Metallothionein 2A expression and its relation to different clinical stages [600917]

Research paper
Metallothionein 2A expression and its relation to different clinical stages
and grades of breast cancer in Egyptian patients ☆
Noha A. Rezka,⁎, Haidy E. Zidana,M o h a m e dR i a db, Wael Mansyb, Samya A. Mohamadc
aMedical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
bSurgery Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
cPathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
abstract article info
Article history:
Received 24 February 2015Received in revised form 19 May 2015Accepted 15 June 2015Available online 18 June 2015
Keywords:MetallothioneinBreast cancerZincCopperTASTOS and OSIObjective: To assess the relation of blood MT-2A expression, serum zinc, copper, Cu/Zn ratio, total antioxidant sta-
tus (TAS), total oxidant status (TOS) and oxidant status index (OSI) with benign and malignant breast tumors,
also, their relation to different clinical stages and grades of breast cancer.Material and methods: Unrelated 199 female patients with breast tumor and 120 healthy controls were enrolled
in this study. Metallothionein-2A (MT-2A) expression was assessed by quantitative real-time polymerase chain
reaction (RT-PCR). Serum MT-2A levels were measured by ELISA. Serum copper (Cu) and Zinc (Zn) concentra-
tions were determined by atomic absorption spectrophotometry. Serum TOS and TAS levels were measured col-orimetrically.
Results: Our study demonstrated that blood metallothionein-2A mRNA level, serum MT-2A, copper, Cu/Zn ratio,
total oxidant status and oxidant status index were signi ficantly increased, while, serum zinc level and total anti-
oxidant status were signi ficantly decreased in patients with breast cancer and benign breast disease as compared
to controls and in breast cancer group as compared to the benign one.
Conclusions: Blood metallothionein-2A expression and serum MT-2A levels could be important prognostic indi-
ces of less differentiated, more aggressive breast cancer phenotype. Disturbance of copper, zinc and oxidativestress status might contribute to the pathogenesis of breast tumor and could be useful biomarkers for diagnosing
and monitoring such disease.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Breast cancer is a highly heterogeneous disease globally. It is the
most frequently diagnosed cancer in women worldwide affecting 1 in
8 women ( Burson et al., 2010; Kakarala et al., 2010 ).
Metallothioneins (MTs) are ubiquitous metal-binding proteins that
have been highly conserved throughout evolution. Cysteine residues
constitute around 30% of its amino acids. The latter represent a signi fi-
cant component due to their thiol ( −SH) groups, through which
metal ions bound ( Coyle et al., 2002 ). In humans four classes of MTs
were identi fied. MT-1 and MT-2 are the most widely distributed MT iso-
forms ( Ghoshal and Jacob, 2001 ). MT-1 is related to the metabolism or
detoxi fication of toxic metals such as cadmium, in contrast, MT-2 is
responsible for the homeostasis of essential metals such as copper
(Nakamura et al., 2004 ). MT-2A is reported to participate in cellulardifferentiation and proliferation processes in megakaryoblastic leuke-
mia ( Bagheri et al., 2011 ). MT-3 is known as a neuronal growth inhibi-
tory factor. Recently, it was found that MT-3 might regulate the levels
and functions of lysosomal proteins ( Lee et al., 2010 ). MT-4 gene poly-
morphism was reported to be associated with disturbed renal function
in long-term lead-exposed workers ( Chen et al., 2010 ).
MTs are thought to mediate several functions, including the control
of indispensable trace element levels (e.g., zinc, copper) and protection
of cells from oxidative stress ( Thirumoorthy et al., 2011; Theocharis
et al., 2003 ).
The ability of MTs to bind heavy metals and detoxify free radicals can
be both bene ficial and deleterious, as, they can also help cancer cells to
survive by inhibition of apoptosis ( McGee et al., 2010; Dutsch-Wicherek
et al., 2008 ).
Metallothioneien gene transcription is initiated when metal ions as-
sociate with metal-regulatory transcription factor-1 (MTF-1). MTF-1 is
the only known mediator of the metal responsiveness of MTs ( Klassen
et al., 2004 ). MTF-1 binds to metal-responsive elements (MREs) that
regulate MT expression. Moreover, it was found that MTF-1 was elevat-
ed in human breast, lung, and cervical carcinoma-derived cell lines
(Shi et al., 2010 ). This finding supports the importance of MTs in
carcinogenesis.Gene 571 (2015) 17 –22
Abbreviations: (MTs), metallothioneins; (TAS), total antioxidant status; (TOS), total
oxidant status; (OSI), oxidative stress index; (RT-PCR), real time polymerase chainreaction; (ABTS), 2,2 ′- azino-di-3-ethylbenz-thiazoline sulfonate; (TNM), tumor node
metastasis.
☆Declaration of interest: no con flict of interest.
⁎Corresponding author at: Medical biochemistry, Zagazig University, Zagazig, Egypt.
E-mail address: nonnarezk@yahoo.com (N.A. Rezk).
http://dx.doi.org/10.1016/j.gene.2015.06.035
0378-1119/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Gene
journal homepage: www.elsevier.com/locate/gene

Thirumoorthy et al. (2011) found that the expression of MT Zinc (II)
ions contributed to a number of biological processes, such as DNA syn-
thesis, gene expression, enzymatic catalysis, neurotransmission, and
apoptosis.
The suggested role of copper and zinc in tumor development could
be related to their action as enzymatic cofactors involved in carcinogene-
sis. Furthermore, disturbing of zinc homeostasis can lead to formation ofreactive oxygen species, which can result in oxidative stress (OS) causing
alterations in immunity, aging and cancer development ( Jomova and
Valko, 2011 ).
Oxidative stress can cause a release of zinc from proteins containing
zincfingers and cluster motifs and alter its distribution, thereby altering
the functions of those proteins, from which it is released and/or to
which it binds affecting mitochondrial functions and signaling pathways,
including alterations in nuclear factor kappa-light-chain-enhancer of acti-
vated B cells (NF- κB), p53, and activator protein 1(AP-1) activities and
levels ( Krizkova et al., 2012 ).
Previous studies examined the association of MTs with breast tu-
mors and its prognosis, However, their data were divergent, most
works focused on the immunohistochemical determination of MTs as
all not speci fic isoforms in cancerous tissues only not the benign ones
(Woolston et al., 2010; Jin et al., 2004; Gomulkiewicz et al., 2010 );
therefore, further studies are essentials.
Thus this study was constructed to assess the relation of the blood
MT-2A expression, levels, serum zinc, copper, Cu/Zn ratio, Total antiox-
idant status (TAS), Total oxidant status (TOS) and oxidative stress index
(OSI) with benign and malignant breast tumors, also, their relation to
different clinical stages and grades of breast cancer.
2. Materials and methods2.1. Subjects
A total of unrelated 199 age matched female patients with breast
tumor were recruited in this study from the outpatient clinic and inpa-
tient units of the surgery department, Zagazig University, Egypt during
the period from August 2013 to October 2014. They were classi fied
into 2 groups using the histopathological examination: group 1 included
95 patients with benign breast tumor and group 2 included 104 patients
with malignant breast tumor. In breast carcinoma group, 21 patients
were classi fied as stage I, 33 patients as stage II, 37 patients as stage
III, and 13 patients as stage IV. The stage of breast cancer was judged
by the tumor node metastasis (TNM) system ( Hammer et al., 2008 ).
The breast cancer was graded according to Bloom –Richardson scale
(Rosen, 2009 ). Breast cancer group included 3 cases with highly differ-
entiated tumors (G1), 46 cases with moderately differentiated tumors
(G2) and 55 cases with poorly differentiated tumors (G3).
Control group included one hundred and twenty healthy females
(mean age 47.13 ± 7.9 years) with no history of any tumor or breast dis-
ease. They were ethnic origin and age matched to the patients.
All subjects underwent complete history taking, thorough clinical
examination and imaging studies. Clinical and demographic character-
istics of studied subjects are shown in Table 1 . The ethical committee
of Zagazig University approved this study and a written informed con-
sent was obtained from the subjects prior to their inclusion in this work.
2.2. Methods2.2.1. Blood samples
Peripheral blood samples were collected from all subjects after over-
night fast between 8:00 am and 10:00 am the next morning. Approxi-
mately 6 ml blood was collected into a BD Vacutainer SST II ADVANCE
tube (Becton Dickinson, Rutherford, NJ, USA) for analysis of serum
MT-2A, TOS, TAS, copper and zinc. Blood samples were centrifuged at
3000 rpm for 15 min, and sera samples were collected and stored at
−20 °C until analysis. About 2 ml blood was collected on EDTA tubefor RNA isolation. The blood samples were collected from all patients
after their diagnosis but before any surgery or initiation of antitumortherapy.
2.3. Assessment of metallothionein-2A expression by quantitative real-time
PCR (RT-PCR)
Total RNA was isolated from EDTA whole blood sample according to
the manufacturer's instructions (GenElute ™Mammalian Total RNA
Miniprep Kit, Sigma-Aldrich, USA).
One microgram of total RNA was reverse transcribed using the
Roche Transcriptor cDNA synthesis kit, according to the instructions of
the manufacturer (Roche Diagnostics, Almere, Netherlands). Quantita-
tive real-time (RT-PCR) was performed using SYBR green on Stratagene
Mx3005P qPCR instrument (Agilent Technologies, Inc.).
GAPDH served as the constitutive control. Forward and reverse
primers were 5 ′
-CCGGCTCCTGCAAATGCAAAGAGTG-3 ′and 5 ′-CGCTCC
CAGATGTAAAGAACGCGAC-3 ′for MT-2A respectively; 5 ′-GCACCGTCAA
GGCTGAGAAC-3 ′and 5 ′-ATGGTG-GTGAAGACGCCAGT-3 ′for GAPDH,
respectively. All reagents and primers were obtained from Applied
Biosystems (Applied Biosystems, Foster City, CA).
The PCR ampli fication protocol was: denaturation at 95 °C for 10 min
followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C
for 35 s, and polymerization at 72 °C for 60 s ( Kim et al., 2010 ).
We took the average value of the control group as internal control.
Gene expression levels were calculated and determined using the
threshold cycle (CT) method (2- ΔΔCT method) ( Xu et al., 2010 ).Table 1
Clinical and demographic characteristics of studied subjects.
Control
group(N = 120)Benign breast
disease(N = 95)Breast
cancer(N = 104)
Age 47.13 ± 7.9 45.8 ± 9.1 46.4 ± 8.2
≤50n(%) 55 (45.8) 42(44.2) 56(53.8)
N50n(%) 65(54.2) 53(55.8) 48(46.2)
First degree relative with
breast cancer
Yesn(%) 47 (39.2) 48 (50.5) 53 (50.9)
Non(%) 73 (60.8) 47 (49.5) 51 (49.1)
Body mass index (kg/m
2)
mean ± SD26.8 ± 5.07 27.8 ± 4.2 28.1 ± 4.9
Marital status
Lining as married n(%) 75 (62.5) 65 (68.4) 61 (58.6)
Separated n(%) 35 (29.9) 19 (20.1) 35 (33.6)
Non(%) 10 (8.3) 11 (11.5) 8 (7.6)
No. of full-term pregnancies
(mean ±SD)2.51 ± 0.82 2.44 ± 0.74 2.29 ± 0.86
Menopausal status
Premenopausal n(%) 48(40) 39(41) 49(47.1)
Postmenopausal n(%) 72(60) 56(49) 55(52.9)
Breast feeding
Yesn(%) 76(63.3) 58(61.1) 53(50.9)
Non(%) 44(36.6) 37(38.9) 51(49.1)
Oral contraceptive use
Yesn(%) 62(51.6) 54(56.8) 59(62.1)
Non(%) 58(48.4) 41(43.2) 45 (37.9)
Smoking history
Yesn(%) 21 (17.5) 18 (18.9) 30 (28.8)
Non(%) 99 (82.5) 77 (81.1) 74 (71.2)
Clinical stage
Stage I n(%) 21(20.2)
Stage II n(%) 33 (31.8)
Stage III n(%) 37(35.5)
Stage IV n(%) 13(12.5)
Histological grade
Grade 1 n(%) 3(2.9)
Grade 2 n(%) 46(44.3)
Grade 3 n(%) 55(52.8)
Quantitative data were presented as mean ± standard deviation; qualitative data were
presented as numbers and percentages.18 N.A. Rezk et al. / Gene 571 (2015) 17 –22

2.4. Measurement of serum metallothionein-2A level
Serum concentrations of MT-2A were determined using a double an-
tibody sandwich ELISA kit (Biosource Europe S.A., Belgium). The proce-
dure was performed following the manufacturers' recommended
protocols.
2.5. Determination of serum copper and zinc levels
Serum samples were diluted 1:10 with deionized water. Serum
copper (Cu) and Zinc (Zn) concentrations were determined by atomic ab-
sorption spectrophotometry (GBC 932 plus) on wavelength 324.8 nm
(Cu) and 213.9 nm (Zn); slit 0.7 nm and the read time was 2 s. The instru-
ment was calibrated using stock standards solutions diluted with 10%
glycerol for Cu and 5% glycerol for Zn ( Silverio Amancio et al., 2003 ).
2.6. Total antioxidant status (TAS) determination
TAS was measured colorimetrically using the Total Antioxidant Sta-
tus kit according to the instructions of the manufacturer (Biodiagnostic,
Egypt). It depended on the ability of the sample antioxidants to
inhibit ABTS+formation from the oxidation of ABTS (2,2 ′-azino-di-
3-ethylbenz-thiazoline sulfonate) by metmyoglobin (a peroxidase).
After 10 min incubation with the chromogen at 37 °C, the amount of
ABTS+formation was measured at 600 nm, and the decrease in this
value was proportional to the antioxidant concentration in the sample.
An antioxidant of known concentration (1.65 mmol/L) was used as
a standard to calculate the antioxidant levels in the samples. Values
of TAS were expressed as mmol Trolox equivalent/L (mmol Trolox
equiv./L).
2.7. Total oxidant status (TOS) determination
Serum TOS was measured using TOS kit according to the instructions
of the manufacturer (Rel Assay Diagnostic, Turkey). With this kit, oxi-
dants present in the sample oxidized the ferrous ion-o-dianisidine com-
plex to ferric ions. The oxidation reaction was enhanced by glycerolmolecules, which were abundant in the reaction medium. The ferric
ions made a colored complex with xylenol orange. The color intensity,
measured spectrophotometrically at 530 nm, was related to the total
quantity of oxidant molecules in the sample. The assay was calibrated
with hydrogen peroxide and the results were expressed as (mmol
H
2O2equiv./dL) ( Erel, 2005 ).
2.8. Oxidative stress index
OSI was de fined as TOS to TAS ratio and was calculated as follows:
OSI (arbitrary unit) = [(TOS, 1 mmol H 2O2equiv./dL) ×10/(TAS,
l mmol Trolox equiv./L)] ( Aycicek and Erel, 2007 ). Values of TOS were
expressed as (mmol H 2O2equiv./dL), then they were multiplied by 10
to be changed to mmol H 2O2equiv./L in order to find OSI.
2.9. Statistical analysis
Data were analyzed with SPSS version 15.0 (statistical package for
the Social Science, Chicago, IL). Quantitative data were presented as
mean ± standard deviation; signi ficant differences between the differ-
ent studied groups were determined using one-way analysis of variance
(ANOVA) test. Differences of the frequencies in the studied groups were
analyzed using chi square ( χ2) test. A difference was considered signif-
icant at Pvalue b0.05.3. Results
3.1. Clinical and demographic characteristic of the studied subjects
There was no signi ficant difference between the patient groups
and controls as regards age, body mass index, first-degree relative
with breast cancer, menopausal status, marital status, number offull-term pregnancies, breast feeding, oral contraceptive use, and
smoking ( Table 1 ).
3.2. Blood metallothionein-2A expression, serum MT-2A, copper, zinc,
Cu/Zn, TOS, TAS and OSI in the studied subjects
We found that blood metallothionein-2A mRNA level, serum MT-2A
levels, serum copper, Cu/Zn ratio, total oxidant status and oxidant status
index were signi ficantly increased in patients with breast cancer ( P
value 0.023 for MT-2A mRNA and b0.001 for other parameters) and be-
nign breast disease ( Pvalues 0.016, b0.001, 0.002, 0.007, b0.001, b0.001
respectively) as compared to controls and in breast cancer group as
compared to benign one ( Pvalues b0.001, 0.005, 0.003, 0.003, 0,002,
0.041 respectively). However serum zinc level and total antioxidant sta-
tus were found to be signi ficantly lower in patients with breast cancer
(Pvalues, b0.001 for both) and benign breast disease ( Pvalues 0.002
and 0.033 respectively), as compared to controls and in breast cancer
group as compared to benign one ( Pvalues 0.004 and b0.001 re-
spectively) ( Fig. 1 ).
3.3. Blood metallothionein-2A expression, serum MT-2A, copper, zinc,
Cu/Zn, TOS, TAS and OSI in relation to the stage of breast cancer
There was no signi ficant difference of blood metallothionein-2A ex-
pression and serum MT-2A levels between different stages of breast can-
cer. Whereas mean values of serum copper, Cu/Zn ratio, total oxidant
status and oxidant status index were higher in stage II, III, IV as compared
to stage I and in stage III, IV as compared to stage II as well as in stage IV as
compared to stage III, while, there was a signi ficant decrease of serum zinc
level, total antioxidant status in stag e II, III, IV as compared to stage I and in
stage III, IV as compared to stage II as well as in stage IV as compared to
stage III ( Fig. 2 ).Pvalues between different stages of breast cancer were
recorded in (Table 2 not shown).
3.4. Blood metallothionein-2A expression, serum MT-2A, copper, zinc,
Cu/Zn, TOS, TAS and OSI in relation to the grade of breast cancer
Breast cancer patients were classi fied into 3 grades according to the
degree of breast cancer differentiation. We found signi ficant higher
values of blood metallothionein-2A mRNA ( Pb0.001), serum MT-2A
levels ( P= 0.008), Cu/Zn ratio (
P= 0.005) and OSI ( P= 0.021) with
lower value of serum zinc ( P= 0.016) in patients with grade 3 (poorly
differentiated malignant cells) as compared to those with grade I and II.
As regard serum copper level, TAS and TOS, there was no statistical
significant difference between both groups ( Fig. 3 ).
4. Discussion
Breast cancer is the most common type of cancer in women and
is a leading cause of cancer related deaths worldwide ( Coughlin and
Ekwueme, 2009 ).
Many studies documented the presence of metallothionein in vari-
ous tumor cells, including breast cancer ( Jin et al., 2004; Ostrakhovitch
et al., 2007; Yap et al., 2009 ).
The results of this study revealed a signi ficant increase of blood
MT-2A expression by RT-PCR and MT-2A levels in patients with breast
cancer and benign breast disease when compared to controls and in
breast cancer group as compared to benign one. This agreed with a
study done by Eckschlager et al. (2009) who found elevated serum19 N.A. Rezk et al. / Gene 571 (2015) 17 –22

levels of metallothioneins in cancer breast. Similar results were found in
studies done by Fabrik et al. (2007) andAdam et al. (2008) who found
that the level of MT in patients with cancer was three times higher
than controls. Furthermore, Gomulkiewicz et al. (2010) ,Yap et al.
(2009) ,Gallicchio et al. (2005) ,Floria ńczyk et al. (2011) assessed the
concentration of metallothionein in breast tissues and found that the
breast cancer group contained more metallothionein in comparison to
mastopathic lesions group. In contrast with our results, El Sharkarvy
and Farrag (2008) described non-signi ficant changes of metallothionein
in breast cancer patients.
Our results reported that blood MT-2A expression and MT-2A levels
were increased signi ficantly with increasing the grade of breast malig-
nancy. In line with our results, Gomulkiewicz et al. (2010) ,Yap et al.
(2009) ,Jin et al. (2001, 2002) found that blood MT-2A expression was
increased signi ficantly with increasing the grade of breast cancer.
As regards the relation of blood MT-2A expression, serum MT-2A
levels to the stage of breast malignancy, we found non-signi ficantassociation between them. Similar to our results, Surowiak et al.
(2006) showed non signi ficant association between tissue MT staining
and the breast cancer stage. In disagreement with our results, Jin et al.
(2002) ,Ioachim et al. (2003) reported a numerical tendency for breast
tumors of poorer stage to be more MT expressing.
Previous studies done by Vasak (2005) andTheocharis et al. (2003)
stated that the unique property of MTs, which signi ficantly determines
their role in the body, involved its ability of binding heavy metals ions,
such as zinc, copper maintaining these metal homeostasis and detoxi fi-
cation processes. There might be a functional link between MT and p53
tumor suppressor gene ( Hainaut and Mann, 2001 ). In the presence of
zinc, MT facilitates normal functional p53 activity by zinc transfer
between MT and p53, resulting in the maintenance of DNA-binding con-
formation ( Meplan et al., 2000 ). However, this transfer might be in the
reverse direction under conditions of zinc depletion ( Jacob et al., 1998 )
resulting in the disruption of the conformation of the DNA-binding do-
main and a phenotype similar to many mutant forms of p53. By this
Fig. 1. Blood metallothionein-2A mRNA expression, serum metallothionein-2A, copper, zinc, Cu/Zn, total oxidant, antioxidant status and oxidant status r atio in the studied subjects. MT2A;
metallothionein 2A, Cu/Zn; copper/zinc, total antioxidant status; TAS, total oxidant status; TOS, oxidant status index; OSI. * Pb0.05 when compared to control group.§Pb0.05 when com-
pared to benign breast disease group.
Fig. 2. Blood metallothionein-2A mRNA expression, serum metallothionein-2A, copper, zinc, Cu/Zn, total oxidant, antioxidant status and oxidant status r atio in relation to the stage of
breast cancer. MT2A; metallothionein-2A, Cu/Zn; copper/zinc, total antioxidant status; TAS, total oxidant status; TOS, oxidant status index; OSI .*Pb0.05 when compared to stage I
group.§Pb0.05 when compared to stage II group.£Pb0.05 when compared to stage III group.20 N.A. Rezk et al. / Gene 571 (2015) 17 –22

way, MT control several intracellular processes, including proliferation,
apoptosis and cell differentiation ( Cherian et al., 2003 ).
Moreover, metallothionein synthesis is induced by metals ions, hor-
mones, in flammatory factors, free radicals, physical stress and some
pharmacological agents ( Floria ńczyk and Stryjecka-Zimmer, 2001 ).
The presence of metal responsive element (MRE) sequence in MT
gene, enables direct induction of its transcription with metal participa-
tion ( Suzuki et al., 2007 ).
The role of trace elements in carcinogenesis had been investigat-
ed in previous studies ( MacDonald, 2000 ), (Clegg et al., 2005 )a n d
(Kelleher et al., 2009 )(Shams et al., 2012 ), however, their results
were not consistent.
We found a signi ficant increase of serum copper level, Cu/Zn ratio
and a signi ficant decrease of serum zinc level in patients with breast
cancer and benign breast disease in comparison to controls and in breast
cancer group as compared to benign one. In line with our results, a study
done in Europe on 4035 patients, demonstrated that a combination of
low serum zinc and high copper values had increased cancer risk syner-
gistically ( Leone et al., 2006 ). Similar results were demonstrated by
Ding et al. (2014) .
In contrast to our results, Siddiqui et al. (2006) showed that blood
zinc level was signi ficantly higher in malignant group as compared to
benign and control groups. This discrepancies could be explained by
the small sample size (25 cases and 25 controls) taken in their study.
Also, Siddiqui et al. (2006) stated that trace element concentration in bi-
ological fluids might vary depending on subjects' characteristics as age,
diet, geographic/climatic conditions, or genetic factors and might fluctu-
ate in circadian rhythm.
The mechanism of Cu elevation in malignancies is not clear. Our
results of elevated copper can be explained by Haddad et al. (2010)
who stated that the elevation in serum Cu concentration might be due
to the destruction and necrosis of the involved tissues, leading to the re-
lease of Cu into circulation. MacDonald (2000) ,Clegg et al. (2005) and
Kelleher et al. (2009) tried to explain the role of zinc in cancer breast.
Kelleher et al. (2009) stated that the normal mammary gland develop-
ment and function was highly dependent on Zn homeostasis which was
important for the cell proliferation ( MacDonald, 2000 ) and programmed
cell death ( Clegg et al., 2005 ).
Grattan and Freake (2012) postulated that the role of Zn as a tumor
promoter might be enhanced by the capability of tumor tissues to incor-
porate Zn. Furthermore, Kelleher et al. (2011) reported that the process
underlying tumor development lead to an uptake of trace elements by
the neoplastic cells which was supported by many studies done by Cui
et al. (2007) ,Al-Ebraheem et al. (2014) , and Shams et al. (2012) who
reported average increase in the tissue levels of Zn in tumor regions
compared to the normal samples.
We found that Cu/Zn ratio was signi ficantly increased and serum Zn
was signi ficantly decreased with increasing the stage and the grade of
breast malignancy, while, serum copper was signi ficantly increasedwith increasing the stage of breast cancer only. In agreement with our
results, Memon et al. (2007) andAbdel-Salam et al. (2011) found that
the concentrations of serum Cu and Cu/Zn were signi ficantly increased,
however, serum Zn was signi ficantly decreased with each successive
stage of breast cancer and with metastatic breast cancer compared to
non metatstatic one. Moreover, Payne et al. (2007) reported that some
Cu dependent factors were involved in the breakdown of cell junctionsleading to cancer metastasis.
Furthermore, Haddad et al. (2010) demonstrated a signi ficantly
higher Cu/Zn ratio with lower value of serum zinc in poorly differentiat-
ed breast malignancy and added that serum copper also was higher in
breast cancer of grade 3 but it did not reach the degree of statistical
significance.
Excessive reactive oxygen species (ROS) cause oxidative damages to
biomolecules and lead to cellular alterations, tumorigenesis and neoplas-
tic transformation ( Cooke et al., 2003 ). Oxidative stress is operationally
defined as an increase in oxidants and/or a decrease in antioxidants
(Serefhanoglu et al., 2009 ). Thus, assessment of the overall or net oxida-
tive status in the body is very important.
Concerning oxidative stress markers, we found high total oxidant
status (TOS) and oxidant status index with low serum total antioxidant
status (TAS), in patients with breast cancer and benign breast disease as
compared to controls and in breast cancer group as compared to benign
one with their signi ficant association with each successive stage of breast
cancer. In agreement with our results, ( Sener et al., 2007 ), (Badid et al.,
2010 )a n d( Gönenc et al., 2006 ) demonstrated similar results.
To our knowledge, this is the first study conducted in breast cancer
patients to investigate the relationship between the oxidative stress
markers and the grade of breast malignancy. Our study showed that
only oxidant stress index was signi ficantly increased in poorly differen-
tiated breast cancer.
In conclusion, Blood metallothionein-2A expression and serum MT-
2A levels could be important prognostic indices of less differentiated,
more aggressive breast cancer phenotype. Disturbance of copper, zinc
and oxidative stress status might contribute to the pathogenesis of
breast tumor and could be useful biomarkers for diagnosing and moni-
toring such disease.
Further studies are recommended to understand the role of other
MT isoforms in the neoplastic process and to de fine its clinical signi fi-
cance in breast cancer.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2015.06.035 .
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