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

doi:10.4236/jct.2013.47A010

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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)

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.

Email: *[email protected], wericabernar[email protected], [email protected], sshimut[email protected], [email protected],

[email protected].

Received May 20th, 2013; revised June 22nd, 2013; accepted June 30th, 2013

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

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

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

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.

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