000859983

Transcription

000859983

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Gabriela Bottaro Gelaleti
MODULAÇÃO DA ATIVIDADE DAS INTERLEUCINAS 17B E 25
ASSOCIADAS À MELATONINA COMO INDUTORES DE
APOPTOSE EM CULTIVO CELULAR DE NEOPLASIAS
MAMÁRIAS
São José do Rio Preto
2015
u
MAMÁRIAS
Tese apresentada como parte dos
requisitos para obtenção do título de Doutor
em Genética, junto ao Programa de PósGraduação em Genética, do Instituto de
Biociências, Letras e Ciências Exatas da
Universidade Estadual Paulista “Júlio de
Mesquita Filho”, Campus de São José do
Rio Preto.
Orientador: Profª. Drª. Debora Ap. Pires de
Campos Zuccari
Co-orientador: Dr. Alicia Viloria-Petit
2015
Gelaleti, Gabriela Bottaro.
Modulação da atividade das interleucinas 17B e 25 associadas à
melatonina como indutores de apoptose em cultivo celular de neoplasias
mamárias / Gabriela Bottaro Gelaleti. -- São José do Rio Preto, 2015
137 f. : il., tabs.
Orientador: Debora Aparecida Pires de Campos Zuccari
Coorientador: Alicia Viloria-Petit
Tese (doutorado) – Universidade Estadual Paulista “Júlio de
Mesquita Filho”, Instituto de Biociências, Letras e Ciências Exatas
1. Genética. 2. Câncer – Aspectos genéticos. 3. Mamas - Câncer.
4. Interleucina-17. 5. Melatonina. 6. Apoptose. 7. Neovascularização.
I. Zuccari, Debora Aparecida Pires de Campos. II. Viloria-Petit, Alicia.
III. Universidade Estadual Paulista "Júlio de Mesquita Filho". Instituto de
Biociências, Letras e Ciências Exatas. IV. Título.
CDU – 616-006.6
Ficha catalográfica elaborada pela Biblioteca do IBILCE
UNESP - Campus de São José do Rio Preto
MAMÁRIAS
Tese apresentada como parte dos
requisitos para obtenção do título de Doutor
em Genética, junto ao Programa de PósGraduação em Genética, do Instituto de
Biociências, Letras e Ciências Exatas da
Universidade Estadual Paulista “Júlio de
Mesquita Filho”, Campus de São José do
Rio Preto.
Comissão Examinadora
Profª. Drª. Debora Ap. Pires de Campos Zuccari
UNESP – São José do Rio Preto
Orientador
Profª. Drª. Ana Elizabete Silva
UNESP – São José do Rio Preto
Profª. Drª. Dorotéia Rossi Silva Souza
FAMERP – São José do Rio Preto
Prof. Dr. Alexandre Lima de Andrade
UNESP – Araçatuba
Profª. Drª. Ana Paula Girol
UNESP – São José do Rio Preto / FAMECA - Catanduva
05 de agosto de 2015
Esse trabalho foi desenvolvido no Laboratório de Investigação Molecular do Câncer
(LIMC) na Faculdade de Medicina de São José do Rio Preto (FAMERP), SP, Brasil,
sob orientação da Profa. Dra. Debora Ap. Pires de Campos Zuccari e no Laboratory for
Integrated Study of the Mechanisms of Breast Cancer Invasion and Metastasis –
University of Guelph, Guelph, Ontario, Canadá, com a co-orientação da Dr. Alicia
Viloria-Petit. Os recursos para o desenvolvimento do projeto foram obtidos na forma de
Auxílio à Pesquisa (Proc. 2012/06098-0) e bolsa de estudos (Proc. 2012/02128-1/Proc.
2012/25191-0) da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
Dedicatória
Dedico esse trabalho aos meus pais Jorge e Mirtes e ao meu irmão Rafael, os
maiores incentivadores da minha vida. Agradeço pelo apoio, amor incondicional e
constante torcida em cada vitória minha.
Dedico também ao meu noivo Andre, por todo o carinho, incentivo e apoio.
Agradecimentos
Agradeço a minha orientadora, Profa. Dra. Debora Zuccari, pela amizade, confiança e todo
apoio durante a realização desse trabalho. Agradeço pelos ensinamentos transmitidos e
principalmente por ter confiado que eu seria capaz, estando ao meu lado nos momentos de
dificuldade e superação.
Aos grandes amigos do LIMC: Thaíz F. Borin, Bruna V. Jardim-Perassi, Marina G. Moschetta,
Lívia C. Ferreira, Camila Leonel, Larissa B. Maschio, Gustavo R. Martins, Naiane N.
Gonçalves, Giovanna R. Varallo, Juliana R. Lopes, Jucimara Colombo, Tialfi Castro, Nathália
Sonehara, Rubens P. Junior, Jéssica Z. Lacerda, Alexandra P. Fantinatti e André L. Mota, pela
amizade e companheirismo todos os dias. Agradeço por muitos se tornarem amigos para a vida
toda e por estarem sempre presentes nos momentos profissionais e pessoais durante todos esses
anos. Meu carinho por vocês é eterno.
A Dr. Alicia M. Viloria-Petit, minha co-orientadora, por permitir a realização do estágio no
exterior na University of Guelph no Laboratory for Integrated Study of the Mechanisms of
Breast Cancer Invasion and Metastasis. Agradeço pelo auxílio no desenvolvimento do trabalho e
também pela amizade e a de todos os integrantes do grupo de pesquisa, permitindo meu
crescimento pessoal e profissional. Agradeço também a Camila Leonel que compartilhou comigo
essa experiência maravilhosa.
A banca examinadora do exame geral de qualificação, Profa. Dra. Ana Paula Girol e Profa. Dra.
Patrícia S. L. Vilamaior pelas sugestões que permitiram melhorar meu trabalho.
Aos Professores: Profa. Dra. Ana Elizabete Silva, Profa. Dra. Dorotéia R. S. Souza, Profa. Dra.
Ana Paula Girol e Prof. Alexandre Lima de Andrade por aceitarem fazer parte da banca de
defesa.
Aos professores, alunos e funcionários da Pós-Graduação em Genética do Ibilce que contribuíram
com meu aprendizado. Agradeço especialmente a Profa. Patricia S. L. Vilamaior e ao Prof.
Eduardo A. Almeida por me aceitarem no estágio de docência, complementando minha formação.
A FAPESP, pela concessão da bolsa de estudos no país (Proc. 2012/02128-1) e exterior (Proc.
2012/25191-0) e Auxílio Financeiro concedido (Proc. 2012/06098-0), indispensáveis para a
realização desse projeto.
A UNESP/IBILCE e a FAMERP pela infra-estrutura e profissionais que possibilitaram a
realização do projeto.
Aos meus pais Jorge Luiz Gelaleti e Mirtes Ap. Bottaro Gelaleti. Obrigada pela dedicação, pelo
exemplo de vida e companheirismo e por sempre acreditarem em minhas escolhas e que essas me
fariam uma pessoa melhor.
Ao meu irmão, Rafael Bottaro Gelaleti, pela demonstração de companheirismo e amor a vida
toda. Obrigada pelos conselhos, por compartilhar do mesmo ideal, pelas conversas de “Biólogos” e
por fazer parte dos momentos felizes da minha vida.
Ao meu noivo, Andre Murari Boatto, por todo amor e companheirismo nessa fase da minha vida
e em todos os anos que estamos juntos. Agradeço pela compreensão, por acreditar em mim sempre
e por me mostrar que a felicidade só aumenta quando compartilhamos nossos sonhos com quem
amamos.
A minha avó Hermínia Bottaro, pelo amor e exemplo de força, e também a minha avó Dornélia
Inês Gelaleti e meus avôs Hilário Bottaro e Jorge João Gelaleti, pelos ensinamentos que levo
comigo para toda a vida, com a certeza de que sempre olham por mim.
Aos meus tios, tias, primos e primas. Obrigada por estarem sempre presentes na minha vida e
desejarem o meu sucesso.
Aos meus sogros Nilo Boatto e Márcia Murari Boatto e toda a família Murari pelo carinho e
dedicação, sempre torcendo por minha vitória.
Aos amigos da faculdade, pela amizade de tantos anos. Em especial a Ana Beatriz M. Tenália e
Ariane Zanchetta minhas “BFFs”, obrigada pelo carinho e dedicação sempre. A Tamirys
Manzatti, Adauto O. Borguetti e Danilo G. da Silva, amigos queridos.
As amigas, Ana Carolina S. Camargo e Natalie P. Cruz pela amizade da vida toda, que a cada
ano se renova, agora com a chegada da Theodora e Victoria, lindas da “titia”. E agradeço também
a Ingrid Dutra, amiga querida.
As amigas de república Ariane Zanchetta, Rafaela R. Brito e Gisele M. Martins pela amizade.
A Deus, pela proteção, conduta aos caminhos certos e por me permitir conviver com pessoas tão
maravilhosas.
Muito obrigada!!!
"Não é o que você faz, mas quanto amor você dedica no que faz que realmente
importa."
Agnes Gonxha Bojaxhiu (Madre Teresa de Calcutá)
“Agradeço todas as dificuldades que enfrentei, não fosse por elas, eu não teria saído do
lugar. As facilidades nos impedem de caminhar. Mas as críticas nos auxiliam muito.”
Chico Xavier
Sumário
SUMÁRIO
LISTA DE FIGURAS............................................................................................ 14
LISTA DE TABELAS........................................................................................... 17
LISTA DE ABREVIATURAS E SÍMBOLOS ..................................................... 19
RESUMO............................................................................................................... 23
ABSTRACT .......................................................................................................... 26
I.
INTRODUÇÃO............................................................................................ 29
1.
CÂNCER DE MAMA ................................................................................. 29
1.1 Aspectos gerais ................................................................................................ 29
1.2 Microambiente tumoral ................................................................................... 31
2. PROCESSOS ATUANTES NA TUMORIGÊNESE .................................... 34
2.1 ANGIOGÊNESE ............................................................................................. 34
2.2 APOPTOSE ..................................................................................................... 38
2.2.1 Via extrínseca ............................................................................................... 39
2.2.2 Via intrínseca ................................................................................................ 40
3. NOVOS ALVOS TERAPÊUTICOS NO CÂNCER DE MAMA ................ 42
3.1 INTERLEUCINA-25 ..................................................................................... 42
3.2 MELATONINA ............................................................................................. 45
3.2.1 Síntese e degradação ..................................................................................... 45
3.2.2 Receptores da melatonina ............................................................................. 47
3.2.3 Melatonina e câncer ...................................................................................... 48
II.
OBJETIVOS ................................................................................................ 52
III. CAPÍTULOS ................................................................................................ 54
Artigo I ................................................................................................................. 56
Artigo II ................................................................................................................ 94
IV. CONCLUSÕES .......................................................................................... 123
V.
REFERÊNCIAS......................................................................................... 125
VI. ANEXOS..................................................................................................... 136
13
Lista de figuras
14
Lista de figuras
Figura 1. Taxas de mortalidade das cinco localizações primárias de neoplasias mais
frequentes em 2012, ajustadas por idade, pela população mundial por 100.000 mulheres,
Brasil, entre 1979 e 2012. .............................................................................................. 29
Figura 2. Capacidades biológicas adquiridas durante o desenvolvimento de múltiplos
passos dos tumores. ....................................................................................................... 32
Figura 3. Representação do tecido inflamatório e do microambiente tumoral invasivo.
........................................................................................................................................ 33
Figura 4. Representação da ativação da angiogênese a partir de células quiescentes.
........................................................................................................................................ 35
Figura 5. Representação das etapas do processo de angiogênese com a participação de
inúmeros fatores pró-angiogênicos. ............................................................................... 37
Figura 6. Representação das vias extrínseca e intrínseca da apoptose. ........................ 40
Figura 7. Via extrínseca e intrínseca da ativação da caspase-3. ................................... 42
Figura 8. Esquema representativo da família IL-17 e seus receptores. ........................ 43
Figura 9. Diagrama esquemático da atividade citotóxica da IL-25 em células do câncer
de mama que expressam IL-25R. .................................................................................. 44
Figura 10. Esquema da sinalização IL-17RB/IL-17B em células do câncer de mama. 45
Figura 11. Esquema representativo da via de síntese da melatonina. ........................... 46
Figura 12. Esquema da via extrínseca e intrínseca da apoptose. .................................. 49
Figura 13. Ação da melatonina na inibição da proliferação de células tumorais. ........ 50
Artigo 1
Fig. 1. Effect of melatonin and interleukin-25 on viability of cell lines. …….……….. 28
Fig. 2. Gene silencing of IL-17B. .................................................................................. 29
Fig. 3. Cleaved caspase-3 expression in MDA-MB-231. …………………………….. 30
Fig. 4. Cleaved caspase-3 expression in MCF-7. …………………..…...…………… 31
15
Fig. 5. Cleaved caspase-3 expression in MDA-MB-231 and MCF-7 3D structures.
……………………………………………………………………………………….... 32
Fig. 6. Differentially apoptotic protein expression in MDA-MB-231 cells treated with 1
mM of melatonin. ………………………………………………..…………………… 33
Fig. 7. VEGF-A expression in MDA-MB-231. ............................................................. 34
Fig. 8. VEGF-A expression in MCF-7. ……………………………………...……….. 35
Artigo 2
Fig. 1. Effect of melatonin on viability of canine mammary tumor cell lines. ……... 115
Fig. 2. Cleaved caspase-3 expression in CF-41 cells. ……………….………….…. 116
Fig. 3. Cleaved caspase-3 expression in CMT-U229 cells. ……………...…………. 117
Fig. 4. Cleaved caspase-3 expression in CF-41 and CMT-U229 3D structures. ...… 118
Fig. 5. Differentially apoptotic protein expression in CF-41 cells treated with 1 mM of
melatonin. …………………………………………………………………………… 119
Fig. 6. VEGF-A expression in CF-41 cells. ……………………………………….... 120
Fig. 7. VEGF-A expression in CMT-U229 cells. …………………………………… 121
16
Lista de tabelas
17
Lista de tabelas
Artigo 1
Table 1. Human Apoptosis Array C1 (RayBiotech) contening 43 different factors for
analysis of protein expression profile of MDA-MB-231 cells after melatonin treatment.
……………………………………………………………………………………..….. 26
Artigo 2
Table 1. Apoptosis Array C1 (RayBiotech) contening 43 different factors for analysis of
protein expression profile of CF-41 cells after melatonin treatment. …………......... 114
18
Lista de abreviaturas e símbolos
19
Lista de abreviaturas e símbolos
µL
3D
5-HTP
AA-NAT
ACTB
AIF
ANOVA
APAF-1
APO-1
ASK
BAD
BAR
BCA
Bcl-2
Bax
BCLW
BID
bFGF
BIM
BIRC
cAMP
CARD
Caspase
cDNA
CFI
cm2
CO2
CYTO-C
DAPI
DD
DED
DISC
DMEM
DMSO
DR
EGF
ELISA
FADD
FAPERP
FAPESP
FAS
FBS
FoxO3A
microlitros
tridimensional
triptofano em 5-hidroxitriptofano
N-acetiltransferase
gene beta actina
fator de indução de apoptose
análise de variância
do inglês apoptotic protease activating factor 1
do inglês accumulation of photosystem one 1
do inglês apoptosis signal-regulating kinase 1
do inglês Bcl-2-antagonist of cell death
do inglês bifunctional apoptosis regulator
do inglês bicinchoninic acid
do inglês B-cell lymphoma 2
do inglês Bcl-2-associated X protein
do inglês Bcl-2-like protein 2
do inglês BH3 interacting-domain death agonist
do inglês basic fibroblast growth factor
do inglês bisindolylmaleimide-based protein kinase C (PKC) inhibitors
do inglês baculoviral IAP repeat-containing protein 3
do inglês adenosine 3′,5′-cyclic monophosphate
do inglês caspase activation and recruitment domains
do inglês cysteine-aspartic proteases or cysteine-dependent aspartatedirected proteases
DNA complementar
do inglês Canadian Foundation for Innovation
centímetros quadrados
fórmula química do gás carbônico
do inglês cytochrome c
do inglês 4',6-diamidino-2-phenylindole
domínio de morte
domínio efetor de morte
complexo de sinalização de morte
do inglês Dulbecco’s modified Eagle’s medium
do inglês dimethylsulfoxide
do inglês death receptor
do inglês endothelial growth factor
do inglês Enzyme Linked Immunosorbent Assay
do inglês fas-associated protein with death domain
Fundação de Amparo à Pesquisa de São José do Rio Preto
Fundação de Amparo à Pesquisa do Estado de São Paulo
do inglês first apoptosis
soro fetal bovino, do inglês fetal bovine serum
do inglês Forkhead box O3
20
GAPDH
GPCR
HIF-1α
HER2
HIOMT
HRP
HSP
hTRA
HuMEC
IAPs
IARC
IGF-1
IGFBP
IL
INCA
iNOS
JNK
kDa
LIMC
M.O.D.
MAPK
MEC
mg
mL
mM
MMPs
MPT
MRI
mRNA
MTNR1A/MT1
MT2
MTT
Na3 VO4
NAS
NF-kB
ng
NRP2
ºC
P21
P53
PARP
PBS
PDGF
PIGF
PKA
do inglês glyceraldehyde-3-phosphate dehydrogenase
do inglês G-protein coupled receptor
do inglês hypoxia-inducible factor 1-alpha
receptor do fator de crescimento epidermal 2
hidroxi- indol-Ometiltransferase
do inglês horseradish peroxidase
do inglês heat shock protein
do inglês high-temperature requirement A serine peptidase
do inglês Basal Human Mammary Epithelial Cell
inibidores de proteínas apoptóticas
do inglês International Agency for Research on Cancer
do inglês insulin-like growth factor 1
do inglês growth factor insulin-binding protein
interleucina
Instituto Nacional do Câncer
espécies reativas de oxigênio
do inglês c-Jun N-terminal kinase
kilodaltons
Laboratório de Investigação Molecular no Câncer
densidade óptica média
gene da família MAPK quinase
matriz extracelular
miligrama
mililitro
milimolar
matrix metaloproteinases
transição de permeabilidade mitocondrial
do inglês Ministry of Research Infrastructure
RNA mensageiro
do inglês melatonin receptor-1
do inglês melatonin receptor-2
do inglês 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
assay
do inglês phosphatase inhibitor cocktail
N-acetilserotonina
do inglês nuclear factor-κB
nanograma
do inglês neuropilin 2
graus celsius
do inglês cyclin-dependent kinase inhibitor 1
do inglês tumor protein p53
do inglês poly (ADP-ribose) polymerase
tampão fosfato salino
do inglês platelet-derived growth factor
fator de crescimento placentário
do inglês protein kinase A
bromide
21
PKC
PLAU
qPCR
RE
RNA
ROR
ROS
RP
RPS19
RPS5
RZR
RZR/ROR
SMAC
siRNA
TGF-b
Th2
Tie-1
Tie-2
TNF
TNFRII
TNFR-II
TNM
TPH1
TRADD
TRAIL
TRAF
u.a.
VEGF
VEGFR
XIAP
do inglês protein kinase C
do inglês urokinase-plasminogen activator
do inglês quantitative polimerase chain reaction
receptor de estrogênio
ácido ribonucléico
do inglês receptor orphan receptor
do inglês reactive oxygen species
receptor de progesterona
gene ribosomal protein-19
gene ribosomal protein-5
receptor Z para retinóide
receptor Z para retinóide/receptor órfão para retinóide
do inglês second mitochondria-derived activator of caspases
do inglês silence RNA
do inglês transforming growth factor beta
do inglês t helper-2
do inglês receptor tyrosine kinase of angiopoietin-1, tyrosine kinase with
immunoglobulin-like and EGF-like domains 1
do inglês receptor tyrosine kinase of angiopoietin-1
do inglês tumor necrosis factor
do inglês tumor necrosis factor receptor II
do inglês Tumor necrosis factor receptor 2
sistema de estadiamento clínico, T= tumor, N=linfonodo (do inglês node),
M= metástase
triptofano hidroxilase 1
do inglês TNFRSF1A-associated via death domain
do inglês TNF-related apoptosis-inducing ligand
do inglês TNF receptor-associated factor
unidades arbitrárias
do inglês vascular endothelial growth factor
do inglês vascular endothelial growth factor receptor
do inglês X-linked inhibitor of apoptosis protein
22
Resumo
23
RESUMO
O desenvolvimento da carcinogênese mamária envolve a neovascularização,
desregulação da diferenciação e apoptose nas células neoplásicas e no
microambiente que estão inseridas. Está estabelecido que tais efeitos podem
ser controlados pela melatonina, um hormônio produzido pela glândula pineal,
que tem ação oncostática em células tumorais e modula a expressão de
interleucinas (ILs). A IL-25, também conhecida como IL-17E, é uma citocina
ativa em processos inflamatórios, capaz de induzir a apoptose em células
tumorais devido à expressão diferencial do seu receptor (IL-17RB). Sabe-se
que a IL-17B compete com a IL-25 pela ligação ao receptor IL-17RB em células
tumorais, promovendo a tumorigênese. O objetivo deste estudo foi verificar a
ação da melatonina e da sinalização IL-25/IL-17B no processo de apoptose e
angiogênese em células tumorais mamárias. Linhagens tumorais mamárias
humanas, metastática (MDA-MB-231) e não-metastática (MCF-7) e caninas,
metastática (CF-41) e não-metastática (CMT-U229), e a linhagem epitelial
mamária
humana
não
tumorigênica
(MCF-10A)
foram
cultivadas
em
monocamada e em estruturas tridimensionais em matriz extracelular ricas em
laminina e tratadas por 48 horas com melatonina, IL-25, siIL-17B e os três
tratamentos combinados. A viabilidade celular foi mensurada pelo ensaio de
MTT, a expressão gênica e proteica por qRT-PCR e imunofluorescência,
respectivamente. Além disso, uma análise quantitativa de proteínas presentes
na via apoptótica foi realizada após o tratamento com melatonina nas células
MDA-MB-231 e CF-41. Os tratamentos com 1 mM de melatonina e 1 ng/mL de
IL-25 reduziram significativamente a viabilidade das linhagens tumorais
humanas (p < 0,05) e não alteraram a viabilidade celular da linhagem MCF-10A
(p > 0,05). Nas linhagens caninas, apenas 1 mM de melatonina reduziu
significativamente a viabilidade celular (p < 0,05). Todos os três tratamentos
independentes e combinados foram capazes de aumentar significativamente a
24
expressão proteica da caspase clivada-3 em todas as linhagens cultivadas em
monocamada e em modelo 3D (p < 0,05), o que confirma o potencial próapoptótico dos tratamentos nas linhagens tumorais mamárias. A análise semiquantitativa das proteínas envolvidas na via apoptótica, mostrou aumento das
proteínas pró-apoptóticas CITO-C, DR6, IGFBP-3, IGFBP-5, IGFPB-6, IGF-1,
IGF-1R, LIVINA, P21, P53, TNFRII, XIAP e hTRA (p <0,05) nas células MDAMB-231 e redução da caspase-3 (p = 0,04). Para as células CF-41, o
tratamento com melatonina foi capaz de aumentar a expressão das proteínas
BCLW e IGF-1 e -2 (p < 0,05) e reduzir as proteínas BAD, IGFBP1 e TNFRII (p
< 0,05). Todos os tratamentos reduziram a expressão da proteína VEGF-A nas
linhagens tumorais mamárias humanas e caninas (p < 0,05). Nossos achados
sugerem o potencial terapêutico, com eficácia oncostática, propriedades próapoptóticas e anti-angiogênicas da melatonina e da sinalização IL-25/siIL-17B
no câncer de mama.
Palavras-chave: câncer de mama, melatonina, interleucina-25, interleucina17E, interleucina-17B, apoptose, VEGF-A, angiogênese.
25
Abstract
26
ABSTRACT
The development of mammary carcinogenesis involves neovascularization,
deregulation
of differentiation and apoptosis in tumor cells and the
microenvironment. It is established that such effects may be controlled by
melatonin, a hormone produced by the pineal gland that has oncostatic action
on tumor cells and modulates interleukins (ILs) expression. IL-25, also known
as IL-17E, is an active cytokine in inflammatory processes, capable of inducing
apoptosis in tumor cells because of differential expression of its receptor (IL17RB). It is known that IL-17B competes with IL-25 for binding to receptor IL17RB on tumor cells, promoting tumorigenesis. The aim of this study was to
investigate the action of melatonin and IL-25/IL-17B signaling in apoptosis and
angiogenesis in breast tumor cells. Metastatic (MDA-MB-231 and CF-41), nonmetastatic (MCF-7 and CMT-U229) human and canine mammary tumor cell
lines and human non-tumorigenic mammary epithelial cells (MCF-10A) were
cultured in monolayer and three-dimensional structures on extracellular matrix
rich in laminin (ECM) and treated for 48 hours with melatonin, IL-25, siIL-17B
and the three combination therapies. Cell viability was measured by MTT assay,
gene
and
protein
expression by qRT-PCR and immunofluorescence,
respectively. Also, a quantitative analysis of proteins present in the apoptotic
pathway was performed after treatment with melatonin in the MDA-MB-231 and
CF-41 cells. The treatment with 1 mM melatonin and 1 ng/mL of IL-25
significantly reduced the viability of human tumor cell lines (p < 0.05) and did
not alter the cell viability of MCF-10A cells (p > 0.05). In canine cell lines, only 1
mM melatonin significantly reduced cell viability (p < 0.05). All three
independent and combined treatments were able to significantly increase the
protein expression of cleaved caspase-3 in all the tumor cells in monolayer and
3D structures (p < 0.05), which confirms the pro-apoptotic potential of the
treatments in mammary tumor cell lines. Likewise, all treatments reduced the
27
expression of VEGF-A protein in human and canine mammary tumor cell lines
(p < 0.05). The semi-quantitative analysis of proteins involved in the apoptotic
pathway showed an increase of pro-apoptotic proteins DR6, IGFBP-3, IGFBP-5,
IGFPB-6, IGF-1sR, LIVIN, P21, P53, TNFRI, xIAP and hTRA in MDA-MB-231
(p < 0.05) and reduction of caspase-3 cells (p = 0.04). For the CF-41 cells,
treatment with melatonin was able to increase expression of proteins BCLW
and IGF-1 and -2 (p < 0.05) and reduce proteins BAD, IGFBP1 and TNFRII (p <
0.05). Our findings suggest the oncostatic efficacy, pro-apoptotic and antiangiogenic properties of melatonin and IL-25/IL-17B signaling as therapeutic
potential in breast cancer.
Keywords: Breast cancer, melatonin, interleukin-25, interleukin-17E, interleukin17B, apoptosis, VEGF-A, angiogenesis.
28
Introdução
29
I. INTRODUÇÃO
1. CÂNCER DE MAMA
1.1 Aspectos gerais
Dados do IARC (Agência Internacional de Pesquisa no Câncer; Julho, 2015)
estimam 14 milhões de novos casos de câncer no mundo para este ano, com previsão de
8.2 milhões de óbito (IARC). O câncer de mama representa a maior causa de morte por
câncer em mulheres e, no Brasil, segundo dados do INCA (Instituto Nacional do
Câncer; Julho 2015), estimou-se a ocorrência de 57.120 novos casos para o ano de 2014
e a mesma freqüência é esperada para o ano de 2015 (INCA).
As taxas de mortalidade por câncer de mama continuam elevadas, sendo a
capacidade de invasão das células tumorais e consequente desenvolvimento de
metástases, as principais causas de mortalidade em mulheres portadoras desta neoplasia
(Santos et al., 2010). Na população mundial, a sobrevida média das pacientes após
cinco anos de acompanhamento é de 61 % e, apesar do progresso no diagnóstico e
tratamento nos últimos 30 anos, essa doença é, ainda, responsável por quase meio
milhão de mortes por ano no mundo (Snoussi et al., 2010) (Figura 1).
Figura 1. Taxas de mortalidade das cinco localizações primárias de neoplasias mais
frequentes em 2012, ajustadas por idade, pela população mundial por 100.000 mulheres,
Brasil, entre 1979 e 2012. Fonte: INCA, 2014.
Dentre todos os mamíferos, a espécie canina apresenta a maior incidência de
neoplasias mamárias,
representando
aproximadamente 52
%
do total (Brodey,
Goldschmidt e Roszel, 1983; Sleeckx et al., 2013), e, quando comparada à mulher,
possui três vezes mais chances de desenvolvê-la (Krol et al., 2009; Gelaleti et al., 2012;
30
Michel et al., 2012; Pawłowski et al., 2013). A prevalência de câncer de mama nessa
espécie tem aumentado ao longo dos anos, sendo que a incidência de lesões malignas
está em torno de 26 a 73 % (MacEwen, 1990; Gelaleti et al., 2012; Zuccari et al., 2012).
Tumores da glândula mamária partilham características comuns entre cães e
seres humanos. Por esta razão, são excelentes modelos para o estudo da biologia do
câncer assim como para testes de agentes terapêuticos, já que animais de estimação têm
tumores com apresentação histopatológica similares àqueles que acometem a mulher e
por apresentarem uma resposta semelhante à cirurgia e a esquemas de quimioterapia
(Andrade et al., 2010; Phillips, Lembcke e Chamberlin, 2010; Rasotto et al., 2014;
Rismanchi et al., 2014).
O grau histológico do tumor e o estadiamento clínico pelo sistema de graduação
TNM, que abrange a avaliação do tamanho tumoral, presença de metástase em
linfonodos regionais (do inglês node) e de metástase à distância permitem estabelecer o
prognóstico e o planejamento terapêutico do câncer de mama (Pedersen et al., 2004).
Além dos fatores clínico-patológicos, o conhecimento das características moleculares
dos tumores tem contribuído com a avaliação mais precisa do prognóstico, e com o
desenvolvimento e incorporação de novos agentes e estratégias terapêuticas (GonzalezAngulo, Morales-Vasquez e Hortobagyi, 2007; Gralow et al., 2008; Hicks e Kulkarni,
2008; Duffy, O'Donovan e Crown, 2011).
A classificação prognóstica atual considera além dos subtipos histológicos os
subtipos moleculares (Perou, 2011), caracterizados nos seguintes fenótipos: Luminal A
(receptor de estrogênio (RE) positivo e/ou receptor de progesterona (RP) positivo e
receptor do fator de crescimento epidermal 2 negativo (HER2 -); Luminal B (RE+ e/ou
RP+, HER2+); Basal like (RE-, RP-, HER2-, citoqueratina 5/6+ e/ou HER1+); HER2
superexpresso (RE-, RP-, HER2+).
Esses subtipos moleculares apresentam comportamentos distintos relacionados
com a sobrevivência, prognóstico e resposta à terapêutica específica. Os subtipos
luminais A tem baixa taxa de proliferação e são acompanhados de um bom prognóstico.
Os luminais B tem alta taxa de proliferação e um prognóstico mais pobre que o luminal
A. Portanto, o status do HER2 e dos receptores hormonais, RE e RP, são fatores
prognósticos e preditivos que foram incorporados à rotina clínica e permitem que se
31
estabeleça um tratamento individualizado
(Hsiao et al., 2010). A imunoexpressão do
RE e RP, por exemplo, está associada ao melhor prognóstico da doença (Duffy,
O'Donovan e Crown, 2011). O HER2 tem expressão alterada em aproximadamente 1015 % dos casos. Essa alteração está diretamente associada com o pior prognóstico,
resistência à quimioterapia e terapia hormonal e aumento da proliferação celular
(Jukkola et al., 2001).
Quanto ao tratamento personalizado, o uso de trastuzumabe (Herceptin) em
pacientes com câncer de mama que apresentam superexpressão da proteína HER2 é um
exemplo de sucesso da terapia-alvo com anticorpos monoclonais. Já a positividade para
o RE é um fator preditivo de resposta ao quimioterápico Tamoxifeno
(Hsiao et al.,
2010). Porém, os tumores chamados triplo-receptor-negativos (RE-, RP- e HER2-) são
caracterizados por um pior prognóstico, uma vez que não respondem a tratamentos
específicos, relacionando-se a ocorrência de metástases e menor sobrevida das
pacientes. Esses dados mostram a importância da expressão de um ou mais
imunomarcadores como uma informação útil, e muitas vezes conclusiva, na prática
clínica (Thomas e Berner, 2000), permitindo que estratégias de tratamento sejam
definidas de maneira mais eficaz e com menor toxicidade (Gonzalez-Angulo, MoralesVasquez e Hortobagyi, 2007; Hicks e Kulkarni, 2008; Duffy, O'Donovan e Crown,
2011).
1.2 Microambiente tumoral
O câncer é um processo complexo que se desenvolve em várias etapas
determinadas por perturbações genéticas,
como
a ativação
de oncogenes ou
silenciamento de genes supressores tumorais, além de eventos epigenéticos que podem
ocorrer no interior das células (Hahn e Weinberg, 2002; Baylin, 2005; Bartels e
Tsongalis, 2009; Pierce et al., 2009). Além disso, a perda de mecanismos reguladores
que controlam o comportamento normal das células tem importante ação no
microambiente tumoral através da atuação de diversos fatores (Hahn e Weinberg, 2002;
Baylin, 2005; Bartels e Tsongalis, 2009).
As influências do microambiente tumoral que relacionam os fatores pró e antiinflamatórios atuantes tem importância crescente (Baylin, 2005; Hahn e Weinberg,
32
2002; Pierce et al., 2009). Os tumores sólidos são reconhecidos por um microambiente
complexo que envolve interações de diferentes componentes celulares e moleculares
(Joyce e Pollard, 2009; Egeblad, Nakasone e Werb, 2010), reforçando a necessidade de
estudos que esclareçam sua influência no desenvolvimento tumoral e resposta a
tratamentos.
Em 2011, Hanahan e Weinberg postularam diretrizes agrupando 10 principais
características das células tumorais, dentre elas a alta taxa de proliferação celular e
replicação contínua (ultrapassando o controle o ciclo celular), resistência a morte celular
(evadindo o processo de apoptose), capacidade de invasão e metástase (ativando a
neovascularização), instabilidade genômica com acúmulo de mutações, desregulação do
metabolismo energético, evasão do sistema imune e infiltração inflamatória, sendo este
um dos fatores contribuintes para o pior prognóstico nas neoplasias mamárias (Figura
2).
Figura 2. Capacidades biológicas adquiridas durante o desenvolvimento de
múltiplos passos dos tumores. Adaptado de Hanahan; Weinberg, (2011).
O microambiente tumoral é um sistema dinâmico e, portanto, formado em
grande parte por células inflamatórias que liberam suas citocinas. A interação desses
33
fatores pode estimular a proliferação e sobrevivência de células com danos no DNA, e o
acúmulo dessas alterações genéticas permite a evolução para a malignidade (Condeelis
e Pollard, 2006) (Figura 3). Algumas células, que regulam a expressão de citocinas e
quimiocinas para ajudar no recrutamento de células inflamatórias e agir contra as
células tumorais, atuam de maneira contrária no microambiente tumoral, utilizando
esses fatores para promover o crescimento e a progressão tumoral (Richmond e
Thomas, 1986). Além disso, as células tumorais são capazes de evadir os principais
mecanismos de ação do sistema imunológico, se beneficiando com a inflamação no
microambiente tumoral (Coussens e Werb, 2002).
Figura 3. Representação do tecido inflamatório e do microambiente tumoral
invasivo. A. Tecido em processo inflamatório, representação da arquitetura
desorganizada. B. Tecido neoplásico de carcinoma invasivo com arquitetura
desorganizada, neovascularização, presença de tecidos linfáticos e matriz extracelular
remodelada que interagem com outros tipos celulares, como citocinas e quimiocinas
pró-inflamatórias. Adaptado de Coussens e Werb (2002).
Apesar dos avanços terapêuticos, a taxa de mortalidade por câncer de mama
continua crescendo. Os padrões de recidiva são relacionados à resistência a múltiplas
terapias (tais como combinações de quimioterapia, radiação ionizante e imunoterapia)
que refletem as respostas multifatoriais desse tipo tumoral (Mohammad et al., 2015).
Nesse sentido, um importante foco das pesquisas no câncer de mama tem sido melhorar
34
o entendimento da relação dos componentes moleculares com a doença, a fim de
identificar possíveis marcadores preditivos e prognósticos, bem como, novos alvos
terapêuticos e, assim, desenvolver novas estratégias terapêuticas (Ali et al., 2010;
Levashova et al., 2010; Park et al., 2010).
2. PROCESSOS ATUANTES NA TUMORIGÊNESE
2.1 ANGIOGÊNESE
A angiogênese é um processo de formação de novos vasos sanguíneos, a partir
de um endotélio vascular preexistente, que visa o fornecimento de nutrientes e oxigênio
e, assim, permitir a proliferação das células e consequente crescimento e progressão do
tumor (Takahashi e Shibuya, 2005; Gavalas et al., 2013). Além disso, permite a retirada
do gás carbônico (CO 2 ) e dos resíduos metabólicos do leito neoplásico, representando
ainda, uma importante via de disseminação metastática (Zhang et al., 2010; Liu e
Ouyang, 2013).
Nesse contexto, o crescimento tumoral se inicia lentamente e, à medida que a
disponibilidade de oxigênio e nutrientes diminui, ocorre a ativação do “interruptor
angiogênico” (angiogenic switch), que induz o aumento dos fatores pró-angiogênicos e
diminuição dos anti-angiogênicos, garantindo o crescimento exponencial do tumor.
Assim, as células endoteliais quiescentes passam a responder a estímulos de
proliferação e migração para formação de novos vasos (Bergers e Benjamin, 2003)
(Figura 4).
35
Figura 4. Representação da ativação da angiogênese a partir de células
quiescentes. Adaptado de http://www.clinicaloptions.com/
Durante seu crescimento, o tumor pode alcançar aproximadamente 1-2 mm3
antes que suas demandas metabólicas sejam restritas devido ao limite de difusão de
oxigênio e nutrientes no local (Carmeliet e Jain, 2011). A hipóxia ou baixa oxigenação
pode ocorrer em decorrência da proliferação descontrolada das células e do rápido
crescimento tumoral e, também, da perfusão inadequada em parte do tecido resultante
da estrutura caótica dos novos vasos sanguíneos formados (Carmeliet e Jain, 2000;
Harris, 2002; Vordermark, 2010).
De acordo com a intensidade, a hipóxia pode resultar em apoptose, ou induzir
respostas adaptativas de sobrevivência celular (Sadri e Zhang, 2013; Zhang e Liu,
2013). Assim, ao contrário das células normais, para manter a sobrevivência em
situações de hipóxia, as células tumorais são capazes de promover mecanismos
adaptativos, como a indução de fatores envolvidos no processo de angiogênese (Kallergi
et al., 2009).
A formação de novos vasos sanguíneos pela angiogênese envolve um número
sequencial de passos e se inicia a partir de capilares pré-existentes no tumor. Além
disso, diversas moléculas participam, como as angiopoitinas 1 e 2 e seus receptores Tie1 e Tie-2, angiogenina, metaloproteinases (MMPs), fator de crescimento placentário
36
(PIGF), fator de crescimento de fibroblastos básico (bFGF), fator de crescimento
endotelial (EGF), fator de crescimento semelhante a insulina-1 (IGF-1), citocinas próinflamatórias, entre outros (Arbab, 2012).
Inicialmente, as células endoteliais quiescentes são ativadas por fatores liberados
pelas células tumorais em resposta a condições adversas como privação de nutrientes e
oxigênio.
Posteriormente,
fatores de crescimento
pró-angiogênicos produzidos e
liberados pelas células tumorais, como o fator de crescimento endotelial vascular
(VEGF) e angiopoitina-2 atuam na desestabilização inicial dos vasos pré-existentes e
aumento da permeabilidade vascular, levando a degradação da membrana basal e matriz
extracelular, pela sua capacidade de induzir a síntese de enzimas proteolíticas tais como
as MMPs, permitindo a remodelação da matriz, e eventual liberação de fatores próangiogênicos. Em seguida, ocorre a proliferação e migração das células endoteliais, bem
como a formação do tubo, recrutamento e diferenciação de periquitos e células de
suporte perivascular. Nessa etapa inúmeros fatores estão envolvidos como VEGF,
angiogenina, PDGF, bFGF, EGF, entre outros. Ao final, ocorre a maturação e
estabilização dos novos vasos formados, com a participação de fatores como a
angiopoitina-1 e seu receptor Tie-2 (Bergers e Benjamin, 2003; Pradeep, Sunila e
Kuttan, 2005; Milkiewicz et al., 2006; Clapp et al., 2009; Coulon et al., 2011; Weis e
Cheresh, 2011) (Figura 5).
37
Figura 5. Representação das etapas do processo de angiogênese com a participação
de inúmeros fatores pró-angiogênicos. Adaptado de Gacche; Meshram, 2013.
O VEGF é um potente mitógeno que atua em diferentes etapas do processo
angiogênico, promovendo o aumento da permeabilidade vascular, estimulação da
migração, proliferação e invasão de células endoteliais (Shibuya, 2011). Esse fator foi
primeiramente descrito em células endoteliais e, portanto, denominado “fator de
crescimento endotelial vascular”, no entanto, o VEGF pode exercer ação mitogênica em
outros tipos celulares (Ferrara e Davis-Smyth, 1997).
O VEGF é composto por uma família de cinco isoformas denominadas VEGFA, VEGF-B, VEGF-C, VEGF-D e PIGF, os quais ligam-se a receptores específicos do
tipo tirosina quinase, promovendo uma cascata de eventos intracelulares (Brito et al.,
2011; Finley, Dhar e Popel, 2013). Cada isoforma pode ativar um ou mais receptores
conhecidos, como VEGFR1, localizado na superfície de células hematopoiéticas,
macrófagos e monócitos, VEGFR2, encontrado no endotélio vascular e linfático e o
VEGFR3, localizado predominantemente no endotélio linfático (Stefanini et al., 2008;
Taneja et al., 2010). Receptores de VEGF também são encontrados em células tumorais,
38
e podem estimular o crescimento celular de maneira autócrina (Weis e Cheresh, 2011;
Arbab, 2012).
O VEGF–A se liga a dois receptores específicos, o VEGFR1 e o VEGFR2
enquanto o VEGF–B e PGF são reconhecidos apenas pelo receptor VEGFR1. O VEGFC e VEGF-D se ligam ao VEGFR2 e também são reconhecidos pelo VEGFR3. A
ligação entre o VEGF-A e VEGFR2 é considerada o mais importante passo do processo
de angiogênese (Woolard et al., 2004; Coulon et al., 2011), enquanto a ligação de
VEGF-C com VEGFR3 está envolvida no processo de linfangiogênese (Brito et al.,
2011).
Nesse contexto, dada a variedade de sinais envolvidos no processo angiogênico,
diversos fatores podem ser considerados alvos terapêuticos, auxiliando no bloqueio da
progressão do câncer (Arbab, 2012).
2.2 APOPTOSE
A morte celular pode ocorrer por um espectro de vias morfologicamente e
bioquimicamente distintas, incluindo a apoptose, necrose e autofagia. O termo
"apoptose", descrito por Kerr, Wyllie e Currie (1972) caracteriza-se por alterações
morfológicas,
incluindo
retração
celular,
condensação
da cromatina,
perda da
integridade da membrana nuclear, formação de vesículas na membrana plasmática e
corpos apoptóticos.
Os mecanismos de apoptose são altamente complexos e sofisticados e envolvem
uma cascata de eventos moleculares dependentes de energia. Existem duas principais
vias apoptóticas descritas: extrínseca ou via do receptor de morte e intrínseca ou via
mitocondrial. Há evidências de que ambas as vias são vinculadas e que moléculas de
uma via podem influenciar na outra. Existe ainda uma via adicional que envolve
citotoxicidade mediada por células T e é considerado um processo independente da
caspase (cysteine-aspartic proteases) que pode ser iniciado pelo fator de indução de
apoptose (FIA) (Proietti et al., 2014). As três vias convergem para o mesmo terminal,
ou via de execução – a apoptose celular.
As vias intrínseca e extrínseca da apoptose podem ser desencadeadas por
diversos estímulos extracelulares e intracelulares que resultam na ativação coordenada
39
de uma família de proteases de cisteína chamada caspases. Estímulos externos podem
ocorrer através da interação de receptores e ligantes da família do fator de necrose
tumoral (TNF, Fas ligand, TRAIL) e estímulos internos, como privação de fatores de
crescimento, danos no DNA, hipóxia ou ativação de oncogenes (Grivicich et al., 2007).
Caspases desempenham um papel crítico na execução da apoptose em variados
tipos celulares. A maioria das caspases são sintetizadas como pró-enzimas inativas e
posteriormente ativadas (adquirindo sua forma clivada) e tornam o processo de morte
celular irreversível. A clivagem ocorre por auto-proteólise e/ou a partir de ações de
outras proteínas, consistindo em subunidades grandes (17-20 kDa) e pequenas (10-12
kDa). Aproximadamente 14 caspases foram descritas em mamíferos, classificadas como
iniciadoras (exemplo: caspase-2, 8, 9, 10), efetoras ou executoras (exemplo: caspase-3,
6, 7) e inflamatórias (exemplo: caspase-1, 4, 5) (Elmore, 2007).
2.2.1 Via extrínseca
A via de sinalização extrínseca da apoptose envolve a interação de receptores
transmembrana. Estes fatores incluem receptores de morte (death receptors DRs),
membros da superfamília de genes receptores de TNF como TNF receptor-1 (TNFRa),
CD95 (também chamado Fas e APO-1), receptor de morte 3 (death receptor DR3), TNF
indutor de apoptose receptor-1 ligante (TRAIL-R1, também chamado DR4) e TRAILR2 (também chamado DR5). Os membros da família de receptores TNF compartilham
domínios extracelulares ricos em cisteína e tem um domínio citoplasmático de cerca de
80 aminoácidos denominado "domínio de morte" (DD). Estes domínios de morte
desempenham papel fundamental na transmissão do sinal de morte a partir da superfície
celular para as vias de sinalização intracelulares. A ligação de ligantes DR a receptores
faz com que a proteína pró-caspase-8 seja recrutada via DED (domínio efetor de morte)
ao complexo de sinalização de morte (DISC) que também inclui proteínas Adaptadoras
de Sinalização de Receptores de Domínio de Morte (FADD) ou TNFR-associada ao
domínio de morte (TRADD). FADD então associa-se a pró-caspase-8 por meio da
dimerização do DED e, uma vez ativada a caspase-8, a apoptose é desencadeada
(Elmore, 2007; McIlwain, Berger e Mak, 2013; Mohammad et al., 2015) (Figura 6).
40
Figura 6. Representação das vias extrínseca e intrínseca da apoptose. Adaptado de
Elmore (2007).
2.2.2 Via intrínseca
A via de sinalização intrínseca que inicia a apoptose envolve um conjunto
diversificado
de estímulos não
mediados
por receptores que produzem sinais
intracelulares e atuam diretamente em alvos mitocondriais no interior da célula. Os
estímulos que iniciam a via intrínseca da apoptose produzem sinais intracelulares que
podem atuar de forma positiva ou negativa. Sinais negativos envolvem a ausência de
alguns fatores de crescimento, hormônios e citocinas que podem levar à insuficiência de
fatores de supressão de morte celular, desencadeando a apoptose. Sinais positivos
incluem radiação, toxinas, hipóxia, hipertermia, infecções virais e presença de radicais
livres (Elmore, 2007; McIlwain, Berger e Mak, 2013; Mohammad et al., 2015).
Todos estes estímulos causam alterações na parte interna da membrana
mitocondrial,
resultando
na abertura do
poro
de transição de permeabilidade
mitocondrial (MPT). A perda do potencial mitocondrial transmembrana ocasiona a
liberação
de
dois
grupos principais de proteínas pró-apoptóticas do
espaço
41
intermembranas para o citosol (Elmore, 2007; McIlwain, Berger e Mak, 2013;
Mohammad et al., 2015).
A caspase iniciadora da via intrínseca da apoptose é a caspase-9, que é ativada
por dimerização induzida quando o domínio de recrutamento de caspase (CARD) ligase à Apaf-1 (proteína humana fator de ativação de protease associada à apoptose 1),
formando o “apoptossomo”. Além disso, a ativação da via intrínseca da apoptose é
dependente da inativação das IAPs (inibidores de proteínas apoptóticas) (Elmore, 2007;
McIlwain, Berger e Mak, 2013; Mohammad et al., 2015).
O segundo grupo de proteínas pró-apoptóticas, FIA, endonuclease G e CAD, são
liberadas a partir da mitocôndria durante a apoptose. Esse grupo de proteínas participam
do evento tardio da apoptose. O controle e a regulação destes eventos apoptóticos
mitocondriais ocorrem através de membros da família de proteínas Bcl-2 (proteína da
célula B linfoma 2), que regulam a permeabilidade da membrana mitocondrial e podem
atuar tanto a favor como contra a apoptose. O principal mecanismo de ação da família
Bcl-2 é a regulação da liberação do citocromo c das mitocôndrias através da alteração
da permeabilidade da membrana mitocondrial. Alguns mecanismos têm sido estudados,
mas nenhum foi comprovado definitivamente. Assim, a elucidação destas vias tem
implicações importantes na tumorigênese (Elmore, 2007; McIlwain, Berger e Mak,
2013; Mohammad et al., 2015) (Figura 6).
A caspase-3 é denominada mediador chave na apoptose, desempenhando papel
crucial no sistema nervoso, sendo ativada por ambas as vias extrínseca e intrínseca da
apoptose. Em resposta a vários sinais de morte, a proenzima caspase-3 é ativada pela
clivagem proteolítica de Asp28-Ser29 e Asp175-Ser176 através das ligações catalíticas
pela granzima B, caspase-6, caspase-8, caspase-9 e caspase-10 gerando duas
subunidades (p17) e (p12), formando um heterodímero ativo. Embora o precursor da
caspase-3 esteja localizado no citoplasma, a caspase-3 desempenha funções essenciais
no núcleo das células apoptóticas (Elmore, 2007; McIlwain, Berger e Mak, 2013;
Mohammad et al., 2015) (Figura 7).
42
Figura 7. Via extrínseca e intrínseca da ativação da caspase-3. Visão geral da
ativação da caspase-3. Setas indicam conversões químicas ou reações catalisadas
enquanto setas com bloqueio representam inibição. Adaptado de Harrington et al.
(2008).
A apoptose é alvo de potencial uso na prática clínica e para a compreensão dos
mecanismos de resistência à radioterapia e à quimioterapia (Grivicich et al., 2007).
Assim, várias tentativas têm sido feitas na última década para desenvolver moléculas
capazes de direcionar a ativação da caspase-3 para utilização na terapia do câncer
(McIlwain, Berger e Mak, 2013).
3. NOVOS ALVOS TERAPÊUTICOS NO CÂNCER DE MAMA
3.1 INTERLEUCINA-25
No câncer de mama invasivo, as células mioepiteliais e a membrana basal são
invadidas, deixando as células tumorais em contato direto com o estroma intersticial
remodelado, compreendendo fibroblastos e miofibroblastos, células vasculares tumorais
e um número considerável de células-imune infiltradas, como linfócitos, macrófagos e
mastócitos (Inman e Bissell, 2010). Um estudo de Furuta et al. (2011) mostrou que
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células mioepitelias não malignas secretam fatores que inibem o crescimento e/ou
sobrevivência de células malignas, bem como acionam mecanismos subjacentes a esta
atividade antitumoral intrínseca, sendo constatada a liberação da interleucina (IL)-25,
também conhecida como IL-17E, denominada citocina antitumoral.
O papel das células Th17 no câncer permanece incerto, com ações controversas
na inibição e progressão de tumores (Lyon et al., 2008). As ILs-17 mediam seus efeitos
pró-inflamatórios através de seus receptores, expressos em todos os tipos celulares
(Aggarwal e Gurney, 2002; Korn et al., 2009). A família IL-17 compreende seis
citocinas, IL-17A, B, C, D, E e F, produzidas pela ativação de células T de memória, e
compartilham ações semelhantes na maquinaria intracelular (Lyon et al., 2008). Cinco
receptores foram descritos, sendo que IL-17A liga-se aos receptores IL-17RA/IL-17RC,
enquanto a IL-17E, liga-se ao heterodímero IL-17RA/RB (Figura 8).
Figura 8. Esquema representativo da família IL-17 e seus receptores. Lyon et al.
(2008).
A IL-25 é uma citocina pró-inflamatória, altamente expressa em alguns órgãos,
tais como testículos, próstata e baço, e expressa em baixas quantidades em outros,
incluindo o epitélio mamário. É o membro mais distante da família das proteínas IL-17,
partilhando apenas 16 a 30 % de homologia com os demais membros da família (Furuta
et al., 2011). Ao contrário dos efeitos pró-inflamatórios associados com a família de IL17, a IL-25 parece ser uma citocina pleiotrópica que desempenha respostas sistêmicas
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do tipo t helper-2 (Th2) com alterações imunológicas e patológicas tecido-específicas,
incluindo, mas não se limitando, a alteração na expressão das interleucinas IL-4, 5 e 13.
Estudo de Furuta et al. (2011) definiu que a interação entre IL-25 e seu receptor
IL-25R (também chamado IL-17RB) envia um sinal de morte celular, promovendo a
indução da apoptose em células neoplásicas enquanto células não malignas apresentam
resistência a essa ligação, conferida pela expressão diferencial do receptor. No entanto,
embora células cancerosas expressem o receptor, elas não expressam o ligante
apoptótico (IL-25), podendo expressar outro ligante que contribui para seu potencial
tumorigênico. O candidato à ligação é a IL-17B, super-expressa em 30 % das células
neoplásicas de mama e não detectada em tecidos normais (Figura 9).
A
B
Figura 9. Diagrama esquemático da atividade citotóxica da IL-25 em células do
câncer de mama que expressam IL-25R. A. MEC não malignas não expressam IL25R e são resistentes a apoptose induzida por IL-25. Células do câncer da mama
expressam IL-25R e são susceptíveis a apoptose induzida por IL-25. B. Ligante IL-17B
competidor do receptor IL-25R em células do câncer de mama tornam-se resistentes a
apoptose induzida. Adaptado de Furuta et al. (2011).
Embora a ligação da IL-17B ao receptor apresente menor afinidade, contribui
para o potencial tumorigênico do câncer de mama, traduzindo sinais através do receptor
associado à TNF, ativando o NF-kB e levando a superexpressão de genes
antiapoptóticos como Bcl2 (Huang et al., 2014) (Figura 10).
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Figura 10. Esquema da sinalização IL-17RB/IL-17B em células do câncer de
mama. Quando a sinalização de IL-17RB/IL-17B é ativada, TRAF6 é recrutado para
IL-17RB e ativa a via de sinalização NF-kB para regular positivamente a expressão do
gene anti-apoptótico Bcl-2. Alvos IL-17B (anticorpos azuis), receptores de IL-17RB
(anticorpos verdes). Adaptado de Huang et al. (2014).
Compreender o papel destas interleucinas na progressão tumoral e durante a
imunoterapia é fundamental para melhorar a eficácia das terapias cujo alvo é a
modulação imunológica.
3.2 MELATONINA
3.2.1 Síntese e degradação
A melatonina, conhecida como N-acetil-5-metoxitriptamina, é um hormônio
naturalmente produzido e secretado pela glândula pineal além da síntese extra-pineal
que ocorre na retina, pele, trato gastrointestinal, células tronco e linfócitos (AcuñaCastroviejo et al., 2014). Diferentes mecanismos de ação da melatonina têm sido
propostos, regulando uma variedade de vias celulares, dentre as quais se destaca a
cronobiológica. Esse hormônio é considerado um “tradutor neuroendócrino” do ciclo
circadiano, controlando os padrões secretórios de diversas substâncias, como o cortisol
e adrenalina, atuando sobre os ciclos de atividade-repouso e vigília-sono (Maganhin et
al., 2008). Além disso, a melatonina atua sobre o sistema reprodutor, cardiovascular,
sistema imunológico, crescimento e envelhecimento (Maganhin et al., 2008).
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Sua produção segue um padrão rítmico, com pico secretório no período noturno,
atingindo níveis plasmáticos máximos entre 03:00 e 04:00 horas em humanos, e quase
nenhuma produção no período diurno (Ravindra, Lakshmi e Ahuja, 2006; Espino,
Pariente e Rodríguez, 2012). É sintetizada a partir da conversão do aminoácido
triptofano em 5-hidroxitriptofano (5-HTP) pela enzima triptofano hidroxilase 1 (TPH1).
O 5-HTP é descarboxilado pela 5-HTP descarboxilase em serotonina, a qual é acetilada
em N-acetilserotonina (NAS) na reação catalisada pela enzima arilalquilamina Nacetiltransferase (AA-NAT). Então, a NAS é convertida em melatonina pela enzima
hidroxi-indol-Ometiltransferase (HIOMT) (Espino, Pariente e Rodríguez, 2012; AlOmary, 2013) (Figura 11).
Figura 11. Esquema representativo da via de síntese da melatonina. Início na
conversão do aminoácido triptofano até o último passo para a formação de melatonina
pela enzima HIOMT.
A enzima AA-NAT apresenta ritmo diário, atingindo concentrações 100 vezes
superiores na fase escura, quando comparado à fase clara. Esta variação cíclica da AANAT faz com que a redução dos níveis de serotonina na fase escura seja acompanhada
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pelo aumento das concentrações de NAS e melatonina. A melatonina é secretada
durante a noite em todas as espécies de mamíferos, independente de possuírem hábito
diurno ou noturno (Korf e Von Gall, 2006; Bilu e Kronfeld-Schor, 2013). Sua
degradação ocorre principalmente no fígado, envolvendo a hidroxilação em 6hidroximelatonina [6(OH)M], seguida dos processos de sulfatação ou glicuronidação, e
posterior excreção na urina (Reiter, 1991; Facciolá et al., 2001).
3.2.2 Receptores da melatonina
Os mecanismos de ação da melatonina incluem a ligação a receptores
localizados
na
membrana
celular,
ligação
a
proteínas
intracelulares,
como
a
calmodulina, interações com receptores nucleares e atividade oxidante (Jablonska et al.,
2013).
A melatonina pode ligar-se e ativar os receptores de membrana MT1 e MT2
em uma variedade de tecidos (Hill et al., 2009). Receptores MT1 pertencem a
superfamília de receptores acoplados à proteína G e, quando mediados à subunidade G,
os receptores MT1 inibem a atividade da adenil-ciclase, diminuindo a produção de
adenosina 3',5'-monofosfato cíclico (cAMP), tornando possível controlar a atividade das
proteínas quinases (PKC, PKA, MAPK), bem como a expressão de genes específicos,
envolvidos nos processos de proliferação, angiogênese, migração e diferenciação celular
(Jablonska et al., 2013). Um terceiro receptor com menor afinidade pela melatonina é
denominado MT3 e sua ativação ainda não possui papel fisiológico definido, no entanto,
apresenta 95 % de homologia com a enzima quinona redutase II, envolvida na
detoxificação de radicais livres (Foster et al., 2000).
A melatonina também pode atuar por mecanismos independentes de seus
receptores de membrana, exercendo atividade antioxidante diretamente na redução de
radicais livres ou pelo aumento de enzimas antioxidantes. Ainda, por ser lipossolúvel, a
melatonina pode atravessar diretamente a membrana celular e interagir com proteínas
intracelulares, como a calmodulina, e com receptores nucleares RZR/ROR (receptor Z
para retinóide/receptor órfão para retinóide). Apesar dessa capacidade de interação
ainda ser controversa, a ligação da melatonina com receptores nucleares explica muitas
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de suas funções, inclusive relacionadas à alteração de genes envolvidos na proliferação
celular e apoptose (Sánchez-Barceló et al., 2003).
Uma vez produzida, a melatonina, por ser altamente lipossolúvel, difunde-se
para a corrente sanguínea e, posteriormente, para órgãos distantes, bem como do fluído
cerebroespinhal para o hipotálamo e sistema nervoso central (Proietti et al., 2013).
3.2.3 Melatonina e câncer
Além de estar envolvida nas funções fisiológicas, a melatonina tem importante
papel em processos patológicos, incluindo o câncer. Os primeiros indícios de que a
melatonina poderia ser útil na terapêutica oncológica surgiram em um estudo realizado
por Cohen, Lippman e Chabner (1978). Os autores propuseram que a diminuição da
função da glândula pineal poderia aumentar o risco de desenvolvimento do câncer de
mama, sugerindo que a ausência da síntese de melatonina poderia induzir a exposição
prolongada ao estrógeno resultando no desenvolvimento do tumor mamário. Em 1981,
Bartsch et al. demonstraram que as concentrações de melatonina são diminuídas em
pacientes com câncer de mama. Desde então, diversas pesquisas confirmaram que os
pacientes com câncer de mama estabelecido têm níveis mais baixos de melatonina
mensurável.
Estudos epidemiológicos têm revelado o elevado risco de câncer de mama em
sociedades industrializadas, sendo que o risco aumenta em mulheres com trabalho
noturno, com consequente falta de exposição à luz e bloqueio da síntese de melatonina
(Proietti et al., 2013). Na década de 90 alguns estudos clínicos foram realizados,
demonstrando que a utilização da melatonina, em conjunto com terapias convencionais,
apresenta efeitos benéficos em diferentes tipos de tumores avançados e intratáveis ou
em pacientes com câncer metastático, quer seja aumentando a eficácia do tratamento
(Lissoni et al., 1995; Lissoni et al., 1996), ou diminuindo os efeitos colaterais
contribuindo, portanto, para a melhora da qualidade de vida desses pacientes (Barni et
al., 1990; Lissoni et al., 1992).
Desde então, têm sido demonstrado que a melatonina inibe o desenvolvimento e
a progressão de diferentes tipos de câncer, e muitos mecanismos de ação estão sendo
investigados (Di Bella et al., 2013). Estes incluem efeitos oncostáticos e anti-
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inflamatórios, aumento da imunidade local (Luchetti et al., 2010; Alvarez-García et al.,
2012; Di Bella et al., 2013; Ordoñez et al., 2014), da capacidade antioxidante celular
(Fic et al., 2007; Mirunalini e Dhamodharan, 2010), regulação da expressão de genes
supressores tumorais (Mediavilla et al., 2010), controle da diferenciação e proliferação
celular (Sánchez-Barceló et al., 2005) e indução da apoptose (Proietti et al., 2013).
Alguns
estudos
demonstram,
ainda,
que a melatonina minimiza a inflamação,
bloqueando fatores de transcrição como o fator nuclear NF-kB, inibindo a expressão de
citocinas, além de espécies reativas de oxigênio (EROS), MMPs e do VEGF (Li et al.,
2005; Korkmaz, Rosales-Corral e Reiter, 2012). Além disso, Alvarez-García et al.
(2012) e Ordoñez et al. (2014) confirmam que a melatonina também modula citocinas
pró-inflamatórias, e isto pode influenciar o microambiente do tumor.
A melatonina pode atuar em ambas as vias extrínseca e/ou intrínseca da
apoptose (Figura 12), sendo que ambas convergem à clivagem da caspase-3.
Figura 12. Esquema da via extrínseca e intrínseca da apoptose. *modulada pela
melatonina. Adaptado de Rodriguez et al. (2013).
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Além disso, no trabalho de Proietti et al. (2013) é proposto um terceiro
mecanismo de atuação da melatonina na fase inicial da apoptose, envolvendo processos
independentes da caspase com atuação do FIA (Figura 13).
Figura 13. Ação da melatonina na inibição da proliferação de células tumorais. Em
destaque a ação da melatonina na apoptose na fase inicial, processo independente da
caspase, envolvendo fator de indução de apoptose (FIA). Ação da melatonina na fase
tardia da apoptose, processo dependente da caspase, com ativação da caspase-9 e 7 e
clivagem de PARP, concomitante a subregulação de Bcl/Bax. Adaptado de Proietti et
al. (2013).
Nesse contexto, a melatonina pode ser considerada importante alvo no controle
da progressão tumoral criando, assim, um caminho promissor para sua utilização como
agente terapêutico no câncer.
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Objetivos
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II. OBJETIVOS
Objetivo geral
 Avaliar a eficácia da melatonina, da IL-25 e do silenciamento gênico da
IL-17B na modulação da apoptose e angiogênese no câncer de mama.
Objetivos específicos
 Verificar a viabilidade celular das linhagens tumorais mamárias humanas
MDA-MB-231 (triplo negativa e metastática) e MCF-7 (REα positiva e nãometastática) e da linhagem epitelial mamária não-tumorigênica (MCF-10A), bem como
das linhagens tumorais mamárias caninas CF-41 (metastática) e CMT-U229 (nãometastática) após os tratamentos com melatonina e IL-25 em diferentes concentrações
por 48 horas;

Estabelecer o silenciamento gênico da IL-17B nas linhagens MDA-MB-
231, MCF-7, CF-41 e CMT-U229;

Avaliar a expressão gênica e proteica da caspase-3 e sua forma clivada e
do VEGF-A por PCR em tempo real e imunofluorescência, respectivamente, em cultivo
em monocamada e tridimensional nas linhagens MDA-MB-231, MCF-7, CF-41 e
CMT-U229 após os tratamentos com melatonina, IL-25 e siIL-17B por 48 horas;

Quantificar a expressão de 43 proteínas envolvidas na via de sinalização
da apoptose em cultivo em monocamada nas linhagens tumorais mamárias metastáticas
MDA-MB-231 e CF-41 após tratamento com melatonina por 48 horas;

Demonstrar a eficácia da melatonina, isolada, e em associação à via de
sinalização da IL-25 e seu competidor IL-17B como potenciais alvos terapêuticos no
controle da tumorigênese mamária.
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Capítulos
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III. CAPÍTULOS
Os resultados referentes aos objetivos desta Tese serão apresentados a seguir na
forma de dois artigos científicos, conforme as normas de publicações específicas de
cada periódico.
Artigo I
Título: Efficacy of melatonin, IL-25 and siIL-17B in apoptosis and angiogenesis
response of breast cancer cell lines
Autores: Gabriela Bottaro Gelaleti, Thaiz Ferraz Borin, Larissa Bazela Maschio,
Marina Gobbe Moschetta, Bruna Victorasso Jardim-Perassi, Guilherme Berto Calvinho,
Mariana Castilho Facchini, Alicia M. Viloria-Petit, Debora Aparecida Pires de Campos
Zuccari*
Periódico: Journal of Pineal Research, submetido.
Artigo II
Título: Melatonin and IL-25 induces pro-apoptotic and anti-angiogenic factors in
canine mammary tumor cell lines
Autores: Gabriela Bottaro Gelaleti, Thaiz Ferraz Borin, Larissa Bazela Maschio,
Marina Gobbe Moschetta, Eva Hellmén, Alicia M. Viloria-Petit, Debora Aparecida
Pires de Campos Zuccari*
Periódico: Breast Cancer Research, a ser submetido.
55
Artigo I
Journal of Pineal Research
Efficacy of melatonin, IL-25 and siIL-17B in apoptosis and
angiogenesis response of breast cancer cell lines
Journal:
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Manuscript ID:
Manuscript Type:
Date Submitted by the Author:
Complete List of Authors:
Draft
Original Manuscript
n/a
er
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Gelaleti, Gabriela; Universidade Estadual Paulista “Júlio de Mesquita Filho”
(UNESP/IBILCE), Programa de Pós-Graduação em Genética
Borin, Thaiz; Faculdade de Medicina de São José do Rio Preto (FAMERP),
Laboratório de Investigação Molecular do Câncer (LIMC)
Maschio, Larissa; Universidade Estadual Paulista “Júlio de Mesquita Filho”
(UNESP/IBILCE), Programa de Pós-Graduação em Genética
Moschetta, Marina; Faculdade de Medicina de São José do Rio Preto
(FAMERP), Laboratório de Investigação Molecular do Câncer (LIMC)
Jardim-Perassi, Bruna; Faculdade de Medicina de São José do Rio Preto
Calvinho, Guilherme; Faculdade de Medicina de São José do Rio Preto
Facchini, Mariana; Faculdade de Medicina de São José do Rio Preto
Viloria-Petit, Alicia; University of Guelph, Ontario Veterinary College,
Department of Biomedical Sciences
Zuccari, Debora; Faculdade de Medicina de Sao Jose do Rio Preto FAMERP, Molecular Biology
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Keywords:
Breast Cancer, Melatonin, Interleukin-25, Interleukin-17E, Interleukin-17B,
Apoptosis, Angiogenesis
Page 1 of 35
Efficacy of melatonin, IL-25 and siIL-17B in apoptosis and angiogenesis response
of breast cancer cell lines
Gabriela Bottaro Gelaleti 1,2
gabi_b_g@yahoo.com.br
Thaiz Ferraz Borin 2
thaiz80@yahoo.com.br
Larissa Bazela Maschio 2
larissa_maschio@hotmail.com
Marina Gobbe Moschetta 2
marinagobbe@hotmail.com
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Bruna Victorasso Jardim-Perassi 2
brunavj@hotmail.com
Guilherme Berto Calvinho 2
gbc1991@gmail.com
Mariana Castilho Facchini 2
vinagretefamerp@yahoo.com.br
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Alicia M. Viloria-Petit 3
aviloria@uoguelph.ca
er
Pe
Debora Aparecida Pires de Campos Zuccari 1, 2*
debora.zuccari@famerp.br
1
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Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP/IBILCE), Programa
de Pós-Graduação em Genética, São José do Rio Preto, SP, Brasil.
2
Faculdade de Medicina de São José do Rio Preto (FAMERP). Laboratório de
Investigação Molecular do Câncer (LIMC), São José do Rio Preto, SP, Brasil.
3
Department of Biomedical Sciences, Ontario Veterinary College, University of
Guelph, Guelph, Ontario, Canada.
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*Corresponding author. Profa. Dra. Debora Ap. Pires de Campos Zuccari, Laboratório
de Investigação Molecular do Câncer (LIMC), Faculdade de Medicina de São José do
Rio Preto (FAMERP), Avenida Brigadeiro Faria Lima, 5416, Vila São Pedro, São José
do Rio Preto (SP) 15090-000, Brasil. Tel.: 55 (17) 32015885. e-mail:
1
ABSTRACT
Mammary tumors involve cell autoregulation and tumor microenvironment changes,
including insufficient apoptosis, cell differentiation and neovascularization. Such effects
can be modulated by melatonin, which has oncostatic action and modulates interleukins
(ILs) expression. IL-25, also known as IL-17E, is an active cytokine that induce
apoptosis in tumor cells due to differential expression of its receptor (IL-17RB). IL-17B
competes with IL-25 for binding to IL-17RB in tumor cells, promoting tumorigenesis.
This study was to address the possibility of engagement of IL-25/IL-17RB signaling as
an enhancer of melatonin effects on breast cancer cells. Breast cancer cell lines were
cultured in monolayer and as tridimensional structures on matrigel and treated with
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melatonin, IL-25, siIL-17B and all combined treatments. Cell viability, gene and protein
expression, immunofluorescence, and apoptosis membrane array was performed.
Treatments with melatonin and IL-25 significantly reduced the tumor cells viability at 1
mM and 1 ng/mL, respectively, but did not alter MCF-10A cell viability. All treatments
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alone and combined significantly increased caspase-3 cleavage in tumor cells grown as
monolayers and 3D structures. Semi-quantitative analysis of proteins involved in the
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apoptotic pathway showed an increase of CYTO-C, DR6, IGFBP-3, IGFBP-5, IGFPB6, IGF-1, IGF-1R, Livin, P21, P53, TNFRII, XIAP and hTRA proteins and reduction of
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caspase-3 (p<0.05) after melatonin treatment. Also, all treatments reduced VEGF-A
protein expression in tumor cells (p<0.05), which confirms the anti-angiogenic potential
of the treatments in mammary tumor cells. Our results suggest therapeutic potential,
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with oncostatic effectiveness, pro-apoptotic and anti-angiogenic properties for
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Page 2 of 35
melatonin and IL-25-driven signaling in breast cancer cells.
Keywords: Breast Cancer, Melatonin, Interleukin-25, Interleukin-17E, Interleukin-17B,
Apoptosis, Angiogenesis.
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Page 3 of 35
INTRODUCTION
The breast tumor microenvironment is composed by several cell types, including
inflammatory and endothelial cells, fibroblasts and adipocytes, among others [1], [2],
[3]. Mutual interactions between the malignant epithelial cells and the surrounding
microenvironment, in part mediated by cytokines and their receptors, modulate the
behavior of malignant cells and drive tumor progression [4], [5], [6] and [3]. Thus, it is
important to identify these microenvironmental mediators of tumor progression as well
as strategies to successfully target them [7].
Melatonin (N-acetyl-5-methoxytryptamine), an endogenous molecule, is an
evolutionary conserved indolamine synthesized from tryptophan that is mainly
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produced by the pineal gland and other nonendocrine organs such as skin, gut and
immune system, among others [8]. Different mechanisms of melatonin action have been
proposed, regulating a variety of cellular pathways. These include oncostatic and antiinflammatory effects, increase local immunity [9], [10], [11], [8], cellular antioxidant
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capacity [12], [13], upregulation of tumor suppressor gene expression [14], control of
cell differentiation and proliferation [15], [16] and induction of apoptosis [17]. Besides
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this, melatonin blocks the activity of transcription factors, such as nuclear factor-κB
(NF-κB), inhibiting the expression of cytokines, metalloproteinases and the vascular
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endothelial growth factor (VEGF) [18], [19], [20]. Further, Alvarez-García et al. [10]
and Ordoñez et al. [8] showed that melatonin also modulate pro-inflammatory
cytokines, and this might impact the tumor microenvironment.
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A family of six pro-inflammatory cytokines comprised of interleukin (IL) 17A,
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B, C, D, E and F [21], [22], [23], [24] have been considered as potent activators of
innate immunity, promoting a protective tumor immunity [25]. Unlike the proinflammatory effects associated with IL-17 family, the IL-17E (also known as IL-25)
appears to be a unique pleiotropic cytokine that engages a systemic Th2-like response
with tissue-specific immunological and pathological changes, which include but are not
limited to, expression of IL-4, 5 and 13 [21], [22] once overexpression of IL-17E
resulted in profound alterations of the immune system.
IL-25 is secreted from non-malignant mammary epithelial cells and referred as
anti-tumoral cytokine [26]. Anti-inflammatory effects has been associated with IL-25 in
both in vitro and in vivo studies [21]; besides, this cytokine can induces breast cancer
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apoptosis by differential expression of its receptor, IL-25R (also called IL-17RB),
which is composed of IL-17RB and IL-17RA heterodimer [26], [27].
Furuta et al. [26] observed that another ligand, IL-17B, compete for the same
receptor site of IL-25 in malignant tissues, which contributes to tumorigenic potential.
IL-17B was reported to bind to IL-17RB with an affinity of 7.6 nM, while IL-25 binds
to IL-17RB with an affinity in the range of 1.1–1.4 nM. The IL-17RB/IL-17B
transduces pro-survival signaling, and their combined expression has been associated
with poor prognosis in breast cancer patients [27].
No studies have previous addressed whether melatonin in combination with IL25 could be a better strategy to target breast cancer cells. Here we explore the
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therapeutic potential of such combinatory approach by assessing the effect of melatonin,
IL-25, and IL-17B gene silencing, alone or in combination, on cell viability and the
differential mRNA and protein expression of apoptosis and angiogenesis mediators in
normal versus malignant human breast cells.
MATERIAL AND METHODS
Cytokines and Antibodies
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Melatonin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Purified IL-25 was
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from ProSpec (East Brunswick, NJ, USA).
Primary antibodies included: cleaved caspase-3 (Sigma-Aldrich, St. Louis, MO, USA),
IL-17RB and VEGF-A (both from Santa Cruz Biotechnology, Dallas, TX, USA).
Cell lines culture
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Triple negative (MDA-MB-231) (ATCC, Manassas, VA, USA) and estrogen
receptor (ER) positive (MCF-7) (ATCC, Manassas, VA, USA) human breast cancer cell
lines were cultured in 75 cm2 culture flasks (Sarstedt, Nümbrecht, Germany) with
Dulbecco’s modified Eagle’s medium (DMEM) (Cultilab, Campinas, SP, Brazil)
supplemented with 10 % fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil),
penicillin (100 IU/mL) and streptomycin (100 mg/mL) (Sigma-Aldrich, St. Louis, MO,
USA) in a humidified incubator at 5.0 % CO2 at 37 ºC until they were 80-90 %
confluent.
The human non-tumorigenic breast epithelial cell line (MCF-10A) (ATCC,
Manassas, VA, USA) was cultured in 1:1 DMEM: Ham's F-12 (Cultilab, Campinas, SP,
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Brazil) media supplemented with 5 % donor equine serum (Thermo Fisher Scientific,
Waltham, MA, USA), epidermal growth factor (20 ng/mL) (Sigma-Aldrich, St. Louis,
MO, USA), hydrocortisone (500 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA), insulin
(0.01 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA), cholera toxin (100 ng/mL)
(Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 IU/mL) and streptomycin (100
mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator at 5.0 % CO2
at 37 ºC until they were 80-90 % confluent.
Three-dimensional (3D) Matrigel culture assay
Sparse tumor single-cell suspensions were plated on individual wells of 8-well
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chamber slides (Sarsted, Newton, NC, USA) previously covered with a 1-mm thick
layer of laminin-rich extracellular matrix (Matrigel® - Becton Dickinson, Franklin
Lakes, NJ, USA), at a concentration of 3.5 x 104 cells/0.5 mL Basal Human Mammary
Epithelial Cell (HuMEC) medium (Life Technologies, Eugene, OR, USA)
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supplemented with the HuMEC Supplement Kit (Gibco® - Life Technologies, Eugene,
OR, USA) and 2 % Matrigel®. The cells were maintained in a humidified incubator at 5
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% CO2 at 37 ºC for eight days until the formation of established 3D structures, with
treatments replenished every two days. The 3D morphogenesis was monitored and
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analyzed using immunofluorescence and confocal microscopy.
Cell viability assessment by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
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For MTT assay, individual wells of 0.31 cm2 (96-well plate) were inoculated
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with 100 µL of regular growth medium containing 5 x 104 cells and incubated in this
media overnight, after which the media was changed to 2.0 % FBS, containing
increasing concentrations of melatonin (0.001 mM, 0.01 mM, 0.1 mM and 1 mM) and
different concentrations of purified IL-25 (1 ng/mL, 10 ng/mL and 50 ng/mL). Control
wells, corresponding to 0 ng/mL of either treatment, contained the highest concentration
of the vehicle for the corresponding treatment, which was 1 % dimethylsulfoxide
(DMSO) (Sigma-Aldrich, St. Louis, MO, USA) for melatonin, and 0.001 % e-pure
water for IL-25. Following 48 hours of the aforementioned treatments, 10 µL of MTT
solution from the Vibrant MTT Cell Proliferation Assay Kit (Invitrogen - Life
Technologies, Eugene, OR, USA) were added to each well and the plates were
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incubated at 37 ºC for an additional 4 h. To solubilize the MTT formazan crystals, the
cells were incubated with 50 µL of DMSO (100 %) and then incubated again at 37 °C
for 10 minutes. Absorbance was measured at 540 nm using an ELISA plate reader
(Thermo Fisher Scientific - Waltham, MA USA). Medium alone was used as a blank
and the corresponding optical density was subtracted from the samples. Cell viability
(%) was calculated for all groups relative to control samples. All treatments were done
in triplicate.
Gene silencing of interleukin-17B
For IL-17B gene silencing four different siRNA were initially tested
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(SI00106127, SI00106134, SI02640652 and SI02640659) (ProSpec, East Brunswick,
NJ, USA), selected from preserved gene regions and thermodynamic stability according
by inventoried assay (Qiagen, Valencia, CA, USA). Individual wells of 1.88 cm2 (24well plate) were inoculated with 500 µL of normal growth medium containing 8 x 104
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cells. Subsequently the cells were transfected using the siRNA Human/Mouse Starter
Kit (Qiagen, Valencia, CA, USA), which included the positive control MAPK-1 siRNA
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(Qiagen, Valencia, CA, USA), a siScramble negative control (Qiagen, Valencia, CA,
USA) and the Gene Kit Solution targeting siIL-17B (Cat No. 1027416 - Qiagen,
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Valencia, CA, USA), all at 10 nM in a 0.5 % HiPerfect solution (Qiagen, Valencia, CA,
USA). Cells were incubated with these reagents for 48 hours, after which total cellular
RNA was isolated using the Trizol method (Invitrogen - Life Technologies, Eugene,
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OR, USA) and purified using the RNeasy Kit extraction columns (Qiagen, Valencia,
CA, USA).
Absolute quantification by real-time (qRT-PCR)
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The concentration of RNA from each sample was determined using a NanoDrop
2000 Spectrophotometer (Thermo Fisher Scientific - Waltham, MA USA). cDNA was
obtained by RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction) using the
High Capacity cDNA Kit (Applied Biosystems, Foster City, CA, USA).
The qRT-PCR reaction was performed to assess the efficiency of gene silencing
of IL-17B, as well as treatment effect on caspase-3 and VEGF-A levels, using a
StepOne Plus Real Time PCR System (Applied Biosystems, Foster City, CA, USA).
Specific primers included: IL-17B - sense (5' GCAGCTGTGGATGTCCAACA 3')
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antisense (5' GGGTCGTGGTTGATGCTGTAG 3') and MAPK-1 - sense (5'
TCCAACCTGCTGCTCAACAC 3') antisense (5' TCATGGTCTGGATCTGCAACA
3'),
inventoried
TaqMan
assays
caspase-3
(Hs0023487_m1),
VEGFA
(Hs00900055_m1), and the housekeeping genes beta-actin (ACTB; 4333762F) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 4333764F), all at a concentration
of 100 ng for each cDNA sample.
The amplification was performed in cycles at 95 °C for 10 minutes, followed
by 40 cycles at 95 °C for 15 seconds and 60 °C for one minute. The value of the relative
expression of the genes of interest was determined with DataAssist 3.0 software
(Applied Biosystems, Foster City, CA, USA) by ∆∆Ct method [28]. The samples were
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tested in triplicate and each experiment included a negative control.
Immunofluorescence staining
Previously treated 3D structures grown on matrigel and treated monolayer cells
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were washed once with PBS, fixed in 4.0 % paraformaldehyde solution in PBS for 20
minutes at room temperature, and blocked with 10 % donkey serum solution for 1 hour
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at room temperature. The specific primary antibodies was then added and incubated
overnight at 4 ºC. After washing three times with immunofluorescence (IF) buffer (0.1
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% FSB, 0.2 % triton and 0.05 % Tween 20), a secondary Alexa Fluor 488 anti-rabbit
IgG (Sigma-Aldrich, St. Louis, MO, USA) for both cleaved caspase-3 and VEGF-A
was added per 1 hour at room temperature. Following three time washing with IF
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buffer, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) solution
(Life Technologies, Eugene, OR, USA) and mounted with Prolong Gold® (Life
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Technologies, Eugene, OR, USA). 3D structures images were captured and processed
using a confocal microscope and associated software (ZEISS, model LSM 710, software
ZEN 2010, Thornwood, NY, USA) and monolayer cells were captured and processed
using a microscope and associated software (OLYMPUS, model BX53, software
Image-Pro Plus version 7.0, Center Valley, PA, USA).
Evaluation of immunofluorescence staining
For apoptosis analysis, all cleaved caspase-3 positive cells were counted in three
different photomicrographs (100X magnification) per treatment group. Results were
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quantified as percent of apoptotic monolayer and 3D structures. The number of
cleaved caspase-3 positive cells was normalized to the area of photomicrography.
IL-17RB and VEGF-A proteins was quantified according to Jardim-Perassi et
al. [29]. In summary, three different photomicrographs taken at 100X magnification
under bright field and the intensity of the staining was quantified by Image J Software
(NIH, Bethesda, MD, USA). Each photograph was divided into four quadrants and 20
spots (small circular ROI) were randomly selected (avoiding the nucleus) in each
photomicrograph. A negative control section of the corresponding staining was used
for measuring background activity. The values were obtained in arbitrary units (a.u.)
and represented as the mean optical density (M.O.D.) for each sample.
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Protein extraction
In order to reaffirm melatonin action in apoptosis pathway in triple negative
hormone receptors cells it was performed a membrane array analysis in MDA-MB-231
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cells. Cells were plated in individual wells of 1.88 cm2 (24-well plate) at 0.5 x 106 cells
confluence and inoculated with 500 µL of normal growth medium overnight.
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Thereafter, cells were treated with or without 1 mM of melatonin for 48 hours and
performed protein extraction of adherent ad supernatant cells. Cells were washed in ice-
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cold PBS and lysed with MILLIPLEX® MAP lysis buffer supplemented with 1 mM of
phosphatase inhibitor cocktail (Na3VO4) (Sigma-Aldrich, St. Louis, MO, EUA) and
1:10 of protease inhibitor (Sigma-Aldrich, St. Louis, MO, EUA). After incubation for
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30 minutes with intermittent vortexing, the cell lysate was centrifuged and the proteins
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collected on supernatant. Concomitantly the supernatant medium was collected and
used ultrafiltration (Amicon®, EMD Millipore, Billerica, MA) to concentrate the
proteins. The supernatant was included in columns containing filter of three kDa,
centrifuged and posteriorly larger proteins were retained in filter and subsequently
quantified.
Proteins extracts were quantified by the bicinchoninic acid (BCA) protein assay
kit (PIERCE - Thermo Scientific - Thermo Fisher Scientific, Waltham, MA, USA).
Membrane array
The membrane Human Apoptosis Array C1 (RayBiotech, Norcross, GA, EUA)
was incubated with 2 mL of 1X blocking solution buffer (RayBiotech, Norcross, GA,
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EUA) for 30 minutes. Treated and control samples were added in a concentration of 600
µg (300 µg of lisate cells and 300 µg of supernatant cells) and incubated in membrane
at 4 ºC overnight. Membrane was washed three times with wash buffer 1X (RayBio I,
RayBiotech, Norcross, GA, EUA) and two times with wash buffer 1X (RayBio II,
RayBiotech, Norcross, GA, EUA) for five minutes each. Biotin conjugate anticytokines (RayBiotech, Norcross, GA, EUA) was added and the samples incubated at 4º
overnight. The membrane was wash again and then incubated with horseradish
peroxidase (HRP) estreptavidina 1000X (RayBiotech, Norcross, GA, EUA) solution at 4
°C overnight. The membrane was wash and incubated with detection solution
(RayBiotech, Norcross, GA, EUA) for two minutes and exposed to ChemiDoc system
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(BioRad, Hercules, CA, EUA).
Optical density reference to protein expression was normalized with positive
control and quantification was performed using ImageJ Software (NIH, Bethesda, MD,
USA) as image analyzer. The values were obtained in a.u. and represented as the
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M.O.D. for each sample. All samples were included in duplicate plus positive and
negative membrane controls.
Statistical analysis
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All data were expressed as mean ± standard error of mean (SEM). All statistical
analyses were done using GraphPad Prism4 (San Diego, CA, USA). Raw data was
initially subjected to descriptive analysis to determine the normal range. Normal range
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data were analyzed by two-way ANOVA, followed by Bonferroni test for MTT results
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and Student’s t-test was performance for another analysis. A p-value ≤ 0.05 was
considered significant.
RESULTS
Melatonin and IL-25 reduce cell viability of breast cancer cells and did not affect
MCF-10A cells
Breast cancer cell lines were subjected to MTT cell viability testing, after
being treated with melatonin and IL-25. Jardim-Perassi et al. [29] and [30] previously
showed that the MDA-MB-231 and MCF-7 cells were sensitive to 1 mM of melatonin
after 24 hours of incubation, showing a statistically significant reduction in cell viability
compared to control groups (p < 0.05). Following 48 hours of treatment, we previously
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found (Borin et al., 2015 submitted on Journal of Pineal Research - JPI-OM-06-150169) that the concentrations of 0.01 to 1 mM melatonin remained significantly
different for both cell lines when compared to control groups (p < 0.05). As the 1
mM concentration
showed
a significant
reduction
of
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was adopted
as the standard dose to other procedures.
Cell viability was tested with IL-25 at 1 ng/mL, 10 ng/mL and 50 ng/mL for 48
hours and were able to reduce cell viability in both breast cancer cell lines. For MDAMB-231 cells, biphasic effect was observed, with 1 ng/mL and 50 ng/mL of IL-25
causing a significant 25 % reduction in viability compared to the control group (73.92 ±
10.25 %; 72.54 ± 8.504 %; p < 0.05). For MCF-7 cells, the lowest dose of 1 ng/mL of
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IL-25 significantly reduced cell viability by 60 % (38.59 ± 8.911 %; p < 0.05) (Fig. 1A
and B).
Neither melatonin or IL-25 treatments caused a significant reduction in viability
compared to control in MCF-10A cells, when the effective doses for MDA-MB-231 and
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MCF-7 cells were chosen (p > 0.05) (Fig. 1C). These results suggest that melatonin and
IL-25 preferentially target transformed cells.
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Effective gene silencing of IL-17B
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In order to test our hypothesis that a reduction in IL-17B level may lead to
increased tumor cell apoptosis by enhancing IL-25 binding to the IL-17RB receptor, IL17B gene silencing was performed, as there is no specific inhibitor for this interleukin.
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Silencing of the positive control, MAPK-1, was carried out to optimize the
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concentration and incubation period of the transient transfection. The positive control
was effective at 10 nM in both breast cancer cell lines with 74.0 % for MDA-MB-231
cells and 67.0 % for MCF-7 cells following 48 hours after transfection (data not
shown). siRNA #2 and #3 were effective for IL-17B silencing for MDA-MB-231 cells
with the best silencing (44 %) obtained with siIL-17B #2 (Fig. 2A). For MCF-7 cells,
the silencing gene was effective for siRNA #2, #3 and #4, with 66 % of silencing for
siIL-17B #2 (Fig. 2B).
Induction of apoptosis after treatment with melatonin, IL-25 and siIL-17B
To test the hypothesis that IL-25 added to melatonin enhances breast cancer
cell apoptosis, breast cancer cell lines were cultured with 1 mM of melatonin, 1 ng/mL
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of IL-25 and 10 nM of siIL-17B #2 as well as its control groups, for 48 hours and the
caspase-3 expression was evaluated.
Those three treatments separated and associated were effective in increasing
the caspase-3 gene expression compared to control groups in MDA-MB-231 cells
(melatonin, p < 0.0001; IL-25, p < 0.0001; siIL-17B, p = 0.0001 and treatments
associated, p = 0.0002). Furthermore, MDA-MB-231 cells treated for 48 hours showed
a 3-fold higher average of positive apoptotic cells compared to control groups for all
treatments (37.1 % versus 12.8 %, p < 0.05; Fig. 3). However, the combined treatments
did not further increase apoptosis compared to individual treatments.
Similarly, melatonin, IL-25 and siIL-17B treatments for 48 hours, alone and
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combined, were effective at increasing the caspase-3 gene expression compared to
control groups in MCF-7 cells (melatonin, p = 0.002; IL-25, p = 0.01; siIL-17B, p =
0.01 and treatments associated, p = 0.02). The average percent of apoptotic cells after
48 hour of individual and combined treatments was 4-fold higher compared to control
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groups (57.5 % versus 13.0 %, p < 0.05; Fig. 4). For MCF-7 cells, combined treatment
showed pro-apoptotic advantage once resulted in an average 87 % enhancement of the
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protein levels of cleaved caspase-3, compared to the 47 % increase observed with each
treatment alone. Although this is largely due to melatonin treatment, that showed 63 %
of apoptotic cells.
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The pro-apoptotic effect of treatments is enhanced in 3D cultures
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As an alternative to classical in vitro studies with cells grown as monolayers
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studies, the 3D culture mimics tissue architecture in vivo, possibly predicting better
cellular response in actual tumors [31] . 3D culture permits cells to explore the three
dimensions of the space thereby increasing cell-cell interactions, as well as interactions
with the microenvironment [31]. Thus, we aimed to characterize the increase expression
of cleaved caspase-3 in 3D structures of breast cancer cells.
An increase of intensity in nuclei labeling of cleaved caspase-3 in different
sequential planes image in MDA-MB-231 and MCF-7 cells was found (Fig. 5A and B).
Treatment with 1 mM of melatonin was capable to enhance apoptotic 3D structures in
31 % for MDA-MB-231 cells and 24 % for MCF-7 (p = 0.05; p = 0.002, respectively),
IL-25 treatment enhanced apoptotic 3D structures in 20 % for MDA-MB-231 cells and
26.4 % for MCF-7 (p = 0.04; p = 0.01, respectively), siIL-17B was more effectively in
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enhanced apoptotic 3D structures for 68 % for MDA-MB-231 cells and 74 % for MCF7 (p < 0.0001; p < 0.0001, respectively) and, treatments combined not shown
significantly increase of apoptotic 3D structures in MDA-MB-231 cells and a minor
enhance was observed in MCF-7 cells (30 % combined treatments versus 11 % control
group; p = 0.01) (Fig. 5C and D).
Melatonin modulate apoptotic proteins in MDA-MB-231 cells
Apoptosis related factors (Table 1) were estimated in MDA-MB-231 cells using
a membrane protein array kit. Treatment with 1 mM of melatonin for 48 hours in this
cells showed increase of pro-apoptotic proteins compared to control group, including
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cytochrome c (CYTO-C), death receptor 6 (DR6), growth factor insulin-binding
protein-3 (IGFBP-3), IGFBP-5, IGFPB-6, insulin-like growth factor 1 (IGF-1), IGF-1
receptor (IGF-1R), Livin, P21, P53, tumor necrosis factor receptor II (TNFRII),
apoptosis inhibitory protein X (XIAP) and high temperature required A (hTRA) (p <
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0.05) and a reduction of caspase-3 was observed (p = 0.04) (Fig. 6).
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Decrease expression of VEGF after treatment with melatonin, IL-25 and siIL-17B
Therefore, we tested the influence of 48 hours treatment with melatonin, IL-25
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and siIL-17B separate or in combination, on VEGF-A RNA and protein expression via
qRT-PCR and immunofluorescence, respectively.
It was observed an increase VEGF-A mRNA expression in MDA-MB-231
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cells after treatment with IL-25, siIL-17B and combined treatments (p = 0.006; p =
0.002; p = 0.006, respectively), though the protein levels of this factor were
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significantly reduced by the same treatments (IL-25 10.67 ± 0.3982 u.a., p < 0.0001;
siIL-17B 13.68 ± 0.4615 u.a., p < 0.0001; treatment combined 21.09 ± 0.7657 u.a., p <
0.0001), as well as melatonin (15.98 ± 0.6371 u.a., p < 0.0001) compared to control
groups (Fig. 7).
For MCF-7 cells, VEGF-A mRNA decreased after treatment with melatonin (p =
002). In contrast, IL-25, siIL-17B and combined treatments increased VEGF-A gene
expression compared to control groups (p < 0.0001; p < 0.0001, p < 0.0001,
respectively). VEGF-A protein levels were reduced after 48 hours of incubation with all
treatments compared to control groups (melatonin 17.11 ± 0.5092 u.a., p < 0.0001; IL-
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25 14.36 ± 0.4136 u.a., p < 0.0001; siIL-17B 17.93 ± 0.7189 u.a., p = 0.004; treatment
combined 14.89 ± 0.5993 u.a., p < 0.0001; Fig. 8).
DISCUSSION
A protective role of melatonin has been showed in various pathophysiological
situations, including numerous tumor types in in vitro as well as in vivo studies [32],
[33], [34], [35], [36], [37], [8], [38], [39], [40], [41], [42], [43]. The concentration of
melatonin in those studies is variable (physiological 10-9 M and pharmacological 10-3 M
or higher) as well as treatment times. The detected concentration of melatonin in plasma
at night is approximately 0.1 nM, however, as an example, concentrations of the
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indoleamine throughout the body are not in equilibrium and intracellular levels may be
several orders of magnitude higher than in blood [42].
Rodriguez et al. [44] found that 1 mM of melatonin did not decrease cell
viability by means of trypan blue assays, suggesting that decrease in cell number is due
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to a cytostatic and not a cytotoxic action of melatonin in this concentration. Besides
that, a systematic review of published randomized control trial studies have
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demonstrated that administration of different dosages of melatonin (20-40mg/day) (as
single agent or in combination with other drugs) is considered to be safe and is
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associated with a significant reduction in risk of death within 1 year in a range, for solid
cancers [15], [45].
In this study we confirm that pharmacological level of melatonin was able to
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reduce cell viability of ER-negative and ER-positive breast cancer cells. Several authors
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have demonstrated the ability of melatonin to inhibit proliferation of MCF-7 cells [46],
[47], [48], [32], [49], [50], [51]. Besides that, the reduction of viable ER-negative cells
found in this study is in agreement with studies of Leman et al. [52] and Jardim-Perassi
et al. [29] that showed significant melatonin decreased proliferation of MDA-MB-231
cells after 24 hours treatment. In contrast, Eck et al. [48] showed that melatonin did not
affect ER-negative breast cancer cells.
The mechanisms of the physiological melatonin action include the binding to
melatonin receptors located in the cell membrane, intracellular proteins, as a
calmodulin, interactions with nuclear receptors and oxidant activity [53]. According to
Dubocovich et al. [54], melatonin’s biological activity can be mediated via two major
mechanisms: G-protein coupled receptor (GPCR) - mediated activity (MT1 and MT2
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receptor) and non-receptor–mediated antioxidant activity, because melatonin is highly
liposoluble [55] . However, the antiproliferative effects of melatonin mediated through
the estrogen-response transactivation by the MT1 activation is the most described [56],
[51], [57].
Melatonin can bind and activate the MT1 and MT2 in a variety of tissues [58].
MT1 receptors belong to the G-protein coupled receptor superfamily and when mediate
of a subunit of G protein, MT1 receptors inhibits the activity of adenyl cyclase,
decreasing the production of adenosine 3′,5′-cyclic monophosphate (cAMP) making
possible to control the activity of selected protein kinases (PKC, PKA, MAPK), as well
as expression of specific genes, involved in proliferation, angiogenesis, cell
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differentiation and migration processes [53]. Both MDA-MB-231 and MCF-7 tumor
cell lines express MT1 receptor, however, Opreas-Ilies et al. [59] and Jablonska et al.
[53] showed lower expression of MT1 receptors in triple negative phenotype breast
cancers when compared to ER-positive cases.
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The activation of the MT1 receptor appears to mediate some of melatonin’s
oncostatic actions in ER-positive MCF-7 human breast cancer cells, according to our
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results. In addition, Ram et al. [60] suggests that pharmacologic concentrations of
melatonin may activate other pathways that are possibly non-receptor mediated and
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which override melatonin’s growth-inhibitory effects, which could explain its action in
MDA-MB-231 cells, supported in our study.
We also found that 1 ng/mL of IL-25 was capable to reduce the viability of both
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MDA-MB-231 and MCF-7 tumor cells. The dose-dependent reduction in cell viability
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Page 14 of 35
of MDA-MB-231 to IL-25 treatment has not been reported until now; although Furuta
et al. [26] showed that MDA-MB-468, a high metastatic and ER-negative cell, and
MCF-7 cells are responsive to treatment with 10 ng/mL of IL-25, a higher concentration
than that used by this study. In addition, Benatar et al. [24] demonstrated that IL-25 has
antitumor efficacy in a xenograft model of human melanoma, suggesting its potential
use as a cancer therapeutic. However, the mechanisms by which IL-25 acts in tumor
inhibition are not yet understood.
On the other hand, we found the absence of an effect of both melatonin and IL25 on MCF-10A viability, indicates no cytotoxic potential of these agents in normal
luminal breast cells. This is in agreement with results by Furuta et al. [26], which
showed virtually absent expression of IL-25R and resistance to IL-25 treatment in
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Page 15 of 35
MCF-10A cells. Whether or not the same is true for other normal cells in the body, thus
indicating a reduced probability of side effects, remains to be determined. Wong et al.
[61] showed that IL-25 enhances the viability of eosinophils for up to 36 hours in an
allergic asthma study, and Benatar et al. [24] reported that IL-25 acts a survival factor in
eosinophils, blocking apoptosis.
Demonstrating the effect of the proposed treatments on apoptosis, we observed
enhanced cleaved caspase-3 protein after each treatment alone and the three combined,
in both MDA-MB-231 and MCF-7 monolayer cells and 3D structures, which confirms
the influence of the treatments in intrinsic (mitochondrial-mediated) and extrinsic
(receptor-mediated) apoptosis pathways. Melatonin was previously shown to reduce
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proliferation of cancer cells by different mechanisms, including cellular differentiation
and apoptotic cell death [62], [63], [51], [42], [43], and corroborating with our results.
Di Bella et al. [11] and Sanchez-Hidalgo et al. [45] showed that the direct anti-tumor
effect of melatonin occurs via caspase activation. Besides that, Rodriguez et al. [42]
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confirms that both apoptosis pathway could be activated by melatonin in cancer cells
but not in normal cells.
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Confirming anti-tumor activity of melatonin as pro-apoptotic agent [109-113] in
breast tumor metastatic cells, we also demonstrating an increase of proteins involved in
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extrinsic signaling apoptosis pathway as TNF-RII and DR6 and in intrinsic, as CYTOC. The CYTO-C has a crucial role in apoptosis. When stimulated, it is released into the
cytosol where it binds to apoptotic protease activating factor 1 (APAF-1) and pro-
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caspase-9 to the apoptosome formation [64] [65] . Wang et al. [66] demonstrated that
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melatonin induces the expression of APAF-1 and stimulates the release of CYTO-C,
causing the activation of caspases and inducing apoptosis in breast cancer cells.
Moreover, melatonin’s treatment was also capable to induce p21 and p53. According by
Hill et al. [55], their review showed that Mediavilla et al. [14] and other authors,
observed that physiologic concentrations of melatonin reduced the in vitro proliferation
of breast cancer cells by elongating cell cycle length by controlling the p53/p21
pathway.
Growth factors as IGF-1, IGF-1R and IGFBP-3, -5 and -6 showed increased
after melatonin treatment, corroborating with our previous results [29]. Kim et al. [67]
suggest that IGFBP plays an important role in cell differentiation, controlling growth
and apoptosis. Butt et al. [68] showed that IGFBP-5 was associated with a
15
transcriptional increase in expression of the proapoptotic regulator BAX and decrease in
the anti-apoptotic BCL-2, being able to activate caspase-8 and -9 in MDA-MB-231
cells. Interestingly, Livin, XIAP and hTRA expression shown to be increased after
treatment with melatonin, which could contribute to the survival of tumor cells for their
apoptosis inhibitors activity, although it is believed that this effect occurs as a
compensatory mechanism to the major apoptotic stimulus of melatonin.
Regarding caspase-3, low protein expression was found in membrane array and,
on the other hand, its cleaved form (active) showed high protein expression by
immunofluorescence. A known explanation for this decrease is the post-translationally
regulation of entire caspase pathway, wherein caspase-3 is converted into its cleaved
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form by melatonin treatment, resulting an increase of this form and reduction of its
inactive structure.
We also observed here enhanced apoptosis in both breast cancer cell types after
addition of IL-25 and siIL-17B, which is in agreement with observations by Huang et
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al. [27], indicating that binding of IL-25 to IL-17RB induces apoptosis in breast cancer
cells, whereas IL-17B binding to IL-17RB transduces pro-survival signaling. Furuta et
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al. [26] confirmed that IL-25 treatment in MDA-MB-468 caused cleavage of caspases3, 8 and PARP, supporting our findings.
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Given that melatonin can suppresses angiogenesis directly or indirectly [69],
[70], [41], and its anti-angiogenic properties have been reported both in in vivo and in
vitro models [71], [72], we also looked at treatments effects of VEGF-A expression.
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VEGF is the most active endogenous pro-angiogenic factor, and it is a specific
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Page 16 of 35
endothelial cell mitogen, also promoting microvascular permeability.
We observed VEGF-A protein reduction after melatonin treatment. Our results
with MCF-7 are in agreement with a study by Dai et al. [69], which evaluated VEGF
concentration in MCF-7 cells culture media, and found a decrease of protein expression
in response to 1 nM and 1 mM of melatonin. Carbajo-Pescador et al. [41] proposed that
melatonin effects occur at a post-transcriptional level, what could explains the high,
possibly compensatory, VEGF-A mRNA expression after melatonin treatment in MDAMB-231 cells, opposed to the reduced VEGF-A protein expression caused by the same
treatments in our study. In addition, to suppressing VEGF protein expression, JardimPerassi et al. [29] reported lower expression of VEGFR2 and VEGFR3 in melatonintreated tumors compared to vehicle-treated tumors. It has been recently suggested by
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Ordoñez et al. [8] that melatonin tumor suppression occurs in part via inhibition of HIF1α-mediated angiogenesis, due to its capacity to suppress HIF-1α transcriptional activity
under hypoxic conditions.
In our study, IL-25 and siIL-17B treatments also reduced VEGF-A protein
expression in breast cancer cells, although addition of these treatments to melatonin did
not significantly enhances the effect of the later. It is noteworthy that there are few
studies looking at IL-25 on angiogenesis, given the impact of angiogenesis suppression
on tumor cell apoptosis in vivo. However, IL-17 family expression has been correlated
with high microvessel density in human ovarian cancer [73], human hepatocellular
carcinoma [74] and Non-small-cell lung carcinoma [75]. According to Liu et al. [76]
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and Maniati and Hagemann [77], IL-17 could modulate angiogenesis directly (through
IL-17R binding on endothelial cells), or indirectly, by stimulating cancer cells to
produce angiogenic factors. In this study was demonstrated that synthetic IL-17 can
induce the expression of the pro-angiogenic factors VEGF, PLAU, NRP2, IL-6 and ID3
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in colorectal cancer cells, as determined by qRT-PCR and ELISA of conditioned media.
Complementing the aforementioned studies, here we demonstrate that enhanced
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IL-17E/IL-25 bioavailability, either by adding IL-25 to the media or reducing IL-17B
expression, induces apoptosis and downregulate VEGF-A protein expression.
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Interestingly, we did not observe any enhancement of apoptosis by combining
engagement of the IL-25/IL-17RB signaling with melatonin. One explanation for this
could be an overlapping mechanism of action of the two approaches, or inadequate
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timing of combination of the different targeting modalities; e.g., depending on the
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mechanisms of action of each targeting modality, consecutive rather than simultaneous
targeting may be more effective. A better understanding of the mechanism of action of
IL-25 will facilitate the design of in vitro and in vivo tumor targeting strategies in the
future. The fact that more robust apoptotic responses were observed when cells were
grown as 3D structures on matrigel suggests that a better recreation of the tumor
microenvironment in vitro will be beneficial to adequately address the therapeutic
potential of an IL-25-melatonin cocktail in breast cancer.
Our study proved the independent efficacy of melatonin and IL-25 in cell
viability of ER-positive and triple negative breast cancer cells and no influence in nontumorigenic cells. In addition, melatonin treatment and the modulation of interleukins
25/17E and 17B promote apoptosis via extrinsic and intrinsic pathways and reduce
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VEGF-A protein expression in breast cancer cells. Taken together, these results show
that combined treatments do not enhance the individual effects, proving the potential
therapeutic effectiveness of the melatonin and IL-25/siIL-17B as individual agents in
breast cancer.
INTEREST CONFLICT
Author declares no interest conflict.
ACKNOWLEDGMENTS
We thank Lívia Carvalho Ferreira, Camila Leonel and Gustavo Rodrigues
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Martins for provided writing services and language editoring. Patricia Simone Leite
Vilamaior for help in acquiring images in confocal microscope. We also thank
Fundacao de Amparo a Pesquisa do Estado de Sao Paulo – FAPESP (grants n°
2012/06098-0 and 2012/02128-1) and Fundacao de Apoio a Pesquisa e Extensao de Sao
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Jose do Rio Preto – FAPERP (grant nº 175/2014) which funded this research,
Laboratory of Molecular Research in Cancer (LIMC) and Laboratory for Integrated
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Study of the Mechanisms of Breast Cancer Invasion and Metastasis for providing
material and structure to carry out this project and Canadian Foundation for Innovation
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(CFI) - Ministry of Research Infrastructure (MRI, Ontario) funds to A.V.P.
TABLE LEGEND
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FIGURE LEGENDS
Fig. 1. Effect of melatonin and interleukin-25 on viability of cell lines. A) MDA-MB231 and B) MCF-7 cells were treated with 1 ng/mL, 10 ng/mL and 50 ng/mL of IL-25
for 48 hours and cell viability was measured by MTT assay. C) MCF-10A cells were
treated with 1 mM of melatonin and 1 ng/mL of IL-25 for 48 hours and cell viability
was measured by MTT assay. The white column corresponds to control group. Each
column represents the mean ± standard error of triplicate experiments. (*p ≤ 0.05).
Statistical significance compared to control group was determine by ANOVA followed
by Bonferroni test.
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Fig. 2. Gene silencing of IL-17B. Gene expression was measured by qRT-PCR at 48
hours post-transfection of cells with 4 different IL-17B siRNAs (#1 to #4) or a
Scramble siRNA (Control: white colum) A) MDA-MB-231 cells showed 44 % gene
silencing with 10 nM of siIL-17B #2. B) MCF-7 cells showed 66 % gene silencing with
10 nM of siIL-17B #2.
Fig. 3. Cleaved caspase-3 expression in MDA-MB-231. A) Increase gene expression of
caspase-3 after treatments compared to control groups. B) Average percentage of
apoptotic cells for each treatment. C) Photomicrographs of immunofluorescence
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staining for cleaved caspase-3 in MDA-MB-231 cells. Magnification= 100X. Cleaved
caspase-3 (green) and nuclei DAPI (blue). Each column represents the mean ± standard
error of triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s ttest. *p<0.01 **p<0.001 ***p<0.0001.
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Fig. 4. Cleaved caspase-3 expression in MCF-7.
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A) Increase gene expression of
caspase-3 after treatments compared to control groups. B) Average percentage of
apoptotic cells for each treatment. C) Photomicrographs of immunofluorescence
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staining for cleaved caspase-3 in MCF-7 cells. Magnification= 100X. Cleaved caspase3 (green) and nuclei DAPI (blue). Each column represents the mean ± standard error of
triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s t-test.
*p<0.01 **p<0.001 ***p<0.0001.
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Fig. 5. Cleaved caspase-3 expression in MDA-MB-231 and MCF-7 3D structures. A)
Photomicrographs of immunofluorescence staining for cleaved caspase-3 in MDA-MB231 3D structures and B) MCF-7 3D structures. C) Average percentage of apoptotic
cells for each treatment in MDA-MB-231 3D structures and D) MCF-7 3D structures.
Magnification= 100X. Each column represents the mean ± standard error of triplicate
experimens (*p ≤ 0.05). Significance was determined by Student’s t-test. *p<0.01
**p<0.001 ***p<0.0001.
Fig. 6. Differentially apoptotic protein expression in MDA-MB-231 cells treated with 1
mM of melatonin. The white column corresponds to control groups. Each column
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represents the mean ± standard error of duplicate experiments. (*p ≤ 0.05). Significance
was determined by Student’s t-test.
Fig. 7. VEGF-A expression in MDA-MB-231. A) Decrease VEGF-A gene expression
after treatments compared to control groups. B) Quantification of VEGF-A protein
expression showing decrease cytoplasm labeling after treatments, compared to control
groups for each treatment. C) Photomicrographs of immunofluorescence staining for
VEGF-A in MDA-MB-231 cells. Magnification= 100X. VEGF-A (green) and nuclei
DAPI (blue). Each column represents the mean ± standard error of triplicate experimens
(*p ≤ 0.05). Significance was determined by Student’s t-test. *p<0.01 **p<0.001
***p<0.0001.
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Fig. 8. VEGF-A expression in MCF-7. A) Decrease VEGF-A gene expression after
treatments compared to control groups. B) Quantification of VEGF-A protein
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expression showing decrease cytoplasm labeling after treatments, compared to control
groups for each treatment. C) Photomicrographs of immunofluorescence staining for
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VEGF-A in MCF-7 cells. Magnification= 100X. VEGF-A (green) and nuclei DAPI
(blue). Each column represents the mean ± standard error of triplicate experimens (*p ≤
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0.05). Significance was determined by Student’s t-test. *p<0.01 **p<0.001
***p<0.0001.
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25
Apoptotic Factors
Melatonin
Treatment
Apoptotic Factors
Melatonin
Treatment
BAD
ns
BAX
ns
CD40
ns
BCL-2
ns
CD40 LIGAND
ns
BCL-W
ns
BIRC-3
ns
BID
ns
CYTO C
↑ p=0.04
BIM
ns
↑ p=0.02
CASPASE-3
↓ p=0.04
ns
CASPASE-8
ns
FAS LIGAND
ns
HSP27
ns
IGFBP-1
ns
HSP60
ns
ns
HSP70
ns
↑ p=0.03
hTRA
↑ p=0.04
IGF-1
↑ p=0.02
ns
DR6
FAS
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IGFBP-2
IGFBP-3
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Pe
IGFBP-4
ns
IGFBP-5
↑ p=0.02
IGF-2
IGFBP-6
↑ p=0.01
IGF-1R
↑ p=0.05
TNF-RII
↑ p=0.04
TNF-α
ns
TNF-β
ns
TRAIL-R1
Re
↑ p=0.04
P53
↑ p=0.04
SMAC
ns
ns
SURVININ
ns
TRAIL-R2
ns
TNF-RI
ns
TRAIL-R3
ns
XIAP
↑ p=0.04
TRAIL-R4
ns
LIVIN
P21
↑ p=0.03
P27
ns
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Page 26 of 35
BAD: Bcl-2-antagonist of cell death; BAX: Bcl-2-associated X protein; BCL-2: B-cell lymphoma 2;
BCL-W: Bcl-2-like protein 2; BID: BH3 interacting-domain death agonist; BIM: Bisindolylmaleimidebased protein kinase C (PKC) inhibitors; BIRC-3: Baculoviral IAP repeat-containing protein 3;
CASPASE-3: Cysteine-aspartic acid protease 3; CASPASE-8: Cysteine-aspartic acid protease 8; CD40:
Cluster of differentiation 40; CD40 LIGAND: Cluster of differentiation 40 ligand; CYTO C:
Cytochrome c; DR6: Death receptor 6; FAS: First apoptosis signal; FAS LIGAND: First apoptosis signal
ligand; HSP27: Heat shock protein 27; HSP60: Heat shock protein 60; HSP70: Heat shock protein 70;
hTRA: High-temperature requirement A serine peptidase; IGF-1: Insulin-like growth factor 1; IGF-2:
Insulin-like growth factor 2; IGFBP-1: Insulin-like growth factor-binding protein 1; IGFBP-2: Insulinlike growth factor-binding protein 2; IGFBP-3: Insulin-like growth factor-binding protein 3; IGFBP-4:
Insulin-like growth factor-binding protein 4; IGFBP-5: Insulin-like growth factor-binding protein 5;
IGFBP-6: Insulin-like growth factor-binding protein 6; IGF-1R: Insulin-like growth factor receptor 1;
Page 27 of 35
Livin; P21: Cyclin-dependent kinase inhibitor 1; P27: Cyclin-dependent kinase inhibitor 1B; P53: Tumor
protein p53; SMAC: Second mitochondria-derived activator of caspases; Survinin; TNF-α: Tumor
necrosis factor alpha; TNF-β: Tumor necrosis factor beta; TNF-RI: Tumor necrosis factor receptor 1;
TNF-RII: Tumor necrosis factor receptor 2; TRAIL-R1: TNF-related apoptosis-inducing ligand receptor
1; TRAIL-R2: TNF-related apoptosis-inducing ligand receptor 2; TRAIL-R3: TNF-related apoptosisinducing ligand receptor 3; TRAIL-R4: TNF-related apoptosis-inducing ligand receptor 4; XIAP: Xlinked inhibitor of apoptosis protein. All antibodies are prepared in duplicate. ns: no significant.
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Fig. 1. Effect of melatonin and interleukin-25 on viability of cell lines. A) MDA-MB-231 and B) MCF-7 cells
were treated with 1 ng/mL, 10 ng/mL and 50 ng/mL of IL-25 for 48 hours and cell viability was measured by
MTT assay. C) MCF-10A cells were treated with 1 mM of melatonin and 1 ng/mL of IL-25 for 48 hours and
cell viability was measured by MTT assay. The white column corresponds to control group. Each column
represents the mean ± standard error of triplicate experiments. (*p ≤ 0.05). Statistical significance
compared to control group was determine by ANOVA followed by Bonferroni test.
94x92mm (300 x 300 DPI)
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Page 29 of 35
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Fig. 2. Gene silencing of IL-17B. Gene expression was measured by qRT-PCR at 48 hours post-transfection
of cells with 4 different IL-17B siRNAs (#1 to #4) or a Scramble siRNA (Control: white colum) A) MDA-MB231 cells showed 44 % gene silencing with 10 nM of siIL-17B #2. B) MCF-7 cells showed 66 % gene
silencing with 10 nM of siIL-17B #2.
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Fig. 3. Cleaved caspase-3 expression in MDA-MB-231. A) Increase gene expression of caspase-3 after
treatments compared to control groups. B) Average percentage of apoptotic cells for each treatment. C)
Photomicrographs of immunofluorescence staining for cleaved caspase-3 in MDA-MB-231 cells.
Magnification= 100X. Cleaved caspase-3 (green) and nuclei DAPI (blue). Each column represents the mean
± standard error of triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s t-test.
*p<0.01 **p<0.001 ***p<0.0001.
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Fig. 4. Cleaved caspase-3 expression in MCF-7. A) Increase gene expression of caspase-3 after treatments
compared to control groups. B) Average percentage of apoptotic cells for each treatment. C)
Photomicrographs of immunofluorescence staining for cleaved caspase-3 in MCF-7 cells. Magnification=
100X. Cleaved caspase-3 (green) and nuclei DAPI (blue). Each column represents the mean ± standard
error of triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s t-test. *p<0.01
**p<0.001 ***p<0.0001.
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Fig. 5. Cleaved caspase-3 expression in MDA-MB-231 and MCF-7 3D structures. A) Photomicrographs of
immunofluorescence staining for cleaved caspase-3 in MDA-MB-231 3D structures and B) MCF-7 3D
structures. C) Average percentage of apoptotic cells for each treatment in MDA-MB-231 3D structures and
D) MCF-7 3D structures. Magnification= 100X. Each column represents the mean ± standard error of
triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s t-test. *p<0.01 **p<0.001
***p<0.0001.
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Fig. 6. Differentially apoptotic protein expression in MDA-MB-231 cells treated with 1 mM of melatonin. The
white column corresponds to control groups. Each column represents the mean ± standard error of duplicate
experiments. (*p ≤ 0.05). Significance was determined by Student’s t-test.
207x66mm (300 x 300 DPI)
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Fig. 7. VEGF-A expression in MDA-MB-231. A) Decrease VEGF-A gene expression after treatments
compared to control groups. B) Quantification of VEGF-A protein expression showing decrease cytoplasm
labeling after treatments, compared to control groups for each treatment. C) Photomicrographs of
immunofluorescence staining for VEGF-A in MDA-MB-231 cells. Magnification= 100X. VEGF-A (green) and
nuclei DAPI (blue). Each column represents the mean ± standard error of triplicate experimens (*p ≤ 0.05).
Significance was determined by Student’s t-test. *p<0.01 **p<0.001 ***p<0.0001.
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Fig. 8. VEGF-A expression in MCF-7. A) Decrease VEGF-A gene expression after treatments compared to
control groups. B) Quantification of VEGF-A protein expression showing decrease cytoplasm labeling after
treatments, compared to control groups for each treatment. C) Photomicrographs of immunofluorescence
staining for VEGF-A in MCF-7 cells. Magnification= 100X. VEGF-A (green) and nuclei DAPI (blue). Each
column represents the mean ± standard error of triplicate experimens (*p ≤ 0.05). Significance was
determined by Student’s t-test. *p<0.01 **p<0.001 ***p<0.0001.
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93
Artigo II
94
Melatonin and IL-25 induces pro-apoptotic and anti-angiogenic factors in canine
mammary tumor cell lines
gabi_b_g@yahoo.com.br
1,2
Thaiz Ferraz Borin 2
thaiz80@yahoo.com.br
Larissa Bazela Maschio 2
larissa_maschio@hotmail.com
Marina Gobbe Moschetta 2
marinagobbe@hotmail.com
Eva Hellmén3
Eva.Hellmen@slu.se
Alicia M. Viloria-Petit4
aviloria@uoguelph.ca
Debora Aparecida Pires de Campos Zuccari1, 2 *
1 Universidade
Estadual Paulista “Júlio de Mesquita Filho” (UNESP/IBILCE), Programa
de Pós-Graduação em Genética, São José do Rio Preto, SP, Brasil.
2 Faculdade de Medicina de São José do Rio Preto (FAMERP). Laboratório de
Investigação Molecular do Câncer (LIMC), São José do Rio Preto, SP, Brasil.
3 Department of Anatomy, Physiology and Biochemistry of the Swedish University of
Agricultural Sciences, Faculty of Veterinary Medicine, Uppsala, Sweden.
4 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada.
*Corresponding author. Profa. Dra. Debora Ap. Pires de Campos Zuccari, Laboratório
de Investigação Molecular do Câncer (LIMC), Faculdade de Medicina de São José do
Rio Preto (FAMERP), Avenida Brigadeiro Faria Lima, 5416, Vila São Pedro, São José
do Rio Preto (SP) 15090-000, Brasil. Tel.: 55 (17) 32015885. e-mail:
95
ABSTRACT
Introduction: Mammary tumors are the most prevalent in female dogs and its
development involve changes in tumor microenvironment. Melatonin, a hormone
produced by the pineal gland, has oncostatic action in cancer and can acts in tumor
microenviroment, taking part in interleukins (ILs) modulation. IL-25 is an active
cytokine in inflammatory processes that induce apoptosis in tumor cells by differential
expression of its receptor (IL-17RB) and the ligand (IL-17B), contributing to their
tumorigenic potential. The aim of this study was to evaluate the potential of melatonin
and IL-25 as pro-apoptotic and anti-angiogenic agents in canine mammary tumor cells.
Methods: Metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumor
cell lines were cultured in monolayer and tridimensional structures on extracellular
matrix laminin-rich and treated for 48 hours with melatonin and IL-25. Also, silencing
gene of IL-17B was performed. Cell viability was measured by MTT assay, gene and
protein expression of caspase-3 and VEGF-A by qRT-PCR and immunofluorescence,
respectively. Apoptosis membrane array was performed in CF-41 cells after melatonin
treatment. Results: Treatment with 1 mM of melatonin were able to reduce cell viability
of both tumor cells (p < 0.05). All treatments alone and combined significantly
increased caspase-3 cleaved in tumor cells, growing as monolayers and 3D structures (p
< 0.05). The semi-quantitative analysis of proteins involved in the apoptotic pathway
showed an increase of proteins BCL-W and IGF-1 and -2, and a decrease of BAD,
IGFB-1 and TNF-RII (p < 0.05). For angiogenic marker, all treatments reduced VEGFA protein expression in both tumor cells. When three treatments were combined, no
advance in response for alone treatments was showed. Conclusion: Our data reinforce
the oncostatic, pro-apoptotic and anti-angiogenic effectiveness of melatonin, IL-25 and
siIL-17B modulation in mammary tumor cells as potential promising treatments to
breast cancer. Besides that, this is the first study in canine species evaluating the
potential use of these agents in mammary tumor cells and the results could be useful in
clinical practice.
Keywords: mammary
angiogenesis
tumor
cells,
canine,
melatonin,
interleukin-25,
apoptosis,
96
INTRODUCTION
According to the IARC [1] there are approximately 14.1 million new cancer
cases in world and 8.2 million cancer deaths. Breast cancer is the major cause of women
cancer mortality, and for female dogs, the prevalence of this type of cancer had increase
over the years, with malignant lesions varying from 26 to 73 %
[2-6]. Besides the
importance of this disease to the specie, mammary gland tumors share common features
between canine and humans and they are excellent models for human breast cancer
research and comparative studies in relation to breast cancer prognosis and treatment [6,
7].
Apoptosis or programmed cell death is evolutionarily conserved process that
plays an essential role in organism development and tissue homeostasis. In cancer
development, cells lose their ability to undergo apoptosis induced death leading to
uncontrolled proliferation [7]. Caspase-3 has been widely used as a marker protein in
the investigation of apoptosis in mammalian cells by representing the confluence of
intrinsic and extrinsic mechanisms of apoptosis and, once activated, this process
becomes irreversible [8]. Besides that, the continuous proliferation of the cancer cells
results in stress in the microenvironment of tumor and it causes release of certain
angiogenic factors as vascular endothelial growth factor (VEGF), which initiate the
formation of new blood vessels, playing a significant role in tumor progression [9].
There is a great interest in veterinary medicine to better monitor tumor
development
and
response
to
new
therapies
[2].
Melatonin
(N-acetyl-5-
methoxytryptamine), is a natural hormone primarily synthesized and secreted by
mammalian pineal gland
as by many other extra-pineal organs, including the retina,
gastrointestinal tract, skin, bone marrow and lymphocytes [10]. A large number of
studies support the role of melatonin in many cellular processes, such as regulating
system in haematopoiesis [11], oxidative damage
[12], anti-angiogenic activity [13],
anti-inflammatory activity [14], anticachectic properties [15], [16], apoptosis induction
[17] and immunostimulation [18].
Although various studies demonstrated a clear anticancer effect of melatonin, it
cannot alone cause a complete regression of tumor. Then, melatonin can act
synergistically (or additively) to potentiate the anticancer effects of other agents. The
97
administration of melatonin generates effects on immune non-specific cellular and
tumor response and is directly related to the modulation of immune activity on the
production of cytokines [19].
Cytokines are proteins that play an important role in the regulation of the body’s
inflammatory response to foreign agents and are related with cancer development.
These proteins include interleukins [e.g., interleukin (IL)-17E or 25], interferons and
tumor necrosis factors (TNFs) [20]. IL-25 is produced by neutrophils, promotes
inflammation and induces the production of inflammatory cytokine as IL-6, IL-1 and
TNF-α in endothelial cells. This cytokine is renowned for its role in the immune
response and it is known that the interaction with its receptor IL-25R induces apoptosis.
Little is known about this interaction, only that there is competition for the site of action
with IL-17B in neoplastic cells, contributing to tumor progression of tumor cells [21].
Until the moment, there are no studies evaluating the melatonin effects in
tumorigenesis of CF-41 and CMT-U229 cell lines alone or in association with other
agents. So, the purpose of this study was to determine the action of melatonin and IL-25
in canine mammary tumor cell lines, by the modulation of apoptosis and angiogenesis,
and explore the possible attenuated efficacy in combination with silencing gene of IL17B (siIL-17B), which could represent new therapeutic alternatives to suppress
mammary tumor progression.
METHODS.
Canine tumor cell lines culture
Metastatic canine mammary tumor cell line (CF-41) (ATCC, Manassas, VA,
USA) and mammary tumor cell line established from an atypical benign mixed
mammary tumor (CMT-U229) (provided by Dr. Eva Hellmén from the Department of
Anatomy, Physiology and Biochemistry of the Swedish University of Agricultural
Sciences) were cultured in 75 cm2 culture flasks (Sarstedt, Nümbrecht, Germany).
Dulbecco’s modified Eagle’s medium (DMEM) (Cultilab, Campinas, SP, Brazil) was
used for maintaining culture CF-41 cells and DMEM: Ham's F-12 (Cultilab, Campinas,
SP, Brazil) for CMT-U229 cells, both supplemented with 10 % fetal bovine serum
(FBS) (Cultilab, Campinas, SP, Brazil), penicillin (100 IU/mL) and streptomycin (100
98
mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in a
humidified incubator at 5.0 % CO 2 at 37 ºC until they were 80-90 % confluent.
Three-dimensional (3D) Matrigel culture assay
Sparse tumor single-cell suspensions were plated on individual wells of 8-well
chamber slides (Sarsted, Newton, NC, USA) previously covered with a 1-mm thick
layer of laminin-rich extracellular matrix (Matrigel® - Becton Dickinson, Franklin
Lakes, NJ, USA), at a concentration of 3.5 x 10 4 cells/0.5 mL. The Basal Human
Mammary Epithelial Cell (HuMEC) medium (Life Technologies, Eugene, OR, USA)
supplemented with the HuMEC Supplement Kit (Gibco® - Life Technologies, Eugene,
OR, USA) and 2 % Matrigel® were used. The cells were maintained in a humidified
incubator at 5 % CO 2 at 37 ºC for eight days until the formation of established 3D
structures, with treatments replenished every two days. The 3D morphogenesis was
monitored and analyzed using immunofluorescence and confocal microscopy.
Cell viability assessment by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
For MTT assay, individual wells of 0.31 cm2 (96-well plate) were inoculated
with 100 µL of regular growth medium containing 5 x 104 cells and incubated
overnight. After, the media was changed to 2.0 % FBS, containing increasing
concentrations of melatonin [obtained from Sigma-Aldrich (St. Louis, MO, USA) in
different concentrations 0.001 mM, 0.01 mM, 0.1 mM and 1 mM. Control wells
contained the highest concentration of the vehicle for melatonin treatment [1 %
dimethylsulfoxide
(DMSO) (Sigma-Aldrich, St. Louis, MO, USA)] was added.
Following 48 hours of the aforementioned treatments, 10 µL of MTT solution from the
Vibrant MTT Cell Proliferation Assay Kit (Invitrogen - Life Technologies, Eugene, OR,
USA) were added to each well and the plates were incubated at 37ºC for an additional 4
h. To solubilize the MTT formazan crystals, the cells were incubated with DMSO
(100%) and then incubated again at 37°C for 10 minutes. Absorbance was measured at
540 nm using an ELISA plate reader (Thermo Fisher Scientific - Waltham, MA USA).
Medium alone was used as blank and the corresponding optical density was subtracted
99
from the samples. Cell viability (%) was calculated for all groups relative to control
samples. All treatments were performed in triplicate.
Effective silencing of interleukin-17B
Previously, a search using gene-tool Blast (NCBI) for the mRNA sequence of
IL-17B in Canis lupus familiaris (NM_014443.2) was performed. After analysis, the
sequences showed 89 % homology to IL-17B gene Homo sapiens sapiens with the
canine specie. Then four sequences of mRNA isoforms to canine genome were
compared (Cat No. 1027416 - Quiagen), and the silencing rates found 100% identical to
#1
(CCAGAGAAAGTGTGAGGTCAA),
(CTGCTGTTTCTTCTTACCATT),
(TTGCACCTTTGTGCCAAGAAA)
90%
100%
and
identical
identical
94%
identical
to
to
#2
#3
to
#4
(CGGAATGGACTGGCCTCACAA). Thus, we concluded that the isoforms even being
made from the human genome are compatible with the canine genome.
For IL-17B gene silencing the different siRNA were tested (SI00106127,
SI00106134, SI02640652 and SI02640659) (ProSpec, East Brunswick, NJ, USA),
selected from preserved gene regions and thermodynamic stability according by
inventoried assay (Qiagen, Valencia, CA, USA). Individual wells of 1.88 cm2 (24-well
plate) were inoculated with 500 µL of normal growth medium containing 8 x 104 cells.
Subsequently the cells were transfected using the siRNA Human/Mouse Starter Kit
(Qiagen, Valencia, CA, USA), which included a siScramble negative control (Qiagen,
Valencia, CA, USA) and the Gene Kit Solution targeting siIL-17B (Cat No. 1027416 Qiagen, Valencia, CA, USA).
The concentration of siRNA was defined as 10 nM,
incubation period of 48 hours and siRNA #3 were more effective for IL-17B silencing
for both tumors cell lines. Cells were incubated in a 0.5 % HiPerfect solution (Qiagen,
Valencia, CA, USA) and the siIL-17B #3 for 48 hours, after which total cellular RNA
was isolated using the Trizol method (Invitrogen - Life Technologies, Eugene, OR,
USA) and purified using the RNeasy Kit extraction columns (Qiagen, Valencia, CA,
USA).
Absolute quantification by real-time (qRT-PCR)
100
The concentration of RNA from each sample was determined using a NanoDrop
2000 Spectrophotometer (Thermo Fisher Scientific - Waltham, MA USA). cDNA was
obtained by RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction) using the
High Capacity cDNA Kit (Applied Biosystems, Foster City, CA, USA).
The qRT-PCR reaction was performed to assess the efficiency of gene silencing
of IL-17B, as well as treatment effect on Caspase 3 and vascular endothelial growth
factor (VEGF)A levels, using a StepOne Plus Real Time PCR System (Applied
Biosystems, Foster City, CA, USA). Specific primers included: IL-17B - sense (5'
GCAGCTGTGGATGTCCAACA 3') antisense (5' GGGTCGTGGTTGATGCTGTAG
3'),
inventoried
TaqMan
assays
Caspase-3
(Cf02643290_m1),
VEGFA
(Cf02623449_m1), and the housekeeping genes ribosomal protein-5 (RPS5) sense (5’
TCACTGGTGAGAACCCCCTG 3’) antisense (5’ GCCTGATTCACACGGCGTA 3’)
and ribosomal protein-19 (RPS19) sense (5’ AGCCTTCCTCAAAAAGTCTGGG 3’)
antisense (5’ GTTCTCATCGTAGGGAGCAAGC 3’), all at a concentration of 100 ng
for each cDNA sample.
The amplification was performed in cycles at 95 °C for 10 minutes, followed
by 40 cycles at 95 °C for 15 seconds and 60 °C for one minute. The value of the relative
expression of the genes of interest was determined with DataAssist 3.0 software
(Applied Biosystems, Foster City, CA, USA) by ΔΔCt method [22]. The samples were
tested in triplicate and each experiment included a negative control.
Immunofluorescence staining and analysis
Previously treated 3D structures grown on Matrigel and treated monolayer cells
were washed once with PBS, fixed in 4.0 % paraformaldehyde solution in PBS for 20
minutes at room temperature, and blocked with 10 % donkey serum solution for 1 hour
at room temperature. The specific primary antibodies, cleaved caspase-3 (SigmaAldrich, St. Louis, MO, USA) and VEGF-A (Santa Cruz Biotechnology, Dallas, TX,
USA) were added and incubated overnight at 4 ºC. After washing three times with
immunofluorescence (IF) buffer (0.1 % FSB, 0.2 % triton and 0.05 % Tween 20), a
secondary Alexa Fluor 488 anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), for
both cleaved caspase-3 and VEGF-A, was added per 1 hour at room temperature.
101
Following three time washing with IF buffer, the cells were incubated with 4',6diamidino-2-phenylindole (DAPI) solution (Life Technologies, Eugene, OR, USA) and
mounted with Prolong Gold® (Life Technologies, Eugene, OR, USA). 3D structures
images were captured and processed using a confocal microscope and associated
software (ZEISS, model LSM 710, software ZEN 2010, Thornwood, NY, USA).
Monolayer cells were captured and processed using a microscope and associated
software (OLYMPUS, model BX53, software Image-Pro Plus version 7.0, Center
Valley, PA, USA).
For apoptosis analysis, all cleaved caspase-3 positive cells were counted in three
different photomicrographs (100 X magnification) per treatment group. Results were
quantified as percent of apoptotic monolayer and 3D structures, according to AveryCooper et al. [23]. The number of cleaved caspase-3 positive cells was normalized to
the area of photomicrography.
VEGF-A protein was quantified according to Jardim-Perassi et al. (2014). In
summary, three different photomicrographs taken at 100 X magnification under bright
field and the intensity of the staining was quantified by ImageJ Software (NIH,
Bethesda, MD, USA). Each photograph was divided into four quadrants and 20 spots
(small circular ROI) were randomly selected (avoiding the nucleus) in each
photomicrograph. A negative control section of the corresponding staining was used
for measuring background activity. The values were obtained in arbitrary units (a.u.)
and represented as the mean optical density (M.O.D.) for each sample.
Protein extraction
CF-41 cells were plated in individual wells of 1.88 cm2 (24-well plate) at 0.5 x
106 cells confluence and inoculated with 500 µL of normal growth medium overnight.
Thereafter, cells were treated with or without 1 mM of melatonin for 48 hours and
performed protein extraction of adherent ad supernatant cells. Cells were washed in icecold PBS and lysed with MILLIPLEX® MAP lysis buffer supplemented with 1 mM of
phosphatase inhibitor cocktail (Na3 VO 4 ) (Sigma-Aldrich, St. Louis, MO, EUA) and
1:10 of protease inhibitor (Sigma-Aldrich, St. Louis, MO, EUA). After incubation for
30 minutes with intermittent vortexing, the cell lysate was centrifuged and the proteins
102
collected on supernatant. Concomitantly the supernatant medium was collected and
used ultrafiltration (Amicon®, EMD Millipore, Billerica, MA) to concentrate the
proteins. The supernatant was included in columns containing filter of three kDa,
centrifuged and posteriorly larger proteins were retained in filter and subsequently
quantified.
Proteins extracts were quantified by the bicinchoninic acid (BCA) protein assay
kit (PIERCE - Thermo Scientific - Thermo Fisher Scientific, Waltham, MA, USA).
Membrane array
The membrane Human Apoptosis Array C1 (RayBiotech, Norcross, GA, EUA)
was incubated with 2 mL of 1 X blocking solution buffer (RayBiotech, Norcross, GA,
EUA) for 30 minutes. Treated and control samples were added in a concentration of 600
µg (300 µg of lisate cells and 300 µg of supernatant cells) and incubated in membrane
at 4 ºC overnight. Membrane was washed three times with wash buffer 1 X (RayBio I,
RayBiotech, Norcross, GA, EUA) and two times with wash buffer 1 X (RayBio II,
RayBiotech, Norcross, GA, EUA) for five minutes each. Biotin conjugate anti-cytokines
(RayBiotech, Norcross, GA, EUA) was added and the samples incubated at 4 ºC
overnight. The membrane was wash again and then incubated with horseradish
peroxidase (HRP) streptavidin 1000X (RayBiotech, Norcross, GA, EUA) solution at 4
°C overnight. The membrane was wash and incubated with detection solution
(RayBiotech, Norcross, GA, EUA) for two minutes and exposed to ChemiDoc system
(BioRad, Hercules, CA, EUA).
Optical density reference to protein expression was normalized with positive
control and quantification was performed using ImageJ Software (NIH, Bethesda, MD,
USA) as image analyzer. The values were obtained in arbitrary units (a.u.) and
represented as the mean optical density (M.O.D.) for each sample. All samples were
included in duplicate plus positive and negative membrane controls.
Statistical analysis
All data were expressed as mean
analyses were performed
±
standard error of mean (SEM). All statistical
using GraphPad Prism4 (San Diego, CA, USA). Raw data
103
were initially subjected to descriptive analysis to determine the normal range. Normal
range data were analyzed by two-way ANOVA, followed by Bonferroni test for MTT
results and Student’s t-test analysis for another results. A p-value ≤ 0.05 was considered
significant.
RESULTS
1 mM of melatonin reduce cell viability of canine mammary tumor cell lines
Cell viability was tested with melatonin at 0.001 mM to 1 mM for 48 hours. The
pharmacological concentration of melatonin (1 mM) was able to reduce viability in both
mammary tumor cell lines. For CF-41 cells, 1 mM of melatonin led to a significant
reduction in cell viability about 20% compared to the control group (79.75 ± 2.126; p =
0.001) and, for CMT-U229 cells, the effect was more effective, showing reduction of
40% compared to the control group (60.24 ± 1.985; p = 0.0002) (Figures 1A and B).
Melatonin and IL-25/siIL-17B enhanced cleaved caspase-3 in both tumor cell lines
Treatments with melatonin at 1 mM, IL-25 at 10 ng/mL (previously chosen by
Western Blotting results, data not show) and silence gene its competitor IL-17B#3 were
performed in order to assess apoptosis in both canine mammary tumor cell lines.
CF-41 cells treated with melatonin, siIL-17B and three treatments performed
together showed an increase of caspase-3 mRNA expression (p < 0.0003; p =0.03 and p
< 0.0001, respectively). This increase were reflected in able to all treatments alone
promotes enhance of 51 % of apoptotic cells compared to 10 % of positive cells in
control groups (p < 0.05) (Figure 2).
Similarly to metastatic cells, it was found a high caspase-3 gene expression in
CMT-U229 cells after melatonin, siIL-17B and three combined treatments (p = 0.0003;
p = 0.0003 and p < 0.0001, respectively). In addition, the three treatments alone
promotes an enhanced of 44 % of apoptotic cells, compared to 9 % of control groups (p
< 0.05) and, when treatments were combined, a slight increase in apoptotic cells was
observed, but did not show influence of simultaneous pathways (Figure 3).
Pro-apoptotic effect of treatments in 3D structures
104
Tridimensional culture allows cells to explore the three dimensions of the space
thereby
increasing
microenvironment
cell-cell
interactions,
as
well
as
interactions
with
the
[24]. Besides that, susceptibility of cancer cells to the inhibitory
actions of melatonin appears to be appreciably dependent on several additional factors,
such a fast-running cell cycle, or the cell attachment on different substratum, which
allows cells to be more sensitive to melatonin [25]. Thus, we aimed to characterize the
increase expression of cleaved caspase-3 in 3D structures of mammary cancer cells.
CF-41 and CMT-U229 3D structures were observed after treatments in different
sequential planes image with cleaved caspase-3 labeling (Figure 4A and B). For
metastatic cells, melatonin and IL-25 treatments were able to induces 32 % and 30 % of
apoptosis in 3D structures, compared to control groups, respectively (p = 0.004; p =
0.001, respectively). In contrast, siIL-17B treatment did not induce apoptosis in 3D
structures, whereas control group showed 25 % more apoptotic cells (p < 0.0001).
Combined treatments enhanced 21 % of apoptosis compared to control group (p =0.04)
but was not more efficiently when compared to alone treatments (Figure 4C).
For CMT-U229 cells, melatonin and IL-25 treatments were able to induce 20 %
and 38 % of apoptosis in 3D structures compared to control groups, respectively (p =
0.01 and p = 0.0008, respectively). siIL-17B and combined treatments showed a slight
enhance in percentage of apoptotic cells (p > 0.05 and p = 0.02, respectively) (Figure
4D).
Apoptotic proteins modulation in metastatic CF-41 cells by melatonin
In order to analyze the involvement of melatonin in apoptosis signaling
pathways of mammary cancer cells, we performed the Apoptosis Array after treatment
with 1 mM of melatonin in CF-41 cells. Apoptosis related factors (Table 1) showed
increase of proteins Bcl-like (BCLW, as known as BCL2L2) (p = 0.03) and insulin-like
growth factor-binding (IGF)-1 and 2 (p = 0.04; p = 0.03, respectively), compared to
control group. Besides that, it was observed a decrease in Bcl-2-associated death
promoter protein (BAD) (p 0.01), insulin-like growth factor-binding protein-1 (IGFB-1)
(p = 0.02) and tumor necrosis factor receptor (TNFR)-II (p = 0.04) after melatonin
treatment (Figure 5).
105
Modulation of VEGF-A after melatonin and IL-25/siIL-17B treatments
To test whether angiogenesis could be affect by established treatments we
quantified the VEGF-A expression in mammary tumor cells. Melatonin, IL-25 and
combined treatments resulted in a decrease of VEGF-A mRNA expression in CF-41
cells (p = 0.0004; p = 0.02 and p = 0.0002, respectively). Though the protein level, three
treatments alone reduced VEGF-A (18.3 ± 0.7 u.a., p < 0.0001; 16.1 ± 0.6 u.a., p = 0.03
and 15.4 ± 0.5 u.a., p < 0.0001), however, when performed together, the decrease was
not significantly, compared to control group (Figure 6).
Melatonin treatment decrease VEGF-A gene expression in CMT-U229 cells (±
0.01, p = 0.01) and the same decrease was showed in protein expression (8.3 ± 0.6 u.a.,
p < 0.0001). For IL-25, siIL-17B and combined treatments were observed increase of
VEGF-A gene expression compared to control groups (p = 0.01; p = 0.003 and p =
0.0004, respectively), though, both treatments alone showed reduction
of VEGF-A
protein levels compared to control groups (11.03 ± 0.6 u.a., p < 0.0001 and 10.7 ± 0.5
u.a., p < 0.0001, respectively) (Figure 7).
DISCUSSION
The current study showed that melatonin, in pharmacological concentration,
reduced the viability of both canine mammary tumor cells (CF-41 and CMT-U229),
which is not show in literature until the moment. Antiproliferative activity of melatonin
has been demonstrated in numerous cell systems in in vitro as well as in vivo studies
[17, 26 - 28], besides that, a systematic review of Mills et al. [29] suggests the benefic
effect of melatonin levels in cancer progression. Corroborate our results, Lopes et al.
[30] showed that high levels of melatonin have efficacy in cell proliferation of primary
canine mammary tumor cells.
We previously found that both canine cell lines expressed the melatonin
receptor-1 (MTNR1A) (data not show). In mammals, melatonin’s biological activity can
be mediated via two major mechanisms: G-protein coupled receptor (GPCR), mediated
activity (MT1 and MT2 receptors) and non-receptor–mediated antioxidant activity [3132].Yuan et al. [33] reported that MT1 and MT2 melatonin antagonists can reverse
inhibitory effects of melatonin in growth of breast cancer cells and that overexpression
106
of MT1 receptor can significantly enhance both in vitro and in vivo inhibitory response
of breast tumor cells to melatonin. In addition, Ram et al. [34] showed that melatonin
can suppress estrogen-induced transcriptional activity of the estrogen receptor (ER)-α,
downregulating the expression of a number of mitogenic proteins and pathways,
including the antiapoptotic protein Bcl-2, while inducing the expression of growthinhibitory and apoptotic pathways including TGF-b and Bax.
Melatonin has pleiotropic effects at both physiological and molecular levels, so,
compelling studies has documented anticancer effects of melatonin in experimental
models (cells and animals), as well as in humans [25]. It is know that melatonin at low
concentrations
(generally
referred
as
‘physiological’
concentrations,
i.e.,
in the
nanomolar range equal) could exert only a cytostatic action; meanwhile, apoptotic
effects are often observed at higher concentrations and occur in many tumor cells [25].
In fact, we showed
metastatic
canine
the enhance of percent of apoptotic cells in metastatic and nonmammary
tumor
cells
after
melatonin
at
pharmacological
concentration (1 mM) in both monolayer cell culture and 3D structures.
The enhance of cleaved caspase-3 is supported by a study of Casado Zapico et
al. [35] that performed a report in a wide array of hematological tumors and confirmed
the increase of melatonin-dependent apoptosis, involving both the intrinsic and extrinsic
pathways. Besides that, El-Aziz et al. [36] reported a significant increase of caspase-3
activity and DNA fragmentation in breast cancer-bearing rats treated with melatonin in
association with other agents. The opposite was observed by Kepka et al. [37] that
showed
that melatonin significantly reduced the early and late apoptosis of peritoneal
leukocytes, inhibiting extrinsic apoptosis pathway rather than the intrinsic mitochondrial
pathway.
The uncontrolled proliferation requires the interaction with various molecules
and signaling pathways [7]. We showed that melatonin was capable to change other
factors associated in apoptosis pathway when treated metastatic canine mammary tumor
cells. Melatonin increased proteins BCLW and IGF-1 and 2. In contrast, was observed a
decrease BAD, IGFBP-1 and TNF-RII proteins.
It is known that the control of apoptotic occur mainly through members of the
BCL-2 family and these proteins regulate mitochondrial membrane permeability, which
107
may act as pro-apoptotic (BAD) or anti-apoptotic (Bcl-2) [38]. Another important
mechanism of this protein family is the regulation of the cytochrome C release by
changing
the
permeability
of
the
mitochondrial
membrane
through
BAD
phosphorylation [38]. We believed that decreased levels of BAD, IGFBP-1 and TNFRII, which act as pro-apoptotic factors, is due to no alteration of cytochrome C
expression.
Once
BLC-W,
IGF-1
and
-2,
important
modulators
of cell growth,
differentiation and invasion [39, 40], in high levels are related to the intense cellular
proliferation and susceptibility to the breast cancer development [40, 41]. A possible
reazon may be acting in compensatory mechanisms against the apoptotic stimulus of
melatonin, especially in this initial period of 48 hours.
The IL-17 cytokine family is composed of six members, IL-17A to IL-17F and
five receptors have been described, IL-17RA to IL-17RE. IL-17B and IL-17E (IL-25),
which can plays opposite functions in breast tumor microenvironment [42]. Although
they share the IL-17RB chain as co-receptor, pro-oncogenic features were described for
IL-17B, which is mainly secreted by the breast tumor cells, whereas tumor suppressor
role was associated to the normal breast epithelial cells, which secreted IL-17E [43].
The present study found, for the first time, that IL-25/siIL-17B signaling are able
to enhance apoptotic cells in canine mammary tumor cells (CF-41 and CMT-U229).
According to Furuta et al. [21], IL-25 had the highest anticancer activity without
affecting nonmalignant extracellular matrix and, is capable to activate caspase-mediated
apoptosis in four breast cancer cell lines (MCF7, MDA-MB-468, SKBR3 and T47D). In
contrast, Mombelli et al. [43] not found IL-17E expression by non-transformed
epithelial cells, and, when MCF-7, T47D and MDA-MB-468 cells were treated with
100 or 500 ng/mL of IL-25 was not possible reproduce its potential induction of breast
cancer cell apoptosis by PARP activity.
Besides that, the silence gene of IL-17B, performed in our study, allows IL25/IL-25R interaction, that send a death signal to breast cancer cells, stimulating FAS
receptor and tumor necrosis factor (TNF) receptor-1 [21]. The IL-17B binds to IL-25R
and promotes the recruitment of TRAF6 to IL-25R that activates NF-kB pathway and
exerts anti-apoptosis via up-regulation of Bcl-2. IL-25R/TRAF6 interaction prevents
108
apoptosis in breast cancer cell. Corroborating our results, Younesi and Nejatollahi [42]
reported that IL-25/IL-15R interaction promptly induces cleavage of caspases 8 and 3.
In addition, Huang et al. [44] showed that depletion of IL-17B significantly impaired
the ability of proliferation, invasion and tumor growth in xenograft breast cancer mice
model.
Angiogenesis inhibition is an attractive target in cancer prevention and therapy
as it deprives tumor of oxygen and nutrients, which reduces tumor proliferation and
expansion [45]. Melatonin and IL-25/siIL-17B signaling were capable to reduce VEGFA protein expression, which proves their ability to modulate angiogenesis in both canine
mammary tumor cells. Besides that, Carbajo-Pescador et al. [46] proposed that
melatonin effects occur at a post-transcriptional level, what could explains the high,
possibly compensatory, VEGF-A mRNA expression after siIL-17B treatment in CMTU229 cells, opposed to the reduced VEGF-A protein expression.
The anti-angiogenic therapy alone can not cause complete tumors regression
because it acts indirectly on cancer cells [47]. Talib and Saleh [45], showed that
combined melatonin treatment with bacterial therapy in breast cancer cells causes tumor
regression associated with apoptosis induction, increasing IFN-γ production, low
expression of VEGF, and a decrease in percentage death to 0%. In addition, to
suppressing VEGF protein expression, Jardim-Perassi et al. [28] reported lower
VEGFR2 and VEGFR3 expression in melatonin-treated tumors compared to vehicletreated tumors.
It is noteworthy that there are few studies looking at IL-25 on angiogenesis,
given the impact of angiogenesis suppression on tumor cell apoptosis in vivo, and there
are no studies evaluating VEGF-A after stablished treatments in these cells. According
to Liu et al. [48] and Ding et al. [49] IL-17 could modulate angiogenesis directly
(through IL-17R binding on endothelial cells), or indirectly, by stimulating cancer cells
to produce angiogenic factors. Maniati and Hagemenn [50] demonstrated that synthetic
IL-17 can induce the expression of the pro-angiogenic factors VEGF, PLAU, NRP2, IL6 and ID3 in colorectal cancer cells, as determined by qRT-PCR and ELISA of
conditioned media.
109
CONCLUSION
Taken together, these results confirms the action of melatonin and IL-25/siIL17B in induction of apoptosis by extrinsic and intrinsic pathways and modulation of
VEGF-A, showing an anti-angiogenic potential of these agents in mammary tumor
cells. Besides that, no study with this approach was found in canine species until the
moment. So, we conclude that melatonin and IL-25 signaling as potential agents in
control of tumorigenesis of canine mammary tumor cells could be useful in clinical
practice.
INTEREST CONFLICT
Author declares no interest conflict.
SUPPORTING INFORMATION CAPTIONS
This study was funded by a FAPESP/Brazil – Fundação de Amparo à Pesquisa
do Estado de São Paulo (2012/06098-0) to D.A.P.C.Z, and Canadian Foundation for
Innovation (CFI) -Ministry of Research Infrastructure (MRI, Ontario) funds to A.V.P.
AUTHORS’ CONTRIBUTION
Conceived and designed the experiments: GBG, TFB, LBM, DAPCZ. Performed the
experiments: GBG, TFB, LBM, MGM, BVJP. Analyzed the data: GBG, TFB, LBM,
AVP, DAPCZ. Contributed reagents/ materials/analysis tools: EH, AVP, DAPCZ.
Wrote the paper: GBG, TFB, DAPCZ.
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Table 1. Apoptosis Array C1 (RayBiotech) contening 43 different factors for analysis of
protein expression profile of CF-41 cells after melatonin treatment.
Apoptotic Factors
Melatonin
Treatment
Apoptotic Factors
Melatonin
Treatment
BAD
↓ p=0.01
BAX
ns
CD40
ns
BCL-2
ns
CD40 LIGAND
ns
BCL-W
↑ p=0.03
BIRC-3
ns
BID
ns
CYTO C
ns
BIM
ns
DR6
FAS
ns
ns
CASPASE-3
CASPASE-8
ns
ns
FAS LIGAND
ns
HSP27
ns
IGFBP-1
↓ p=0.02
HSP60
ns
IGFBP-2
ns
HSP70
ns
IGFBP-3
ns
hTRA
ns
IGFBP-4
ns
IGF-1
↑ p=0.04
IGFBP-5
ns
IGF-2
↑ p=0.03
IGFBP-6
ns
LIVIN
ns
IGF-1R
TNF-RII
ns
↓ p=0.04
P21
P27
ns
ns
TNF-α
ns
P53
ns
TNF-β
ns
SMAC
ns
TRAIL-R1
ns
SURVININ
ns
TRAIL-R2
ns
TNF-RI
ns
TRAIL-R3
ns
XIAP
ns
TRAIL-R4
ns
BAD: Bcl-2-antagonist of cell death; BAX: Bcl-2-associated X protein; BCL-2: B-cell lymphoma 2;
BCL-W: Bcl-2-like protein 2; BID: BH3 interacting-domain death agonist; BIM: Bisindolylmaleimidebased protein kinase C (PKC) inhibitors; BIRC-3: Baculoviral IAP repeat-containing protein 3;
CASPASE-3: Cysteine-aspartic acid protease 3; CASPASE-8: Cysteine-aspartic acid protease 8; CD40:
Cluster of differentiation 40; CD40 LIGAND:Cluster of differentiation 40 ligand; CYTO C: Cytochrome
c; DR6:Death receptor 6; FAS: First apoptosis signal; FAS LIGAND: First apoptosis signal ligand;
HSP27:Heat shock protein 27; HSP60:Heat shock protein 60; HSP70:Heat shock protein 70;
115
hTRA:High-temperature requirement A serine peptidase; IGF-1:Insulin-like growth factor 1; IGF2:Insulin-like growth factor 2; IGFBP-1:Insulin-like growth factor-binding protein 1; IGFBP-2: Insulinlike growth factor-binding protein 2; IGFBP-3:Insulin-like growth factor-binding protein 3; IGFBP4:Insulin-like growth factor-binding protein 4; IGFBP-5:Insulin-like growth factor-binding protein 5;
IGFBP-6:Insulin-like growth factor-binding protein 6; IGF-1R: Insulin-like growth factor receptor 1;
Livin; P21: Cyclin-dependent kinase inhibitor 1; P27: Cyclin-dependent kinase inhibitor 1B; P53: Tumor
protein p53; SMAC:Second mitochondria-derived activator of caspases ; Survinin; TNF-α: Tumor
necrosis factor alpha; TNF-β: Tumor necrosis factor beta; TNF-RI: Tumor necrosis factor receptor 1;
TNF-RII: Tumor necrosis factor receptor 2; TRAIL-R1:TNF-related apoptosis-inducing ligand receptor
1; TRAIL-R2:TNF-related apoptosis-inducing ligand receptor 2; TRAIL-R3:TNF-related apoptosisinducing ligand receptor 3; TRAIL-R4:TNF-related apoptosis-inducing ligand receptor 4; XIAP:X-linked
inhibitor of apoptosis protein.All antibodies are prepared in duplicate.ns: no significant.
Fig. 1. Effect of melatonin on viability of canine mammary tumor cell lines. A) CF41 and B) CMT-U229 cells were treated with 0.001 mM to 1 mM of melatonin for 48
hours and cell viability was measured by MTT assay. The white column corresponds to
control group. Each column represents the mean ― standard error of triplicate
experiments. (*p ≤ 0.05). Statistical significance compared to control group was
determine by ANOVA followed by Bonferroni test.
116
Fig. 2. Cleaved caspase-3 expression in CF-41 cells. A) Increase gene expression of caspase-3 after
treatments compared to control groups. B) Average percentage of apoptotic cells for each treatment.
C) Photomicrographs of immunofluorescence staining for cleaved caspase-3 in CF-41 cells.
Magnification= 100X. Cleaved caspase-3 (green) and nuclei DAPI (blue). Each column represents the
mean ± standard error of triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s
t-test. *p<0.01 **p<0.001 ***p<0.0001.
117
Fig. 3. Cleaved caspase-3 expression in CMT-U229 cells. A) Increase gene expression of caspase-3
after treatments compared to control groups. B) Average percentage of apoptotic cells for each
treatment. C) Photomicrographs of immunofluorescence staining for cleaved caspase-3 in CMT-U229
cells. Magnification= 100X. Cleaved caspase-3 (green) and nuclei DAPI (blue). Each column
represents the mean ± standard error of triplicate experimens (*p ≤ 0.05). Significance was determined
by Student’s t-test. *p<0.01 **p<0.001 ***p<0.0001.
118
Fig. 4. Cleaved caspase-3 expression in CF-41 and CMT-U229 3D structures. A)
Photomicrographs of immunofluorescence staining for cleaved caspase-3 in CF-41 3D structures and
B) CMT-U229 3D structures. C) Average percentage of apoptotic cells for each treatment in CF-41 3D
structures and D) CMT-U229 3D structures. Magnification= 100X. Each column represents the mean
± standard error of triplicate experimens (*p ≤ 0.05). Significance was determined by Student’s t-test.
*p<0.01 **p<0.001 ***p<0.0001.
119
Fig. 5. Differentially apoptotic protein expression in CF-41 cells treated with 1 mM of
melatonin. The white column corresponds to control groups. Each column represents the mean ±
standard error of duplicate experiments. (*p ≤ 0.05). Significance was determined by Student’s t-test.
120
Fig. 6. VEGF-A expression in CF-41 cells. A) Decrease VEGF-A gene expression after treatments
compared to control groups. B) Quantification of VEGF-A protein expression showing decrease
cytoplasm labeling after treatments, compared to control groups for each treatment. C)
Photomicrographs of immunofluorescence staining for VEGF-A in CF-41 cells. Magnification= 100X.
VEGF-A (green) and nuclei DAPI (blue). Each column represents the mean ± standard error of
***p<0.0001.
121
Fig. 7. VEGF-A expression in CMT-U229 cells. A) Decrease VEGF-A gene expression after
treatments compared to control groups. B) Quantification of VEGF-A protein expression showing
decrease cytoplasm labeling after treatments, compared to control groups for each treatment. C)
Photomicrographs of immunofluorescence staining for VEGF-A in CMT-U229 cells. Magnification=
100X. VEGF-A (green) and nuclei DAPI (blue). Each column represents the mean ± standard error of
***p<0.0001.
122
Conclusões
123
I.
CONCLUSÕES
O trabalho permite estabelecer as seguintes conclusões:
 A melatonina tem ação citostática e, portanto, não citotóxica nas linhagens
tumorais mamárias metastáticas e não-metastáticas humanas e caninas, na
concentração farmacológica de 1 mM.
 A IL-25 na concentração de 1 ng/mL atua na redução da viabilidade celular das
linhagens tumorais mamárias humanas metastática e não-metastática;
 O silenciamento gênico da IL-17B é eficaz nas linhagens tumorais mamárias
humanas e caninas, podendo ser usado como possível alvo terapêutico no câncer
de mama;
 O tratamento com melatonina a 1 mM é eficaz na ativação da via da apoptose,
bem como de fatores pró-apoptóticos e anti-angiogênicos no cultivo em
monocamada e tridimensional das células tumorais mamárias humanas e
caninas;
 A modulação da IL-25, em adição ao silenciamento gênico da IL-17B induz a
apoptose e reduz a expressão do fator angiogênico VEGF-A no cultivo em
monocamada e tridimensional das células tumorais mamárias humanas e
caninas;
 As vias de ativação da melatonina, IL-25 e siIL-17B não são sinérgicas nas
linhagens tumorais mamárias, porém apresentam resultados satisfatórios quando
em conjunto podendo representar promissores alvos terapêuticos no câncer de
mama.
124
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135
Anexos
136
III. ANEXOS
ANEXO 1: Parecer da Comissão de Ética na Experimental Animal da Faculdade de
Medicina de São José do Rio Preto.
137
ANEXO II: Comprovante de submissão do artigo: Efficacy of melatonin, IL-25 and
siIL-17B in apoptosis and angiogenesis response of breast cancer cell lines ao
periódico Journal of Pineal Research.

000859983

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