Journal of Cancer Therapy, 2013, 4, 65-69
http://dx.doi.org/10.4236/jct.2013.47A010 Published Online August 2013 (http://www.scirp.org/journal/jct)
65
Effects on Cell Viability and on Apoptosis in Tumoral
(MCF-7) and in Normal (MCF10A) Epithelial Breast Cells
after Human Chorionic Gonadotropin and
Derivated-Angiotensin Peptides Treatments
Silvana Aparecida Alves Corrêa de Noronha1*, Werica Bernardo2, Alexandre Jesus Barros3,
Clovis Ryuichi Nakaie3, Suma Imura Shimuta3, Ismael Dale Cotrim Guerreiro da Silva2,
Samuel Marcos Ribeiro de Noronha1
1Laboratorio de Cirurgia Translacional, Departamento de Cirurgia, Universidade Federal De Sao Paulo/Escola Paulista De Medicina,
São Paulo, Brazil; 2Laboratório de Ginecologia Molecular, Universidade Federal de São Paulo, São Paulo, Brazil; 3Departamento de
Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.
Received May 20th, 2013; revised June 22nd, 2013; accepted June 30th, 2013
Copyright © 2013 Silvana Aparecida Alves Corrêa de Noronha et al. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
ABSTRACT
Angiotensin-(1 - 7) [Ang-(1 - 7)] is an endogenous heptapeptide hormone of the renin-angiotensin system that has anti-
proliferative properties. The aim of this work was to evaluate the anti-proliferative and pro-apoptotic properties of
Ang-(1 - 7) and of Ang-(1 - 7)-substituents 9-fluorenylmethyloxycarbonyl (Fmoc) e Ang II-derivatives containing the
TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) in normal (MCF10A) and in tumoral (MCF7)
epithelial mammary cell lines. Both cell lines received an hCG and angiotensin peptides 24-hour treatment, in combina-
tion or alone followed by cell viability, apoptosis and cell cycle assays performed by flow cytometer (GUAVA). After
hCG, Ang-(1 - 7), hCG + Ang-(1 - 7) and hCG + Ang-(1 - 7)-Fmoc treatments, MCF7 displayed cell viability decrease
and mid-apoptosis increase. We also observed cell viability decrease in MCF10A after Ang-(1 - 7), Ang-(1 - 7) Fmoc
and hCG + AngII Toac treatments. These cells had an increase in late apoptosis and necrosis after AngII Toac, hCG +
Ang-(1 - 7) and hCG + Ang-(1 - 7)-Fmoc treatments. Regarding the cell cycle analysis, we did not observed any
changes in cell cycle phases. In summary, cell viability was decreased and apoptosis (initial, mid and late) was in-
creased after hCG and/or Ang-(1 - 7) peptides treatments. These results point out hCG and Ang-(1 - 7) as effective
compounds to inhibit cell proliferation, since they decrease cell viability and increase apoptosis in both normal and in
tumoral breast cells, being the effect more pronounced in the tumoral cell line. Our results support the idea of investi-
gating more closely the putative use of these compounds as novel therapeutic agents for breast cancer.
Keywords: Angiotensin II; Angiotensin 1 - 7; Angiotensin II Type 1 Receptor (AT1R); Breast Cancer; Apoptosis;
Human Chorionic Gonadotropin.
1. Introduction
Breast cancer is the most common cancer among Ame-
rican women, except for skin cancers. About one in eight
(12%) women in the US will develop invasive breast cancer
throughout her lifetime. The American Cancer Society’s
estimates that 226,870 new cases of breast cancer will be
diagnosed in 2012 in the United States; about 63,300 new
cases of carcinoma in situ (CIS) will be diagnosed and
about 39,510 women will die from breast cancer [1].
The renin angiotensin system (RAS) is a hormone
widely known for regulating blood pressure. The prote-
olytic cascade of the RAS begins with the release of
renin (REN), an aspartyl protease which cleaves angio-
tensinogen (AGT) produces Angiotensin I (Ang I), which
is hydrolyzed by angiotensin converting enzyme (ACE)
and releases Angiotensin II (Ang II); this octapeptide ex-
*Corresponding author.

Effects on Cell Viability and on Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A) Epithelial Breast Cells
after Human Chorionic Gonadotropin and Derivated-Angiotensin Peptides Treatments
66
erts its functions through its specific membrane receptors,
Angiotensin II receptor Type 1 and Type 2 (AGTR1 and
AGTR2, respectively) [2]. Most of the physiological ef-
fects are attributed to the signaling pathways activated by
the AGTR1. However, other components of the renin-
angiotensin system (RAS) have been described as having
mitogenic and angiogenic activities [3,4]. Since angio-
genesis and proliferative processes are related to the de-
velopment, progression and metastasis of cancer, we be-
lieve that there might be an association between these
other RAS components with cancer [5]. Recent studies
have shown that at a local tissue level, the components of
the RAS influence tumor growth by changing its micro-
environment [6,7]. Angiotensin-(1 - 7) [Ang-(1 - 7)] is an
endogenous peptide hormone that functions as a vasodi-
lator [8] with antihypertensive [9], antiproliferative [10-
12] and antiangiogenic properties [13]. The formation of
Ang-(1 - 7) from Ang I requires the action of three tissue
endopeptidases: prolyl endopeptidase, neutral endopep-
tidase and thimet oligopeptidase [14-16]. Ang-(1 - 7)
may also be synthesized from Ang II by the action of the
ACE2 (Angiotensin converting enzyme 2) [17,18] or
from Angiotensin-(1 - 9) [19]. Ang-(1 - 7) may be hy-
drolyzed by ACE forming Ang-(1 - 5) and Angiotensin-
(1 - 3) [20,21]. The existence of a receptor for Ang-(1 - 7)
is controversial. Most studies have shown that this Ang-
(1 - 7) exerts its physiologic effects probably through acti-
vation of a unique G protein-coupled Ang-(1 - 7) [AT (1 - 7)]
receptor encoded by the mas oncogene (mas1) [22].
The aim of this work was to evaluate the anti-prolif-
erative and pro-apoptotic properties of Ang-(1 - 7), of
Ang (1 - 7)-FMOC and of Ang II-TOAC analogues in
normal (MCF10A) and in tumoral (MCF7) epithelial
mammary cells. These cell lines were also treated with
Human Chorionic Gonadotropin (hCG), a hormone that
elicits life-long refractoriness to carcinogenesis by dif-
ferentiation of the breast epithelium in order to assess
any possible synergistic effect of these compounds.
2. Materials and Methods
2.1. Peptides
Ang II-TOAC, an Ang II analogue containing the TOAC
(2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxy-
lic acid) spin label synthesized by solid phase method-
ology [23] and Ang-(1 - 7)-FMOC, an Ang-(1 - 7) ana-
logue containing the FMOC (9-fluorenylmethyloxycar-
bonyl) group was used for protection of the amine func-
tion [24]. These peptides were supplied by dr. C.R. Na-
kaie (department of Biophysics/UNIFESP-BRAZIL). hCG
was obtained from Ovidrel®.
2.2. Cell Culture and Treatments
MCF7 cells (HTB-22, ATCC) were grown in Dulbecco’s
Modified Eagle Medium (DMEM), 4.5 g/l of glucose,
supplemented with 5% fetal calf serum, 100 U/mL of
penicillin (PAA), and 100 mg/mL of streptomycin (PAA).
MCF10A cells (CRL-10317, ATCC) were cultured in
DMEM/F-12 medium (PAA, Carlsbad, CA) supplemented
with 10 mg/mL of human insulin (Sigma, St. Louis, MO),
20 ng/mL of epidermal growth factor (Sigma, St. Louis,
MO), 0.5 mg/mL of hydrocortisone (Sigma, St. Louis,
MO), 5% horse serum (Invitrogen), 100 U/mL of peni-
cillin (PAA) and 100 mg/mL of streptomycin (PAA). All
the cells lines employed in this work were cultured at
37˚C in a humidified atmosphere and 5% CO2. The fol-
lowing treatments were performed on these cells: control
(1); hCG (2); Ang II (3); Ang-(1 - 7) (4); Ang-(1 -
7)-FMOC (5); Ang II-TOAC (6); hCG + Ang II (7); hCG
+ Ang-(1 - 7) (8); hCG + Ang-(1 - 7)-FMOC (9); hCG +
Ang II-TOAC (10).
2.3. Flow Cytometer (GUAVATM) Assays
MCF7 and MCF10A cells were seeded into 24-well plates
with DMEM supplemented with 10% FBS in the absence
(control) or presence of hCG. The cells were treated with
20 μM of peptide, the most active concentration value
[25,26], and Angiotensin-deriveted peptides [Ang-(1 - 7)-
Fmoc and Ang II-Toac] in the concentration of 10−6 M.
After 24 hours of treatment, it was possible to determine
viability index, and percentage of apoptosis, and cell cycle
by means of biochemical assays in flow cytometer (Guava
EasyCyteTM-Millipore) using kits ViaCount® Guava,
Guava Nexin® and cell cycle assay®, respectively.
2.4. Statistical Analysis
The results for cell proliferation, apoptosis, and cell cycle
assays were analyzed by descriptive statistics (means and
standard deviation) and inferential statistics through the
Student’s t-test with significance level of 5% (p < 0.05).
3. Results
3.1. Cell Viability Assays
MCF-7 cells showed cell viability decrease while the
mid-apoptosis increased after Ang-(1 - 7), Ang-(1 - 7)-
FMOC and hCG + Ang II-TOAC treatments (Figure
1(a)). On the other hand MCF10A cells showed cell vi-
ability decrease, while the mid-apoptosis increased after
Ang-(1 - 7) and hCG + Ang-(1 - 7)-FMOC treatments
(Figure 1(b)).
3.2. Apoptosis Assays
Late apoptosis and dead cells ratio increase in MCF-7
cells after hCG, Ang-(1 - 7), and hCG + Ang-(1 - 7)-
FMOC treatments (Figure 2(a)). Late apoptosis or

Effects on Cell Viability and on Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A) Epithelial Breast Cells
after Human Chorionic Gonadotropin and Derivated-Angiotensin Peptides Treatments
67
Figure 1. Cell viability analysis after peptides, analogues
and hCG treatments in MCF-7 (a) or MCF10A (b) cells.
control 1); hCG 2); Ang II 3); Ang-(1 - 7) 4); Ang-(1 -
7)-FMOC 5); Ang II-TOAC 6); hCG + Ang II 7); hCG +
Ang-(1 - 7) 8); hCG + Ang-(1 - 7)-FMOC 9); hCG + Ang
II-TOAC (10). *p < 0.05 (compared to controls).
Figure 2. Apoptosis analysis after peptides, analogues and
hCG treatments in MCF-7 (a) and in MCF10A (b) cells.
control 1); hCG 2); Ang II 3); Ang-(1 - 7) 4); Ang-(1 -
7)-FMOC 5); Ang II-TOAC 6); hCG + Ang II 7); hCG +
Ang-(1 - 7) 8); hCG + Ang-(1 - 7)-FMOC 9); hCG + Ang II-
TOAC 10). *p < 0.05 (compared to controls).
dead cells increase after hCG treatment while early apop-
tosis in creases in MCF10A cells after hCG + Ang-(1 -
7)-FMOC treatment (Fig ure 2(b)).
3.3. Cell Cycle Assays
MCF7 and MCF10A cells did not evidenced alterations
for G0/G1 or S cell cycle after treatments. Except for
hCG that increases G2/M in MCF-7 (Figure 3(a)) and
Figure 3. Cell cycle analysis after peptides, analogues and
hCG treatments in MCF-7 (a) and in MCF10A (b) cells.
control 1); hCG 2); Ang II 3); Ang-(1 - 7) 4); Ang-(1 - 7)-
FMOC 5); Ang II-TOAC 6); hCG + Ang II 7); hCG + Ang-
(1 - 7) 8); hCG + Ang-(1 - 7)-FMOC 9); hCG + Ang
II-TOAC 10). *p < 0.05 (compared to controls).
for Ang II-TOAC and hCG + Ang-(1 - 7)-FMOC that
increase G2/M in MCF-10A (Figure 3(b)).
4. Discussion
Ang II stimulates proliferation of AT1R-positive breast
cancer cells through PI3-kinase/Akt pathway activation
[27] and Ang-(1 - 7) has a clinical and pre-clinical activity
in vascular sarcomas by reducing HIF-1alpha and PlGF
genes expression [28]. In the present study, cell viability
was decreased and mid-apoptosis was increased in both
tumoral and in normal cells (MCF-7 and MCF10A) after
Ang-(1 - 7) and analogs containing TOAC or FMOC
treatments. Also, we observed that Ang II containing the
TOAC substituent became an anti-proliferative hormone.
Late apoptosis and cell death were increased after hCG,
Ang-(1 - 7), and Ang-(1 - 7)-FMOC trreatments in MCF-7
cells. Early apoptosis was increased after stimulation with
Ang-(1 - 7) and the effects of the peptides containing an
FMOC group were powered by hCG in MCF10A cells.
Furthermore, cell cycle changes were not observed after
peptide treatments, only hCG increased the stationary
phases of the cell cycle (G2/M) in MCF-7 cells. Moreover
in MCF10A cells, only the peptides containing the groups
FMOC and TOAC triggered this same type of change.
Therefore it is possible to say that the TOAC and FMOC
substituent groups potentiate the anti-proliferative or pro-
apoptotic effect in both cell types. Moreover we could
also observe that Ang II becomes anti-proliferative in
these mammary cells when the TOAC group is added to
the hormone molecule and finally that hCG enhances the
effects of the peptides.

Effects on Cell Viability and on Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A) Epithelial Breast Cells
after Human Chorionic Gonadotropin and Derivated-Angiotensin Peptides Treatments
68
In summary, cell viability was decreased and apoptosis
(initial, mid and late) was increased after hCG and/or
Ang-(1 - 7) peptides treatments. These results point out
hCG and Ang-(1 - 7) as effective compounds to inhibit
cell proliferation, since they decrease cell viability and
increase apoptosis in both normal and tumoral breast cells,
being the effect more pronounced in the tumoral cell line.
Our results support the idea of investigating more closely
the putative use of these compounds as novel therapeutic
agents for breast cancer.
5. Acknowledgements
The work was supported by Grants number 2007 /56480-
0, 2008/54383-0 and 2011/10516-0 from the Sao Paulo
Research Foundation (FAPESP)-Brazil.
REFERENCES
[1] “What Are the Key Statistics about Breast Cancer?” 2013.
http://www.cancer.org/cancer/breastcancer/detailedguide/
breast-cancer-key-statistics
[2] P. M. Frossard, M. J. Malloy, G. G. Lestringant and J. P.
Kane, “Haplotypes of the Human Renin Gene Associated
with Essential Hypertension and Stroke,” Journal of Hu-
man Hypertension, Vol. 15, No. 1, 2001, pp. 49-55.
doi:10.1038/sj.jhh.1001107
[3] M. Fujita, I. Hayashi, S. Yamashina, M. Itoman and M.
Majima, “Blockade of Angiotensin AT1a Receptor Sig-
naling Reduces Tumor Growth, Angiogenesis, and Meta-
stasis,” Biochemical and Biophysical Research Commu-
nications, Vol. 294, No. 2, 2002, pp. 441-447.
doi:10.1016/S0006-291X(02)00496-5
[4] S. Greco, A. Muscella, M. G. Elia, et al., “Angiotensin II
Activates Extracellular Signal Regulated Kinases via Pro-
tein Kinase C and Epidermal Growth Factor Receptor in
Breast Cancer Cells,” Journal of Cellular Physiology,
Vol. 196, No. 2, 2003, pp. 370-377.
doi:10.1002/jcp.10313
[5] F. Deshayes and C. Nahmias, “Angiotensin Receptors: A
New Role in Cancer?” Trends in Endocrinology & Me-
tabolism, Vol. 16, No. 7, 2005, pp. 293-299.
doi:10.1016/j.tem.2005.07.009
[6] E. I. Ager, J. Neo and C. Christophi, “The Rennin-Angio-
Tensin System and Malignancy,” Carcinogenesis, Vol. 29,
No. 9, 2008, pp. 1675-1684. doi:10.1093/carcin/bgn171
[7] F. Deshayes and C. Nahmias, “Angiotensin Receptors: A
New Role in Cancer?” Trends in Endocrinology & Meta-
bolism, Vol. 16, No. 7, 2005, pp. 293-299.
doi:10.1016/j.tem.2005.07.009
[8] R. A. Santos, M. J. Campagnole-Santos and S. P. Andrade,
“Angiotensin-(1 - 7): An Update,” Regulatory Peptides,
Vol. 91, No. 1-3, 2000, pp. 45-62.
doi:10.1016/S0167-0115(00)00138-5
[9] I. F. Benter, D. I. Diz and C. M. Ferrari, “Pressor and
Reflex Sensitivity Is Altered in Spontaneously Hyperten-
sive Rats Treated with Angiotensin-(1 - 7),” Hypertension,
Vol. 26, No. 6, 1995, pp. 1138-1144.
doi:10.1161/01.HYP.26.6.1138
[10] W. B. Strawn, C. M. Ferrario and E. A. Tallant, “Angio-
tensin (1 - 7) Reduces Smooth Muscle Growth after Vas-
cular Injury,” Hypertension, Vol. 33, No. 1, 1999, pp.
207-211. doi:10.1161/01.HYP.33.1.207
[11] E. J. Freeman, G. M. Chisolm, C. M. Ferrario and E. A.
Tallant, “Angiotensin-(1 - 7) Inhibits Vascular Smooth Mus-
cle Cell Growth,” Hypertension, Vol. 28, No. 1, 1996, pp.
104-108. doi:10.1161/01.HYP.28.1.104
[12] E. A. Tallant, C. M. Ferrario and P. E. Gallagher, “An-
giotensin-(1 - 7) Inhibits Growth of Cardiac Myocytes
through Activation of the Mas Receptor,” American Jour-
nal of Physiology—Heart and Circulatory Physiology,
Vol. 289, No. 4, 2005, pp. H1560-H1566.
doi:10.1152/ajpheart.00941.2004
[13] R. D. Machado, R. A. Santos and S. P. Andrade, “Op-
posing Actions of Angiotensins on Angiogenesis,” Life
Sciences, Vol. 66, No. 1, 2000, pp. 67-76.
[14] R. A. Santos, K. B. Brosnihan, D. W. Jacobsen, P. E.
DiCorletto and C. M. Ferrario, “Production of Angio-
tensin-(1 - 7) by Human Vascular Endothelium,” Hyper-
tension, Vol. 19, No. 2, 1992, pp. 56-61.
[15] K. Yamamoto, M. C. Chappell, K. B. Broshinan and C.
M. Ferrario, “In Vivo Metabolism of Angiotensin I by
Neutral Endopeptidase (EC 3.4.24.11) in Spontaneously
Hypertensive Rats,” Hypertension, Vol. 19, No. 6, 1992,
pp. 692-696. doi:10.1161/01.HYP.19.6.692
[16] M. C. Chappell, E. A. Tallant, K. B. Brosnihan and C. M.
Ferrario, “Conversion of Angiotensin I to Angiotensin-(1 - 7)
by Thimet Oligopeptidase (E.C.3.4.24.15) in Vascular
Smooth Muscle Cells,” Journal of Vascular Medicine and
Biology, Vol. 5, No. 4, 1994, pp. 129-137.
[17] S. R. Tipnis, N. M. Hooper, R. Hyde, E. Karran, G. Christie
and A. J. Turner, “A Human Homolog of Angiotensin-
Converting Enzyme. Cloning and Functional Expression
as a Captopril-Insensitive Carboxypeptidase,” The Jour-
nal of Biological Chemistry, Vol. 275, No. 43, 2000, pp.
33238-33243. doi:10.1074/jbc.M002615200
[18] M. A. Crackower, R. Sarao, G. Y. Oudit, C. Yagil, I.
Kozieradzki, S. E. Scanga, et al., “Angiotensin-Convert-
ing Enzyme 2 Is an Essential Regulator of Heart Func-
tion,” Nature, Vol. 417, No. 6891, 2002, pp. 822-828.
doi:10.1038/nature00786
[19] M. Donoghue, F. Hsieh, E. Baronas, K. Godbout, M.
Gosselin, N. Stagliano, et al., “A Novel Angiotensin-Con-
verting Enzyme-Related Carboxypeptidase (ACE2) Con-
verts Angiotensin I to Angiotensin 1-9,” Circulation Re-
search, Vol. 87, No. 5, 2000, pp. E1-E9.
doi:10.1161/01.RES.87.5.e1
[20] M. C. Chappell, N. T. Pirro, A. Sykes and C. M. Ferrario,
“Metabolism of Angiotensin-(1 - 7) by Angiotensin-Con-
verting Enzyme,” Hypertension, Vol. 31, No. 1, 1998, pp.
362-367. doi:10.1161/01.HYP.31.1.362
[21] K. Yamada, S. N. Iyer, M. C. Chappell, D. Ganten and C.
M. Ferrario, “Converting Enzyme Determines the Plasma
Clearance of Angiotensin-(1 - 7),” Hypertension, Vol. 32,
No. 3, 1998, pp. 496-502. doi:10.1161/01.HYP.32.3.496

Effects on Cell Viability and on Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A) Epithelial Breast Cells
after Human Chorionic Gonadotropin and Derivated-Angiotensin Peptides Treatments
69
[22] R. A. Santos, A. C. Simoes e Silva, C. Maric, et al., “An-
giotensin-(1 - 7) Is an Endogenous Ligand for the G Pro-
tein-Coupled Receptor Mas,” Proceedings of the National
Academy of Sciences of the United States of America, Vol.
100, No. 14, 2003, pp. 8258-8263.
doi:10.1073/pnas.1432869100
[23] C. R. Nakaie, E. G. Silva, E. M. Cilli, R. Marchetto, S.
Schreier, T. B. Paiva and A. C. Paiva, “Synthesis and
Pharmacological Properties of TOAC-Labeled Angioten-
sin and Bradykinin Analogs,” Peptides, Vol. 23, No. 1,
2002, pp. 65-70.
[24] G. N. Jubilut, E. M. Cilli, E. Crusca, E. H. Silva, Y.
Okada and C. R. Nakaie, “Comparative Investigation of
the Cleavage Step in the Synthesis of Model Peptide
Resins: Implications for Nalpha-9-Fluorenylmethyloxy-
carbonyl-Solid Phase Peptide Synthesis,” Chemical &
Pharmaceutical Bulletin (Tokyo), Vol. 55, No. 3, 2007,
pp. 468-470.
[25] D. E. Morbeck, P. C. Roche, H. T. Keutmann and D. J.
McCormick, “A Receptor Binding Site Identified in the
Region 81-95 of the Beta-Subunit of Human Luteinizing
Hormone (LH) and Chorionic Gonadotropin (hCG),”
Molecular and Cellular Endocrinology, Vol. 97, No. 1-2,
1993, pp. 173-181.
[26] S. M. R. Noronha, S. A. A. Correa-Noronha, I. H. Russo,
R. L. di Cicco, J. Santucci-Pereira and J. Russo, “Human
Chorionic Gonadotropin and a 15 Amino Acid hCG
Fragment of the Hormone Induce Downregulation of the
Cytokine IL-8 Receptor in Normal Breast Epithelial
Cells,” Hormone Molecular Biology and Clinical Inves-
tigation, Vol. 6, No. 3, 2011, pp. 241-245.
[27] Y. Zhao, X. Chen, L. Cai, Y. Yang, G. Sui and S. Fu,
“Angiotensin II/angiotensin II Type I Receptor (AT1R)
Signaling Promotes MCF-7 Breast Cancer Cells Survival
via PI3-Kinase/Akt Pathway,” Journal of Cellular Phy-
siology, Vol. 225, No. 1, 2010, pp. 168-173.
[28] W. J. Petty, M. Aklilu, V. A. Varela, J. Lovato, P. D. Sa-
vage and A. A. Miller, “Reverse Translation of Phase I
Biomarker Findings Links the Activity of Angiotensin-(1 - 7)
to Repression of Hypoxia Inducible Factor-1Alpha in
Vascular Sarcomas,” BMC Cancer, Vol. 12, No. 1, 2012,
p. 404.