarea rapidă a acestor biomarkeri și
eficacitatea terapiilor moleculare specifice, există o nevoie continuă pentru identificarea de noi markeri.

Fiziologia - Physiology • 2015.25.3(87)35IN VITRO MORPHOLOGICAL CHARACTERIZATION OF
ENDOTHELIAL CELLS DERIVED FROM BOVINE CORNEARADU IVANESCU, MARIUS TOMA, RADU LAZAROVICZ, FLORINA BOJIN, DACIANANISTOR, CARMEN PANAITESCU, VIRGIL PAUNESCU, GABRIELA TANASIE
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTThe monolayer of cells called the corneal endothelium that lines the posterior corneal surface is derived from theneural crest during embryologic development.
In this paper we describe a simple procedure for growing bovine cornea1 endothelial cells (BCEC) in culture,
followed by morphological and ultrastructural analysis of cells. Also, we present data regarding the laser confocal
microscopic images of epithelium, stroma and endothelium of ex vivo bovine cornea. The cultured cells
demonstrate the roughly hexagonal shape and close apposition to one another. That is a distinguishing
characteristic of this endothelial type. There are small nits or depressions on surface caused bv cvtoplasmic
processes of endothelial cell that extend into the membrane. The cells presented a uniform distribution ofcytoskeletal fibers stained for Vimentin, which were present even in the cytoplasmic processes extending fromone cell to the others.
The present study demonstrates the possibility of obtaining primary endothelial cell cultures, and shows anextensive characterization of the cells, with the possibility of using these cells in tissue-engineering strategies.
Key words: corneal endothelial cells, SEM, TEM, confocal microscopy

INTRODUCTION
The cornea1 endothelium is a neural crest-derived,
simple squamous epithelium that covers the posterior
surface of the cornea [l]. The endothelial layer maintains
the clarity of the cornea by pumping salts and water outof the connective tissue stroma and into the anteriorchamber of the eye [2]. Comeal endothelial cells alsoelaborate a thick basement membrane termed
Descemet’s membrane [3]. Descemet’s membraneincreases in thickness throughout life, as a result of theongoing synthesis and deposition of membranouselements by the corneal endothelium [4]. The membranepossesses a distinctive internal substructure which
consists of a series of hexagonally arranged nodesinterconnected by fine filaments. This substructure ispresent to a variable extent in amphibians, birds, andmammals [5].
Successful primary outgrowths of cornea1
endothelium have been observed from explants of a
Descemet’s membrane-endothelial cell complex that had
been manually dissected from the comeal stroma.
Perlman et al. [10] were the first to describe the synthesis
and deposition of a collagen-containing extracellularmatrix by cultured (rabbit) cornea1 endothelial cells.
Subsequent studies by Tseng et al. [11] andGospodarowicz et al. [12, 13] using bovine cornea1
endothelial cells grown in the presence of an
exogenously supplied mitogen, fibroblast growth factor(FGF), demonstrated that endothelial cells from thisspecies also synthesize collagen types III, I, IV and V,
fibronectin and laminin.
In this paper we describe a simple procedure forgrowing bovine cornea1 endothelial cells in culture,
followed by morphological and ultrastructural analysis ofcells. Also, we present data regarding the laser confocal
microscopic images of epithelium, stroma andendothelium of ex vivo bovine cornea.

MATERIALS AND METHODSCorneal endothelial cells harvest and culture
Adult bovine eyes were obtained from slaughteredcattle within 2-5 h after death. Adhering extraocular
tissues were removed from the globes by dissection. Theeyes were then vigorously rinsed in room temperature,running tap water and subsequently covered for 15 mm

Received 12th of May 2015. Accepted 5th of June 2015. Address for correspondence: Ivanescu Radu, Department of
Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041,
Timisoara, phone/fax: +40256 490507, e-mail: radu.ivanescu@yahoo.com

36

Fiziologia - Physiology • 2015.25.3(87)with-gauze wetted with an aqueous solution 2%
Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/ml;
PromoCell, Heidelberg, Germany). The cornea was
excised with an attached l-2 mm wide scleral ring andplaced with the endothelial side up in a Petri dish. The
endothelial layer was peeled off together with Descemet
membrane and further submitted to enzymatic treatmentusing Collagenase I AS for 2 hours (Sigma-AldrichCompany, Ayrshire, UK). The next step in obtaining
endothelial cells suspension was another enzymatic
digestion with
0.25% Trypsin-EDTA solution(Sigma-Aldrich Company) for 5 minutes. The cells werethen rinsed with PBS (Sigma-Aldrich Company),
centrifuged 300 x g for 10 minutes and plated infibronectin-coated culture flasks. Endothelial cell culture
and expansion was performed using F99 culture medium(DMEM F12 + M199 2:1) supplemented with 5% fetal calf
serum (FCS; PromoCell, Heidelberg, Germany), 20 μg /ml ascorbic acid, 1 x ITS, and 10 ng/ml FGF. All culture
media and supplements were purchased from
Sigma-Aldrich Company, unless otherwise specified.

Cells transmission electron microscopy (TEM)
Bovine corneal endothelial cells were employed forinvestigation of ultrastructural details. Cells were prefixedfor 1 h with glutaraldehyde (2.5 % in PBS), rinsed threetimes in PBS, and postfixed for 1 h in osmium acid (2 %in PBS). Dehydration was done in graded acetone indistilled water dilutions, followed by infiltration with Epon
resin. Sections of about 100 nm, obtained on a diamondknife (Diatome) with Leica UC6 ultramicrotome were
post-stained with lead citrate and uranyl acetate. The
grids were examined with a FEI Tecnai 12 transmission
electron microscope (FEI Company).

RESULTS
Cultured bovine corneal endothelial cells, uponreaching confluence form a highly ordered monolayerthat closely resembles the arrangement of the native
endothelium. The cells demonstrate the roughly
hexagonal shape (Figure 1).

Confocal microscopy of the bovine cornea
The entire eye balls of the bovine specimens were
investigated ex vivo by laser corneal confocal
microscope (Heidelberg Retina Tomograph-HRT3,
Rostock Corneal Module) (Heidelberg, Germany).
Immunocytochemical/immunofluorescence analyses
Bovine corneal endothelial cells (BCEC) prepared forthese analyses were fixed with methanol andinvestigated for expression of interest markers,
employing antibodies for vimentin (clone V9)(DakoCytomation, Glostrup, Denmark). Vimentin
expression was revealed by fluorescence method after
coupling the primary antibody with specificfluorochrome-conjugated secondary antibody (AlexaFluor
488, Invitrogen , Carlsbad, CA, USA).
Cells scanning electron microscopy (SEM)
Scanning electron microscopy was performed for
identification of morphological features of bovine corneal
endothelial cells. Cells were cultured at density of 7000
cells/cm2 in 24-well format cell culture inserts (BDLabware Europe, Le Pont De Claix, France). 24 hours
after plating, cells were pre-fixed for 1 hour with 2.5 %buffered glutaraldehyde (in PBS), rinsed three times in
PBS, and the 0.4 μm pore-sized membranes weredetached from the culture inserts. For better image
quality, cells fixed on the membranes weresputter-coated with platinum-palladium and examinedwith a FEI Quanta 3D FEG electron microscope (FEI
Company, Eindhoven, NL) generating digital electron
micrographs.
Fiziologia - Physiology • 2015.25.3(87)Fig. 1. Optic microscopy of BCEC. a. 5 days after harvestingand plating; b. 10 days in culture (Magnification 40x).

When immunocytochemically stained for Vimentin,
BCEC revealed a uniform distribution of cytoskeletal
fibers, which were present even in the cytoplasmic
processes extending from one cell to the others. These
cellular connections makes the endothelial cells tofunction together and send signals even at a larger
distance (Figure 2).

Fig. 2. Immunocytochemical staining for Vimentin of BCEC
after 3 weeks in culture (Magnification 40x and 100x).

The Heidelberg Retina Tomograph (HRT) is a confocal
scanning laser ophthalmoscope. The instrument can beconverted into a confocal corneal microscope using an
optional add-on kit. The kit is composed of an additional
microscope lens which attaches to the standard lens.
Along with corneal analysis software, the HRT is able toimage cells and cell layers within the cornea.
37

To create an image, a beam of light scans the cornea,
creating a 384 x 384 point image in a 400 micron square
at a magnification of 63X. Because the system isconfocal, it has two unique properties: the first is that the
instrument can be focused at varying depths through thefull thickness of the cornea, and second, stray light isblocked, enabling crisp, clear images not available from
specular microscopes using white light sources.
Laser scanning with confocal imaging represents
one of the most significant advances in ophthalmic
imaging because it enables visualization deep within
living tissue. Confocal imaging has several advantages
over white light photography including the ability toimage within tissue at sequential depths, capturing
sharply defined optical sections. This is accomplishedthrough a “pinhole” concept, which only allows light fromthe targeted focal plane to reach the sensor.
Layer-by-Layer Corneal Imaging: most anatomicallayers and cell types may be viewed easily including
epithelial cells (superficial, intermediate and basal),
nerve plexi, stromal layers with keratocytes,
Descemet’s membrane, endothelial cells, and immune
response cells (Figure 3).

Fig. 3. Confocal microscopic corneal images. a. 215 µm superficial epithelium; b. 841 µm - posterior stroma; c.
1049 µm - endothelium.

Corneal confocal microscopy is a novel clinicaltechnique for the study of corneal cellular structure. It
provides images which are comparable to in-vitro
histochemical techniques delineating corneal epithelium,
Bowman’s layer, stroma, Descemet’s membrane andthe corneal endothelium. Because, corneal confocal
microscopy is a non-invasive technique for in vivo
imaging of the living cornea it has huge clinical potential
to investigate numerous corneal diseases.
When viewed with the scanning electron
microscope (SEM), the cultured cells demonstrate theroughly hexagonal shape and close apposition to one
another. That is a distinguishing characteristic of thisendothelial type. There are small nits or depressions on
surface caused bv cvtoplasmic processes of endothelial
cell that extend into the membrane. The smoothsurfaced ridges occasionally deposited beneath oraround individual cells or groups of cells (Figure 4).

Fig. 4. SEM of bovine corneal endothelial cells (BCEC) 3
weeks after plating and culture.

Ultrastructurally, cultured endothelial cells possess
an abundant complement of organelles required for thesynthesis of secretory proteins and glycoproteins. There
are numerous profiles of rough endoplasmic reticulum,
the cistemae of which are filled with a moderately
electron-opaque, granular material. An extensive
juxtanuclear Golgi complex composed of multiple arraysof flattened lamellae and a wide variety of vesicles is alsopresent. Occasional large vesicles filled with a granularor flocculent material together with segments of trilaminarmembrane are also present in the cytoplasm. Theselatter structures are thought to be secondary lysosomes
containing sequestered secretory products. Typicalsecondary lysosomes filled with membranous elements
increase in number with prolonged culture. These
structures could be the result of an intense vesiculartraffic (Figure 5).

Fig. 5. TEM of bovine corneal endothelial cells (BCEC) 3
weeks after plating and culture.

38

Fiziologia - Physiology • 2015.25.3(87)DISCUSSION
Bovine corneal endothelial cells can be easily grownin culture using conventional techniques. The resiliency
and thickness of Descemet’s membrane in the bovine
eye interposes an effective barrier between theendothelium and the underlying stromal fibroblasts(keratocytes) - a barrier that is not breached by gentle
rubbing of the endothelial surface with a rubber spatula.
Therefore, if the separating medium is kept away fromthe cut stromal surface, contamination of the cultures bystromal fibroblasts rarely occurs.
The monolayer of cells called the corneal
endothelium that lines the posterior corneal surface isderived from the neural crest during embryologic
development [14]. Human endothelial cell density isapproximately 6000 cells/mm2 during the first month oflife [15] but decreases to about 3500 cells/mm2 by age 5
years [16]. Growth of the cornea accounts for some ofthis decrease in density, but a decrease in the number ofcells also occurs.2 There is no evidence that humanendothelial cells divide under normal circumstances,although they can be induced to divide in cultured
corneas [17,18].
The cornea is an exquisite example of natural
engineering; the requirement for a living, optically
clear lens on the surface of the eye was solved bypacking collagen and cells in an orderly lamellar
arrangement without blood vessels. The crystalline
organization and critical spacing of collagen fibrils
makes this tissue optically transparent. Any
accumulation of fluid would disrupt this spacing anddegrade the transparency. The endothelium must
serve two functions to maintain the health and clarityof the stroma: it must control hydration (maintainstromal deturgescence) and it must be permeable tonutrients and other molecules.
The present study demonstrates the possibility ofobtaining primary endothelial cell cultures, as well as
characterization of the BCEC, with the possibility of using
these cells in tissue-engineering strategies.

Fiziologia - Physiology • 2015.25.3(87)ACKNOWLEDGEMENT
This work was supported by CNCSIS-UEFISCSU, project
number PNII-IDEI 318/2011 and 259/2011.

REFERENCES
1. Johnson MC, Noden DM, Hazelton RD, Coulombre JL,
Couiombre AJ. Exp Eye Res 1972; 29: 27.
2. Stocker FW. The endotheiium of the cornea and its clinicalimplications 2nd edn. Ch. C. Thomas, Springfield, Ill. (1971).
3. Hay ED, Revel JP. Fine structure of the developing avian
cornea. S. Karger, New York (1969).
4. Cogan DG, Kuwabara T. Trans Ophthalmo 1971; 191: 875.
5. Jakus M. J Cell Bio1. 1956; 2: 243.
6. Matsui, J. Arch Exp Zellforsch. 1929; 8: 553.
7. Stocker FW, Eiring A, Georgiade R, Georgiade N. Am J
Ophthalmol. 1958; 146: 294.
8. Newsome D, Takasugi M, Kenyon K, Stark W, Opelz G.
Invest Ophthalmol Vis Sci. 1974; 13: 23.
9. Yanoff M. Dot Ophthalmol. 1976; 141: 157.
10. Perlman M, Baum J, Kaye G. J Cell Biol. 1974; 63: 306.
11. Tseng S, Savion N, Gospodarowicz D, Stern R. J Biol
Them. 1981; 256: 3361.
12. Gospodarowicz D, Greenburg G, Vlodavsky I, Alvarado J,
Johnson LK. Exp Eye Res. 1979; 29: 485.
13. Gospodarowicz D, Greenburg D, Foidart JM, Savion N. J
Cell Physiol. 1981; 107: 171.
14. Johnson DH, Bourne WM, Campbell RJ. The ultrastructureof Descemet’s membrane. I. Changes with age in normal
corneas. Arch Ophthalmol 1982; 100: 1942-1947.
15. Bahn CF, Glassman RM, MacCallum DK, Lillie JH, Meyer
RF, Robinson BJ et al. Postnatal development of corneal
endothelium. Invest Ophthalmol Vis Sci 1986; 27: 44-51.
16. Nucci P, Brancato R, Mets MB, Shevell SK. Normal
endothelial cell density range in childhood. Arch Ophthalmol
1990; 108: 247-248.
17. Senoo T, Joyce NC. Cell cycle kinetics in corneal
endothelium from old and young donors. Invest Ophthalmol Vis
Sci 2000; 41: 660-667.
18. Senoo T, Obara Y, Joyce NC. EDTA: a promoter ofproliferation in human corneal endothelium. Invest Ophthalmol
Vis Sci 2000; 41: 2930-2935.

39

CARACTERIZAREA MORFOLOGICĂ IN VITRO A CELULELOR
ENDOTELIALE OBŢINUTE DIN CORNEEA BOVINĂREZUMAT
Celulele organizate în monstrat numite endoteliu cornean, care sunt localizate pe partea posterioară a suprafeței
corneei sunt derivate din creasta neurală în timpul dezvoltării embrionare.
În acest articol descriem o procedură simplă de cultivare a celulelor endoteliale corneene bovine (BCEC), urmată
de analiza morfologică și ultrastructurală a celulelor. De asemenea, sunt prezentate date referitoare la imaginileobținute prin tehnici de microscopie confocală ex vivo a corneei bovine, cu 3 dintre straturile corneene – epiteliu,
stroma și endoteliu. Celulele obținute și crescute în condiții de cultură demonstrează forme hexagonale și
apoziția celulelor, aceasta fiind o caracteristică a celulelor endoteliale. Celulele au prezentat o distribuție uniformă
a fibrelor citoscheletice marcate pentru Vimentină, fiind prezente și la nivelul proceselor citoplasmatice care se
extind de la o celulă la alta.
Studiul prezent demonstrează posibilitatea de a obține culturi celulare primare endoteliale și prezintă ocaracterizare extensivă a celulelor obținute, cu posibilitatea de a utiliza aceste celule în strategii de inginerietisulară
Cuvinte cheie: celule endoteliale corneene, SEM, TEM, microscopie confocală

40

Fiziologia - Physiology • 2015.25.3(87)ANTHROPOMETRIC BASIS FOR THE PHYSIOLOGICALDEMAND OF ANAEROBIC POWER AND AGILITY IN YOUNG
INDIAN NATIONAL LEVEL FIELD HOCKEY PLAYERSBARUN HANJABAM1*, KONTHOUJAM KOSANA MEITEI2
1Department of Sports & Exercise Medicine, Sports Sciences & Fitness Centre, North-East Regional Centre, Sports Authority
of India, Imphal, Manipur, India.
2Incharge, Sports Sciences & Fitness Centre, North-East Regional Centre, Sports Authority of India, Imphal, Manipur, India.

ABSTRACTThe study was conducted to assess selected anthropometric correlates & predictors of anaerobic power andagility variables in young Indian field hockey players. 13 female (16.000±2.160 years) & 18 male
(15.000±1.815 years) trainees in NERC, SAI, Imphal were the volunteers. The variables taken for the study
were: demographic and anthropometric [age, gender, height, body weight (BW), lean body mass
(LBM), %body fat (%BF) and body mass index (BMI)]; anaerobic [Running-based Anaerobic Sprint Test (RAST)& vertical jump test (VJ) related power variables; bicycle ergometer based anaerobic test: maximum revolution
per minute (RPMmax) and anaerobic power index (API)]; and agility [505 agility test timing]. The result
showed that male players had better body composition, more anaerobic power, and were more agile. BW was
the single major determinant of anaerobic power among all the anthropometric variables. LBM was the next
major contributor. %BF & BMI were also found to have significant positive correlation with anaerobic power.
However, it was found that those who had higher %BF & BMI also had higher LBM & BW. The power variablesof RAST & VJ were positively correlated, indicating similar underlying energy systems, & were not affected bygender. API was found to be a better correlate of anaerobic power than RPMmax. The data also indicated thatagility might improve with better body composition & more anaerobic power. The study could be used as amonitoring guide for field hockey training, and might also serve as a platform for future research.
Key words: Running-based Anaerobic Sprint Test, Maximum revolution per minute, Anaerobic power index,
Vertical jump and 505 agility test.

INTRODUCTION
In field hockey, not only there is importance ofphysique and body composition [1, 2], but also areaerobic-anaerobic fitness [1, 3], power and strength [1,4]. Agility and technical skills [5] are other major areas
which the elite players need to concentrate.
As quick acceleration and deceleration [6], andrepeated back-to-back sprints [6, 7] are essential partsof the game, a high anaerobic power is very muchneeded [8]. This makes sprint training regimens very
beneficial to field hockey players [6, 7]. Running-based
Anaerobic Sprint Test (RAST) is an anaerobic test for
those athletes where running is the primary method of
movement [9, 10]. Though not so sports specific inhockey [11], anaerobic capability is commonly assessedby bicycle based tests like Wingate Anaerobic Test
(WANT) [12] and its varieties.

Strength training is an important training componentof field hockey [6, 7]. Power output by a muscle is related
to its strength. In field hockey, many activities are forcefuland explosive, measuring strength and power of theconcerned muscles will thus guide training programme.
Strength training is therefore, beneficial, not only for
explosive activities, but also for injury prevention [8, 13].
Vertical jump test (VJ) or Sargent Jump Test is a goodtest for knee and hip extensor muscles’ strength [14].
Explosive or anaerobic power of the lower limbs and hips
can be measured by vertical jump and jump tests [15].
The agility or the ability to rapidly change directions
without losing balance and speed is another importantfactor for successful performance. 505 agility test [16] isgood and reliable in assessing the ability of changing
direction rapidly [17].
The present study tried to assess the selected
anthropometric correlates & predictors of anaerobic

Received 7th of August 2015. Accepted 18th of August 2015. Address for correspondence: Barun Hanjabam, Department ofSports & Exercise Medicine, Sports Sciences & Fitness Centre, North-East Regional Centre, Sports Authority of India, Imphal,
Manipur, India; phone: +91-9450870774; e-mail: dr.barun.hanjabam@gmail.com

Fiziologia - Physiology • 2015.25.3(87)41power and agility test variables in young Indian
national level female and male Manipuri field hockey
players.

duration during which 100 rpm was maintained, wasnoted as total time (TT) in second. Anaerobic powerindex (API) was calculated as TT by DT. The maximum
rpm reached (RPMmax) was also noted.

METHODOLOGY

Vertical jump test was done following the standard
methodology [20]. Three trials were attempted with aminimum of 30sec between attempts. The best score
was used to estimate PVpeak (peak power) and PVavg(average power) [21]. MPP (mean peak power) and MAP(mean average power) were calculated as arithmetic
mean of PRmax & PVpeak, and PRavg & PVavg
respectively.

Participants & Testing:Sports Sciences & Fitness Centre, North-East
Regional Centre (NERC), Sports Authority of India (SAI),
Imphal conducted the study. The subjects who
participated voluntarily in the study were thirteen female(mean age: 16.000±2.160years) & eighteen male(mean age: 15.000±1.815years) healthy Indian national
level field Hockey players from Manipur, who weretrainees in NERC, SAI, Imphal. The age of the subjects
were taken from legal certificates submitted to theauthority. A well informed and written consent was
taken, after explaining the purpose and the proceduresof every test with merit & a possible chance of injury. All
the tests were done in the morning about the same time(±1hours). The players were instructed to come after asound night sleep, and not to have any physicalexertion before 12 hours of the test. They were notallowed to drink any caffeinated drinks or eat solid food
4 hours before the test. They were encouraged andmotivated throughout to give their best. The study wasapproved by the Ethical Committee of the Institute.
Anthropometric variables measurement:
The heights (HT) of all the subjects were measured
to nearest 0.1cm using a stadiometer (Seca220, UK).
Weight (BW) and body composition variables were
measured using TANITA Body Composition Analyzer(TBF310 Model, Japan). A patented foot to foot pressure
contact electrode Bioelectrical Impedance Analysis
technique is the basis of the instrument [18].
Anaerobic variables measurements:
RAST was conducted following the standard
methodology [9, 10, 19]. PRmax (maximum power),
PRmin (minimum power), PRavg (average power) were
calculated as per the standard technique [9, 10, 19].
For the bicycle based anaerobic test, electronicallyoperated computerized bicycle ergo-meter (Jaeger,
LE900, Germany) was used. The subject remained
seated with toe clips on the bicycle ergometer for thewhole duration of the test. Warm up cycling for
5minutes at 60-70 rpm was done at a fixed power of 1
times the body weight in kg. After that the subject was
instructed to pedal as fast as possible till exhaustion
with constant verbal encouragement at a fixed power (5times body weight in kg for females, 6 times bodyweight for males). Delay time (DT) was noted as thetime taken in second to reach 100 rpm. The time
42

Agility variables measurements:
505 agility test was conducted as per the standard
methodology [16, 22]. The minimum timing each for left
(Tl) and right (Tr) foot was recorded after three attempts
for each foot.
Statistical Analysis
SPSS (Statistical Package for Social Science) version
20 software was used for data analysis. Standard
descriptive statistics were determined. One way ANOVA
was used for comparison between female and maleplayers. In case, there was significant violation ofhomogeneity of variances, Welch’s test was appliedinstead of one way ANOVA. To study relationships among
variables measured, Pearson’s correlation coefficients(zero-order) were measured. Partial correlation was used
to study effect of gender & other interested variables on
the associations among various variables. Various
regression models were generated for predicting MPP &MAP using linear regression analysis. Durbin-Watson
statistics were used to detect serial correlation.
Hierarchical multiple regression analysis (includingsemi-partial correlation R2 calculation) was done to assess
the effect of a particular independent variable above &beyond others on predicting MPP & MAP. In each case,statistical significance was chosen at α value of ≤ 5%.

RESULTS AND DISCUSSION
Among the anthropometric parameters, both thefemale and male players were comparable in case of BW,
BMI (body mass index) & age (Table I). The male players
had significantly higher HT, LBM (lean body mass) &lower %BF (%body fat) as compared to their female
counterparts (Table I). As both the groups were given
similar training of field hockey for similar duration (DOT)(Table I), and had similar dietary and physical activity
habits, the difference in their body composition
parameters were due to gender specific difference intheir physiology.
Fiziologia - Physiology • 2015.25.3(87)Table I. Comparison of various parameters between the female
and male players

Table II. Zero-order correlation among various variables offemale players (n=13)*p -value≤0.05: significant;** p-value≤0.01: highly significant (Pearson’s
Correlation). Positive correlations of anaerobic power variables with MPP &MAP, and among MPP, MAP & VJ not shown.

Table III. Zero-order correlation among various variables ofmale players (n=18)*p -value≤0.05: significant;** p-value≤0.01: highly significant (One way
ANOVA).!Welch statistic at df (1, 24.664).

PRmax, PRmin, PRavg, PVpeak, PVavg, MPP &MAP were significantly higher among the male players,
indicating more anaerobic power (Table I). The maleplayers had higher lower limb explosive power as they
had higher vertical jump scores, its associated power
variables & also more RPMmax (Table I). There wassignificant positive correlation between anaerobic
power variables (PRmax, PRmin, PRavg, PVpeak,
PVavg, MPP & MAP) and VJ with BW, LBM and HT inboth the groups (Table II, III & IV, except for VJ among
the female players, Table II). The relationships remain
still significant when the effect of gender wascontrolled for (Table V & VI), and when controlling wasdone for %BF & BMI in addition to gender (Table VI. 1& 2 (g)).

Fiziologia - Physiology • 2015.25.3(87)*p -value≤0.05: significant;** p-value≤0.01: highly significant (Pearson’s
Correlation). Positive correlations of anaerobic power variables with MPP &MAP, and among MPP, MAP & VJ not shown.

43

Table IV. Zero-order correlation among various variables of both female & male players as a whole (n=31)

Variables
PRmax
PRmin
PRavg
RPMmax.

API

VJ
PVpeak
PVavg
MPP
MAP
Tl
Tr

Significantly correlated with (R-value)
Anthropometric-demographic
Anaerobic
HT (.807**), BW (.706**) & LBM
PRmin (.912**), PRavg (.987**), RPMmax(.830**)(.500**), API (.372*), VJ (.775**), PVpeak(.855**) & PVavg (.836**)
HT (.787**), BW (.639**) & LBM
PRmax (.912**), PRavg (.944**), RPMmax(.776**)(.450*), API (.367*), VJ (.716**), PVpeak(.774**) & PVavg (.746**)****
HT (.813 ), BW (.689 ) & LBM
PRmax (.987**), PRmin (.944**), RPMmax**(.819 )(.484**), VJ (.758**), PVpeak (.831**) &PVavg (.807**)*
LBM (.388 )PRmax (.500**), PRmin (.450*), PRavg(.484**), API (.497**), VJ (.419*), PVpeak(.422*), PVavg (.426*), MPP (.445*) &MAP (.459**)**
HT (.418 ), BW (.423 ) & LBM
PRmax (.372*), PRmin (.367*),**(.478 )
RPMmax (.497**), VJ (.406*), PVpeak(.485**), PVavg (.481**), MPP (.475**) &MAP (.461**).
****HT (.733 ), BW (.467 ) & LBM
PRmax (.775**), PRmin (.716**), PRavg**(.652 )(.758**), RPMmax (.419*), API (.406*),
PVpeak (.852**) & PVavg (.852**)
HT (.854**), BW (.856**), LBM
PRmax (.855**), PRmin (.774**), PRavg(.866**) & BMI (.496**)(.831**), RPMmax (.422*), API (.485**), VJ(.852**) & PVavg (.995**)****
HT (.796 ), BW(.834 ), LBM
PRmax (.836**), PRmin (.746**), PRavg****(.829 ) & BMI (.517 )(.807**), RPMmax (.426*), API (.481**), VJ(.852**) & PVpeak (.995**)****
HT (.864 ), BW (.848 ), LBM
RPMmax (.445*) & API (.475**)****(.878 ) & BMI (.474 )HT (.833**), BW (.827**), LBM
RPMmax (.459**) & API (.461**)****(.859 ) & BMI (.472 )LBM (-.394*)
PRmax (-.364*), PRmin (-.444*), PRavg (.420*), MPP(-.363*) & MAP (-.380*)*
HT (-.413 ), LBM
PRmax (-.463**), PRmin (-.471**), PRavg(**(-.395 ) & %BF (.361 ).482**), RPMmax (-.499**), VJ (-.547**),
PVpeak (-.407*), PVavg (-.397*),
MPP (-.426*) & MAP (-.436*)

AgilityTl(-.364*)&
Tr(-.463**)
Tl(-.444*)&
Tr(-.471**)
Tl(-.420*)&
Tr(-.482**)
Tr(-.499**)−

Tr (-.547**)
Tr (-.407*)
Tr (-.397*)
Tl (-.363*)&
Tr (-.426*)
Tl (-.380*)&
Tr (-.436*)
Tr (.558**)
Tl (.558**)*p -value≤0.05: significant;** p-value≤0.01: highly significant (Pearson’s Correlation).
Positive correlations of anaerobic power variables with MPP & MAP, and among MPP, MAP & VJ not shown.

The combined effect of these anthropometricvariables in the form of M, where M= (BW+LBM+HT)/3,
uniquely accounted for 16.16% & 11.97% of the total
variability in MPP & MAP respectively, which were
statistically significant, after controlling for any
overlapped or shared variances accounted for by otherstatistically significant, zero-order correlated predictors &gender (Table VII & VIII model 1). To assess the uniquecontributions of BW, LBM & HT on MPP & MAP,
hierarchical multiple regression analysis was done. BW &44

LBM uniquely accounted for 11.49% & 5.48% of the total
variance in MPP, and 13.84% & 6.25% of the total
variance in MAP respectively over & beyond those
explained by HT & gender, which were statisticallysignificant (Table VII & 8 model 3 (b) & (c)). Whereas BW
contributed a statistically significant unique variance of
8.29% & 9.3% out of the total variance in MPP & MAP
respectively, after controlling the overlapping effect of
LBM & gender (Table VII & VIII model 4 (ii)). The relative
importance of BW over & above HT & LBM also became
Fiziologia - Physiology • 2015.25.3(87)clear from Table VI. 1 & 2 (d). Apart from the above
variables, when BW & gender were controlled, there nolonger was any significant correlation of MPP & MAP withany other variables (Table VI. 1 & 2 (b)). Therefore, theextent of the unique contributions of the above
anthropometric variables on MPP & MAP in decreasingorder was: BM>LBM>HT.

Table VII. Regression models for predicting MPP

Table V. Partial correlation among various variables of both
female & male players as a whole (n=31)*p -value≤0.05: significant;** p-value≤0.01: highly significant. G#(Gender) = 0 for male player &1 for female player. M! =(HT+BW+LBM)/3. ^&^^Hierarchical Multiple Regression Analysis. HT,
BW &LBM couldn’t be used in one equation due to multicollinearity.
*p -value≤0.05: significant;** p-value≤0.01: highly significant (Partial
Correlation). Positive correlations of anaerobic power variables with
MPP & MAP, and among MPP, MAP & VJ not shown.

Table VIII. Regression models for predicting MAP

Table VI. Partial correlation of MPP & MAP with various
variables of both female & male players as a whole (n=31)*p -value≤0.05: significant;** p-value≤0.01: highly significant. G#(Gender)=0 for male player &1 for female player. M! = (HT+BW+LBM)/3.
^&^^Hierarchical Multiple Regression Analysis. HT, BW & LBM couldn’t
be used in one equation due to multicollinearity.
*p -value≤0.05: significant;** p-value≤0.01: highly significant (Partial
Correlation). Positive correlations of anaerobic power variables with
MPP & MAP, and among MPP, MAP & VJ not shown.

Fiziologia - Physiology • 2015.25.3(87)It was found that gender controlled BW hadsignificant positive correlation with MPP & MAP after
additionally controlling for either %BF & %BF+BMI, or
45

LBM (Table VI. 1 & 2 (e), (g) & (c)). But the significant
positive correlation disappeared when both %BF & LBM
were controlled (Table VI. 1 & 2 (h)). This may due to thatfact that BW has both fat component (%BF) & LBM
component, & both the components might havecontributed uniquely to the overall gender controlled
significant positive correlations of BW with MPP & MAP
seen in our result. Since the gender controlled positive
correlations of LBM with MPP & MAP were stronger thanthose of %BF (larger R values, Table VI. 1 & 2 (a)), thecontribution of LBM might be more.
The positive correlation of anaerobic power with BW
has been reported earlier [21, 23, 24]. This isunderstandable since BW is one of the components ofanaerobic power computation [21, 25]. Also, in repeatedsprints, the player with more BW requires to produce
relatively more power to reach a fixed distance in same
duration, producing same average acceleration orvelocity. To jump same vertical height with more BW also,
more power is needed [21]. However, an earlier study
reported that increase in BW due to increase in LBM
alone didn’t adversely affect anaerobic power, whereas
those associated with increase body fat might decrease
anaerobic power [23]. Although the study was done on
cycling, a non weight bearing exercise. In our study, theanaerobic power output analyzed was based on weightbearing exercise.
The contribution by LBM component isunderstandable since LBM reflects muscle mass, where
ATP, phosphocreatine (PC) & glycogen is reserved forquick energy supply. Earlier study also reported
performance dependent strength of correlation between
lean body mass and anaerobic power [26]. Besides
gender difference in muscle mass, relatively more type II
muscle fibers, & hence more ATP, PC reserves plus more
activity of ATP enzymes, creatine phosphokinase,phosphorylase and M subunit of lactate dehydrogenase& phosphofructokinase in males [27] might beresponsible for gender difference in anaerobic power(Table I).
The reason behind the discrepancy of significant
zero-order positive correlations of anaerobic power
variables including MPP & MAP with %BF in female
data, male data, and significant partial correlation
controlled for gender as found in our study (Table II, III,
V & VI) was not fully clear. However, when the
combined data was analyzed, %BF was found not to bea significant correlate of anaerobic power variables
including MPP & MAP (Table IV). Some earlier studies
also reported more anaerobic power output by obese
subjects [28]. Body fat is an inert extra load whichneeds to be carried while running or jumping vertically,hence higher power output may be needed for rapid
movement [28]. Also, among the studied players, thosewith higher %BF also had more LBM & BW (LBM:
zero-order correlation R=.840 ** in female, R=.365 in46

male & R=.054 in combined data, and partial correlation
R, controlling for gender, =.509 **; BW: zero-ordercorrelation R=.937** in female, R=.722** in male &R=.547** in combined data, and partial correlation R,
controlling for gender, =.789**, not given in Tables).
Earlier study also reported a positive & linear
relationship between fat mass and fat-free mass over
the range of body fat extending from 10 to 90 kg [29].
Just like %BF, BMI also had significant positive
correlation with anaerobic power variables including
MPP & MAP in our study (Table II, III, IV, V, VI). The
positive correlation of BMI with anaerobic power was
reported earlier, although the study was done on obese
adult population [30]. BMI presents both LBM & fat
mass, hence it should be used with much caution
among sports populations as a surrogate for %BF forindicating or classifying fatness or obesity [31]. A player
with high LBM & low %BF may have high BMI, & may
be even falsely classified as being obese [31, 32].
Among the studied players, there were significant
positive correlations of BMI with LBM & BW (LBM:
zero-order R=.938** in female, R=.665 ** in male &R=.468** in combined data, and partial correlation R,
controlling for gender, =.763**; BW: zero-order correlation
R=.975** in female, R=.824** in male & R=.794**incombined data, and partial correlation R, controlling for
gender=.890**, not given in Tables). The gender controlled
significant positive correlations of BMI with MPP & MAP
were still maintained after additionally controlling for %BF(Table VI. 1 & 2 (e)), but not after LBM (Table VI. 1 & 2 (c)).
Also, after additionally controlling for BMI, the gender
controlled significant positive correlations of %BF with
MPP & MAP were lost (Table VI. 1 & 2 (f)). Hence, thesignificant positive correlations of BMI with MPP & MAP
could be contributed by two components, one associatedwith LBM & other with %BF, & out of these, the major
share was by that associated with LBM.
Generalization of the above finding of significant
positive correlations of %BF & BMI with anaerobic power
variables, including MPP & MAP should be avoided, as
the finding was valid only for the present studied groupwhich was selected non randomly & didn’t represent the
population of adolescent hockey players. Besides, many
earlier studies reported inverse relation of vertical jump &anaerobic power with %BF (33), and inverse or inverted
J shaped relationship with BMI [33, 34].
The anaerobic power variables of both RAST & VJ
had highly significant positive zero-order & partial
correlation with one another (Table II, III & IV) indicating
both involved similar energy systems & mechanism,
which were also not affected by gender (Table V).
RPMmax was shown to have significant positive
correlation with PRmin (in females), PRavg (in females)and API (in males) (Table II & III). However, when both
the groups were considered as whole, RPMmax was
found to have significant positive correlation with LBM,
Fiziologia - Physiology • 2015.25.3(87)anaerobic power variables, API, VJ, MPP & MAP (Table
IV). The relationships became non significant when theeffect of gender was controlled (Table V & VI). Thus
RPMmax could only give a rough idea of anaerobic powerof a player.
API did not differ significantly between the two groups(Table I). The low API values in both the groups indicatedthat there was need for improvement of speed endurance.
API was found to have significant positive correlation with
LBM (in females) and RPMmax (in males) (Table II & III).
When the two groups were considered as a whole, there
was significant positive correlation of API with HT, BW,
LBM, anaerobic power variables (except PRavg),
RPMmax, VJ, MPP & MAP (Table IV). Even after
controlling for gender, API continued to maintain theabove significant positive relationships (except for HT,
RAST power variables & VJ) (Table V & VI), showing
clearly that API could be used for rough monitoring of
anaerobic power of a player, better than RPMmax.
Male players had significantly lower time scores forboth the feet in 505 agility test, indicating they weremore agile than their female counterparts (Table I).
There was significant negative correlation between Tr
and RPMmax in female players (Table II). However,when the two groups were considered as a whole, there
were significant negative correlations of both Tl & Trwith LBM & RAST power variables; Tr with HT,
RPMmax, VJ & it’s power variables (Table IV). Tr hadsignificant positive correlation with %BF (Table IV).
Hence, the agility seems to improve with improving
body composition & anaerobic power. However, theeffect of gender on agility was very profound & when its
effect was controlled, all the above relationships ofagility became non-significant (Table V).

CONCLUSION
The male players were found to have better bodycomposition; more anaerobic power, RPM max and API.
They were also more agile than their female counterparts.
Out of all the anthropometric variables measured, BW
was the single major determinant of anaerobic power.
LBM was the next major contributor. %BF & BMI werealso found to have significant positive correlation withanaerobic power. However, it was found that those who
had higher %BF & BMI also had higher LBM & BW. The
power variables of RAST & VJ were positively correlated,indicating similar underlying energy systems, & were notaffected by gender. API was found to be a better indicatorof anaerobic power than RPMmax. Our result indicatedthat agility might improve with better body composition &anaerobic power. The study could not only be used as amonitoring guide for training & fitness of the players, butalso might serve as a platform for future research in therelated area.
Fiziologia - Physiology • 2015.25.3(87)ACKNOWLEDGEMENTS
Authors are grateful to North-East Regional Centre(NERC), Sports Authority of India (SAI) for providing thefacilities to conduct the study. The authors would like toexpress their sincere acknowledgement to the Director
Incharge, NERC, SAI for his valuable administrative &academic contribution. They further expressed their
gratitude for the contribution given by the field hockey
coaches, physiotherapy & staff nurse of Sports &Exercise Medicine department of the institute for datacollection, & most importantly by the field hockey traineesof the institute who participated in this study.

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