UNIVERSIDADE FEDERAL DE GOIÁS ESCOLA DE VETERINÁRIA E ZOOTECNIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL EXPRESSÃO DE RECEPTORES DE EGF, INIBIDORES E REGULADORES DO CICLO CELULAR EM LESÕES PROLIFERATIVAS DA PRÓSTATA CANINA Mariana Batista Rodrigues Faleiro Orientadora: Prof.ª Dr.ª Veridiana Maria Brianezi Dignani de Moura GOIÂNIA 2014
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UNIVERSIDADE FEDERAL DE GOIÁS
ESCOLA DE VETERINÁRIA E ZOOTECNIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL
EXPRESSÃO DE RECEPTORES DE EGF, INIBIDORES E
REGULADORES DO CICLO CELULAR EM LESÕES
PROLIFERATIVAS DA PRÓSTATA CANINA
Mariana Batista Rodrigues Faleiro
Orientadora: Prof.ª Dr.ª Veridiana Maria Brianezi Dignani de Moura
GOIÂNIA
2014
ii
MARIANA BATISTA RODRIGUES FALEIRO
EXPRESSÃO DE RECEPTORES DE EGF, INIBIDORES E
REGULADORES DO CICLO CELULAR EM LESÕES
PROLIFERATIVAS DA PRÓSTATA CANINA
Tese apresentada junto ao Programa de Pós-
Graduação em Ciência Animal da Escola de
Veterinária da Universidade Federal de Goiás
para obtenção do título de Doutorem Ciência
Animal.
Área de Concentração:
Patologia, Clínica e Cirurgia Animal
Linha de Pesquisa:
Patobiologia animal, experimental e comparada
Orientadora:
Prof.ª Dr.ª Veridiana Maria Brianezi Dignani de Moura-EVZ/UFG
Comitê de Orientação:
Prof. Dr. Adilson Donizeti Damasceno (EVZ / UFG)
Prof. Dr. Eugênio Gonçalves de Araújo (EVZ / UFG)
GOIÂNIA
2014
iii
MARIANA BATISTA RODRIGUES FALEIRO
Tese defendida e aprovada em 14/11/2014 pela banca examinadora constituída pelos
professores:
iv
Dedico este trabalho:
A vocês que abriram mão de momentos de convívio, que sofreram com a ausência quando o
dever do estudo chamava;
A vocês que muitas vezes receberam-me com silenciosa mágoa ou muda revolta quer pela
ausência, quer por saudade ou impaciência;
A vocês que agora vêem com alívio este fim de etapa e que, por mais que não queiram
demonstrar, estão mais felizes do que eu;
Pois amaram suficientemente para aplaudir, chorar, tolerar e encorajar.
1.1.Morfofisiologia e enfermidades da próstata canina
No cão, a próstata representa a única glândula sexual acessória, compondo-se por
órgão retroperitonial músculo-glandular, ovóide e bilobulado, que circunda a uretra
proximal1. Localiza-se caudal à bexiga, ventral ao reto e dorsal à sínfise púbica
1,2, podendo
variar de posição a depender da idade, distensão da bexiga e enfermidades envolvendo a
glândula3 (Figura 1A). Sua principal função é a produção do líquido prostático, que atua como
meio de transporte, proteção e nutrição para os espermatozóides durante a ejaculação5. Além
disso, auxilia no metabolismo da testosterona em diidrotestosterona3.
A próstata canina possui dois lobos, separados por um septo medial de tecido
fibroso1,2
. Esses lobos subdividem-se em lóbulos, contendo glândulas túbulo-alveolares
ramificadas (ácinos), recobertas por epitélio colunar simples5, sustentadas por delicado
estroma e que se estendem desde sua abertura no ducto uretral até a cápsula fibromuscular
prostática, a qual limita e protege o parênquima glandular1,6
. O epitélio glandular prostático é
composto por dois tipos celulares, as células epiteliais secretoras colunares altas e as células
epiteliaisda camada basal, estas localizadas ao longo da membrana basal. O estroma consiste
de fibroblastos e células musculares lisas, envoltos em colágeno, com vasos sanguíneos e
nervos3,6
(Figura 1B).
FIGURA 1 - A) Vista ventral da cavidade pélvica canina. 1) Bexiga urinária; 2) próstata; 3) reto; 4)
uretra. B) Fotomicrografia da histomorfologia normal da próstata canina. Glândula
túbulo-alveolar ramificada (ácinos), recobertas por epitélio colunar simples (seta preta). Lúmen glandular (asterisco), estroma de sustentação fibromuscular (seta amarela) e
célula basal (seta verde). HE, 200x.
Fonte: Faleiro7,8
1 2
3
4
A B
2
As doenças prostáticas representam um problema comum em cães adultos e
idosos, semelhante ao que ocorre na próstata do homem9-11
. Dentre as enfermidades que
acometem a prostáta dos cães, destacam-se a hiperplasia prostática benigna12
(HPB), as
prostatites3,4
, as lesões displásicas12
e o carcinoma prostático (PC)6,14-17
. Quanto às lesões
displásicas da próstata, consideradas pré-malignas, destacam-se a neoplasia intraepitelial
prostática (PIN)15,18
e a atrofia inflamatória proliferativa (PIA)13,16
.
A caracterização diagnóstica das prostatopatias caninas é um constante desafio,
uma vez que a similaridade dos sinais nas diferentes enfermidades e a substancial
possibilidade de ocorrência concomitante de duas ou mais alterações limitam diagnósticos
precisos, somado ao fato da inexistência de testes bioquímicos apropriados para a espécie,
como biomarcadores de detecção precoce de enfermidades como HPB e câncer. Ressalte-se
que à exceção da prostatite aguda, cães com doenças prostáticas são
comumenteassintomáticos3,5,19
.
Similaridades em relação à ocorrência natural e influência hormonal no
desenvolvimento das afecções prostáticas, principalmente HPB17
, PIN18
, PIA13
e PC6, têm
feito do cão modelo experimental para o estudo de algumas dessas prostatopatias na glândula
humana10,11,17,18
, contribuindo para o conhecimento médico e médico-veterinário acerca das
doenças prostáticas nessas espécies. Considerando essas similaridades e o contexto do estudo,
apresentam-se a seguir as enfermidades relacionadas aos objetivos desta pesquisa.
1.1.1. Hiperplasia prostática benigna (HPB)
A hiperplasia prostática benigna (HPB) é a prostatopatia mais comum no cão20
eestá associada ao envelhecimento e a desequilíbrios hormonais envolvendo a testosterona, a
diidrotestosterona (DHT) e o estrogênio3,12,17
. A HPB no cão envolve preferencialmente o
componente epitelial e ocorre de forma difusa19,21
, enquanto no homem costuma ser estromal
e nodular20
. Apesar disso, o cão é a única espécie não humana que desenvolve HPB
espontaneamente, o que reforça sua utilização como modelo experimental para essa doença
no homem20,22
.
Em decorrência do desequilíbrio entre andrógenos prostáticos, há alteração da
interação entre estroma e epitélio, havendo resposta das células epiteliais aos fatores de
crescimento produzidos pelas células estromais. Observa-se tanto aumento no número de
células (hiperplasia), quanto aumento no tamanho das mesmas (hipertrofia)19
. Essa alteração
resulta em descontrole na proliferação, migração e morte celular, sendo um dos fatores que
3
contribuem para o desenvolvimento da HPB20
. Quanto ao padrão histológico da HPB canina
há dois tipos bem caracterizados, a hiperplasia glandular, com proliferação das células
secretoras; e hiperplasia complexa, com hiperplasia glandular intercalada a focos de atrofia.
Neste último, há proliferação dos elementos estromais em associação a áreas atróficas ou de
ácinos dilatados, císticos e preenchidos por material eosinofílico12,20,21
(Figura 2).
FIGURA 2 - Fotomicrografia da próstata canina com
HPB cística. Evidenciação deácinos
acentuadamente dilatados (asterisco vermelho), irregulares e proliferação
estromal adjacente (asterisco preto).
HE, 200x.
Fonte: Faleiro8
Embora a HPB possa ocorrer concomitantemente a lesões potencialmente pré-
malignas, como a PIN e PIA, e malignas, como o PC22,23
, é improvável que a HPB represente
lesão pré-neoplásica24
.
1.1.2.Atrofia inflamatória proliferativa (PIA)
O termo atrofia inflamatória proliferativa, do inglês proliferative inflammatory
atrophy (PIA), foi proposto para designar focos de epitélio glandular proliferativo com o
aspecto morfológico de atrofia focal simples e inflamação concomitante24
. A atrofia da
próstata é identificada pela redução no volume glandular e estroma pré-existentes, sendo
caracterizada em dois padrões, difuso e focal. A forma difusa resulta da diminuição de
andrógenos circulantes e envolve a próstata como um todo, de maneira relativamente
uniforme. Ao contrário, a atrofia focal não se relaciona unicamente à diminuição de
andrógenos circulantes e ocorre como áreas de epitélio atrófico intercaladas com áreas de
epitélio de aspecto normal e são proliferativas, sendo que a ampla maioria associa-se à
4
inflamação, de maneira que essas lesões podem se originar em um cenário de estresse
oxidativo aumentado, possivelmente derivado das células inflamatórias próximas24-26
.
A inflamação é sugerida como fator etiológico capaz de incitar a carcinogênese
por causar dano celular e no genoma, promover a substituição celular e criar um
microambiente rico em radicais livres tóxicos, ácido aracdônico, proteases, citocinas e fatores
de crescimento que elevam a replicação celular, antiapoptóticos e a angiogênese27,28
. Portanto,
no que refere à carcinogênese, a inflamação pode atuar como agente iniciador, via efeitos
genotóxicos, ou como agente promotor, via efeitos citotóxicos29
. Ainda, segundo Nonomura
et al.30
e Sfanos e De Marzo26
, a inflamação crônica em tecidos benignos prediz o
desenvolvimento de câncer de alto grau (com escore de Gleason entre 7-10).
Sugar31
e Tomas et al.14
sugerem que na PIA ocorre equilíbrio entre proliferação e
perda celular por mecanismos que não os apoptóticos, uma vez que nessas lesões há
proliferação, mas não há perda celular por apoptose, e mesmo assim não há crescimento em
volume. Ainda, acreditam em injúria celular direta e que as células lesadas caem no lúmen
glandular e são eliminadas no ejaculado ou capturadas por macrófagos. Além disso, inferem
que o epitélio em regeneração suprima a morte celular programada, ao menos
temporariamente, para substituir as células perdidas, fato que poderia explicar a expressão
aumentada de B-cell lymphoma/leukemia-2 (Bcl-2) nas células secretoras da PIA, resultando
em níveis muito baixos de apoptose e embasando o conceito de que a PIA é uma lesão
regenerativa. Ainda, os fatores de crescimento poderiam estar sendo liberados tanto pelas
células epiteliais lesadas quanto pelas células inflamatórias presentes.
Há vários critérios para que se considere uma lesão pré-maligna, como a relação
epidemiológica, a presença anterior ao câncer, assemelhanças morfológicas e a proximidade
ou equivalência às suas características de malignidade32
. A PIN é considerada a lesão
precursora mais provável do carcinoma prostático invasivo por atender a tais critérios32
.
Apesar da PIA também ser apresentada como lesão prostática pré-maligna, destaca-se que
Anton et al.34
e Billis e Magna35
, não encontraram associações entre a atrofia prostática e
câncer. Já Wang et al.36
observaram que 70% das lesões de PIN e 28% das de PC eram
margeadas por PIA. Com isso, concluíram sobre a evidência direta entre as lesões e
mostraram transições morfológicas entre PIA e PIN, e PIA e PC na próstata humana. Além
disso, muitos focos de PIA compartilham alterações fenotípicas, genotípicas e moleculares
com PIN e PC, podendo esses focos compor o principal alvo de transformação neoplásica33-37
.
Putzi e De Marzo38
avaliaram a relação entre PIA, PIN e adenocarcinoma
prostático em humanos e observaram concomitância entre PIA e PIN, PIA e PC, e PIN e PC,
5
sendo as combinações de topografia variada, observando-se desde lesões adjacentes a
distantes. Dessa forma, os autores acreditam que embora não caracterize regra, a PIA pode
representar um precursor da PIN e/ou do PC, embora assumam que a relação topográfica não
seja prova definitiva dessa proposta. Enfatizam ainda que nem todas as lesões de PIN ou
pequenas lesões de PC estão associadas à atrofia, assim como nem todas as lesões atróficas
são precursoras de PIN ou PC.
Segundo Palapattu et al.27
, a PIA compreende resposta das células epiteliais
prostáticas normais a um microambiente de estresse, e regiões individuais de PIA que são
inábeis em debelar o dano oxidativo ao genoma podem progredir para PIN ou PC. Assim, as
células epiteliais da PIA são possíveis alvos de transformação neoplásica, passando ou não
por PIN, o que significa dizer que dão origem ao PC indireta ou diretamente. Contudo, alguns
tumores parecem se desenvolver diretamente da PIN enquanto outros ocorrem sem evidência
de lesões precursoras36,37
.
Em cães, alguns autores estudaram a relação da PIA com a evolução do
PC13,15,16,39,40
. Di Santis41
caracterizou morfologicamente e imunofenotipicamente a PIA e a
PIN, validando o potencial pré-maligno dessas lesões na próstata do cão. Rodrigues et
al.15
embora não tenham examinado o aspecto proliferativo da PIA, encontraram
imunomarcação para cicloxigenase-2 (COX-2) e fator de crescimento transformador beta
(TGF-β) em quantidade intermediária entre o tecido normal e o PC, concluindo tratar-se de
lesão pré-neopásica, já que o aumento na expressão desses reguladores de atividades celulares
é observado em processos neoplásicos.
Toledo et al.13
, com base nos critérios de De Marzo et al.25
e Di Santis41
,
propuseram a subdivisão da PIA canina de acordo com o grau de inflamação adjacente aos
focos atróficos, sendo discreta, quando há poucas células inflamatórias e estas são esparsas
em sua maioria; moderada, quando há agregados de células inflamatórias sem destruição
tecidual ou formação de folículos ou nódulos linfoides (Figura 3A); e acentuada, quando da
presença de agregados confluentes de células inflamatórias com destruição tecidual ou
formação de folículos ou nódulos linfoides (Figura 3B).
6
FIGURA 3 - Fotomicrografia de próstata canina com foco de atrofia inflamatória proliferativa
(PIA). A)PIA cominfiltrado inflamatório mononuclear intersticial (asterisco) e
atipias epiteliais com mais de uma camada de células epiteliais (seta), citoplasma marcadamente reduzido, núcleos aumentados e nucléolos evidentes (PIA-moderada),
HE, 400x; B) Atrofia acinar, dupla camada de células epiteliais (seta) e infiltrado
, a expressão aumentada de p21 pode significar bom
prognóstico, uma vez que em testes utilizando modelos animais e o potente agente terapêutico
anticâncer inibidor de tirosina quinase, genisteína, foi observado aumento da expressão do
gene CDKN1A, codificador de p21, com concomitante declínio de ciclinas em cultura de
células prostáticas andrógeno dependentes (LNCaP) e independentes (DuPro).
TambémWangetal.74
, emestudocom o agente anticâncer ácido metilselénico (MSeA) sobre
cultura de células de PChumano (DU145),relataramaumento significativodonívelde RNAm
parap21 ep27, e redução de RNAm para ciclina D1(CD1)apóstratamento com MSeAdurante
24horas. Ainda, esse aumento foi maioremais rápidopara o RNAm de p21 (3horas) em
comparação ao RNAm dep27 (12horas). Segundo os autores, essesdados
sustentamqueoaumentodep21 ep27remetema bomprognóstico, easdiferenças noespaço de
tempodep21 ep27sugeremque a indução dep21seria maisimportante do que a de
p27paraaparada do ciclo celular em G1124,125
.
Aaltomaaetal.135
relataram o oposto, quep21está significativamente relacionado
aprognóstico desfavorável. Concluíram quea expressão da proteínap21associa-se à
proliferação celulare à sobrevida do paciente com PC. Ainda, segundo Huang et al.123
, a p21
pode ter participação na patogênese da HPB, pois uma alteração no gene CDKN1A pode
causar desequilíbrio entre a proliferação de células epiteliais e do estroma da próstata e falha
da apoptose, contribuindo para o acúmulo de células característico da HPB. No entanto, vale
ressaltar que as alterações relacionadas à expressão p21 nem sempre têm origem em mutação
do CDKN1A, podendo também ocorrer alterações epigenéticas, ou seja, aquelas que
25
envolvem metilação, acetilação e aumento ou modificação de histonas, já que CDKN1A é
frequentemente silenciado por metilação no PC humano, reduzindo a síntese de p21. Da
mesma forma, é possível que ocorra aumento na síntese de p21 quando da ativação do
mecanismo de modificação de histonas136
.
O inibidor de ciclina dependentede quinase1B(CDKN1B) é o genequecodifica a
proteína p27, membro dafamíliaCIP/KIP, que atua de forma a inibir a proliferação celularea
carcinogênese136,137
. Contrariamente à p21, que parece ser uma CdkI universal, já que atua
sobre diferentes complexos Cdk/ciclina, a p27 exerce função de modulação inibitória apenas
sobre o complexo Cdk2/ciclina E, o que resulta em bloqueio da entrada da célula na fase S.
Ressalte-se que essa função inibitória do p27 só é possível a partir de sua ligação aos
receptores de TGF-β113,115
. Portanto, a ausência de sinalização de TGF-β contribui para a
proliferação celular anormal e para a malignidade117
Segundo Coqueret138
, aredução da expressão dep27 sugere prognóstico
desfavorável, tendo em vista que em sua ausência ocorre manutenção da célula no ciclo
celular. Essa redução de expressão de p27 tem sido observada em várias neoplasias, incluindo
as da próstata. Yang et al.135
e De Marzo et al.26
observaram menor expressão de p27 em
lesões como HPB, PIN e PC, concluindo que isso contribui para a alta taxa de mitose nessas
lesões proliferativas. Também Wangetal.139
observaram expressãoelevada dep27 em células
normais, comlesões hiperplásicasmuito precoceseem células com PIN e concluíram que o
aumento da expressãodep27 contribui para a prevençãoe inibe aprogressão das lesõesde
PINparaPCinvasivo. Da mesma forma, Majumderetal.140
e DiCristofanoet al.141
observaram
elevada expressão dep27emlesões de PIN experimentalmente induzidas em camundongos.
Tayloretal.142
completaram que na redução ou ausência de expressão de p27 a PIN possui
maior potencial de progressão edesenvolvimento de um fenótipo mais agressivo.
Wang et al.139
demonstraram que o epitélio nas lesões de PIA possui suas próprias
características moleculares, com fenótipo intermediário entre o das células basais e luminais,
isto é, expressa fatores de crescimento que podem aumentar a atividade de proliferação e
regular a expressão de p27, podendo, dessa forma, servir como marcador de evolução tumoral
e prognóstica75,26
.
Em tumores mamários de cadelas, Klopfleisch et al.143
observaram redução da
imunoexpressão de p27, já que as amostras de epitélio mamário normal apresentaram maior
expressão dessa proteína em comparação às amostras de tecido mamário com adenomas e
carcinomas. Em outro estudo de neoplasias mamárias em cadelas, Klopfleische
Gruber126
tambémrelataramredução significativadonível deexpressão de RNAm dep27em40%
26
dos adenomas, 90% de adenocarcinomase80% dasmetástases nos nódulos linfáticos
regionais,quandocomparado àglândulamamárianão neoplásica. Os autores concluíram que o
controle do ciclocelular via p27 é falho na maioria dos tumores mamários caninos e ainda que
essa redução representa papel importante na evolução do processo tumoral, apesar de não
terem observado diferençasignificativa na expressão de p27 entretumoresmamários
benignosemalignos. Isso limita a utilização dessa proteína como marcador de malignidade
para os tumores mamários caninos, mas não descarta o envolvimento da mesma na regulação
do crescimento tumoral79
.
O produto do gene TP53, a proteína p53, é considerada a guardiã do genoma, a
chave reguladora, pois é responsável por regular grande parte dos processos celulares,
incluindo o ciclo celular, reparo do DNA, estabilização do genoma, morte celular
programada, diferenciação, senescência e angiogênese144
. Em condições normais a proteína
p53, conhecida como p53 tipo selvagem, possui meia-vida curta, sendo degradada
imediatamente após o término de sua função. Assim, é sabido que mutações no gene TP53
resultam em falha da proteção celular, desestabilização do genoma e permite a produção de
proteína p53 afuncional, que se acumula na célula e é eliminada lentamente, o que permite
sua detecção nuclear pela técnica da IHQ76
.
O ciclo celular prossegue normalmente sob a vigilância passiva de p53 em níveis
baixos. Quando um erro ocorre na transcrição do DNA, esta interrompe o ciclo na fase S para
a atuação dos mecanismos de reparação, ou seja, os níveis de p53 se elevam e há indução da
expressão de p21, que impedirá a ligação Cdk-ciclina. Consequentemente, pRB não será
fosforilada, impedindo assim a liberação do fator de transcrição (E2F), com parada do ciclo
na fase S. Nesse momento, a célula pode promover reparo do DNA e seguir no ciclo ou
acionar eventos apoptóticos para que ocorra a destruição da célula danificada70,115,122
.
A ação da p53 é inibida quando da ligação à proteína murine double minutes
(Mdm2), impedindo-a de reativar a transcrição da proteína p21. No entanto, o gene que
codifica a proteína Mdm2, gene MDM2, é ativado por transcrição pela p53, evitando o
excesso funcional da mesma. Em células tumorais, os níveis de Mdm2 estão aumentados e há
o decréscimo na concentração de p53117
, mas também pode haver mutação do gene TP53,
seguida de ausência de expressão da proteína p53. Nesses casos, as células com danos no
DNA escapam do reparo ou do processo de apoptose, dando início a um clone
maligno115,122,145,146
.
Entre as neoplasias humanas, o TP53 é considerado o gene mais comumente
alterado146
. Nos animais, alterações nogene TP53foram identificados em vários tipos de
27
tumor, como de tiroide147
, papilomaoral148
, mamário149-150
, osteossarcoma151
, adenomada
glândula hepatoide152
, linfoma153
e próstata55,154
.
Segundo Stricker et al.155
, Cheng et al.156
, Leite et al. 157
e Tsujimoto et al.158
, em
humanos com PIN e PC quanto maior a expressão da p53 maior a probabilidade de
progressão da doença devido a índices proliferativos mais elevados e a um fenótipo mais
agressivo. Ainda, de acordo com Stricker et al.155
, nos casos de carcinoma prostático humano
em que a expressão da p53 foi pouco expressiva o câncer não progredia. Ressalte-se que a
avaliação imunoistoquímica da proteína p53 não deve ser considerada isoladamente na
determinação do prognóstico das alterações prostáticas de caráter maligno, considerando que
devido a meia-vida curta da proteína, é possível reação cruzada entre as formas selvagem e
mutante, como demonstrado por Wang et al.47
.
Na próstata canina, a expressão de p53 foi relatada em alguns trabalhos, mas com
resultados antagônicos. Di Santis58
não detectou a expressão de p53 nas afecções da próstata
canina. De outra parte, Croce et al.55
verificaram sua expressão tanto no tecido prostático
normal quanto com PC, e ainda nas lesões proliferativas e atípicas da próstata canina, sendo
que 95% das amostras de PIA e PC apresentavam-se intensamente marcadas quando
comparadas às amostras de tecido normal. Embora não tenham observado imunomarcação de
p53 no tecido prostático normal, Faleiro e De Moura 154
obtiveram resultados semelhantes aos
de Croce et al.55
, já que observaram imunomarcação de p53 em amostras de próstata canina
com PIA, PIN e PC. Nesse estudo, os autores argumentaram que a elevada expressão de p53
constatada nos casos de PIA se justifica em decorrência do microambiente inflamatório, fator
importante na promoção da alteração da p53. Ainda, segundo Wang et al.47
, a p53 mutante é
frequentemente detectada em tecidos com inflamação crônica, incluindo colangioepatites,
gastrites e colites, assim como Tsujimoto et al.158
também referiram a expressão de p53 na
atrofia inflamatória pós-atrófica da próstata humana.
1.7. Ciclina D1 (CD1)
As ciclinas, produtos do gene CLN (neuronal ceroid lipofuscinosis), são enzimas
reguladoras das subunidades das ciclinas dependentes de quinase (Cdk). Diferentes ciclinas se
associam a diferentes Cdk, podendo associar-se a mais de uma Cdk nas diferentes fases do
ciclo celular. Esse grupo de enzimas, por sua vez, fosforila uma série de substratos-chave que
permitirão a progressão de uma fase à outra do ciclo75-77,159
. Em G0 não há expressão de
ciclinas nem de Cdk. Em G1 há expressão dos complexos Cdk4/ciclina D1 e Cdk6/ciclina D1
e, ao passo que se aproxima o ponto de restrição, ocorre aumento do complexo Cdk2/ciclina
28
E. Já em S há expressão de Cdk2/ciclina A e, em G2, de Cdk1/ciclina B e Cdk1/ciclina A, que
se intensifica em M75-77,161
(Figura 11).
FIGURA 11 - Representação gráfica da atividade dos
complexos Cdk/ciclina durante o ciclo
celular. A largura das faixas coloridas é
aproximadamente proporcional à atividade dos complexos indicados.
Fonte: Lodish et al.76
.
Na fase G1, a CD1 representa um ponto crítico de regulação e, por isso, alterações
na sua expressão estão relacionadas à tumorigênese e à progressão tumoral de diferentes tipos
neoplásicos, incluindo os prostáticos. Ainda, em muitos casos há correlação entre essa
alteração e a expressão e regulação esteróide, com aumento de receptores de andrógenos77,162
.
Dessa forma, a avaliação da expressão da CD1 pode fornecer informações de prognóstico,
tendo em vista que maior expressão foi observada em lesões indiferenciadas e avançadas135
.
Quanto às outras ciclinas, pouco se conhece acerca do seu papel na carcinogênese
prostática77
.
O geneda CD1, CCND1, é reguladoem nívelde transcriçãopela interação entre a
proteínas guanilato quinase (ZO-2), E-box, e c-myc, e recrutamento de histonas
desacetiladas159,160
, sendo essa transcrição reprimida pelas proteínas fosfatase tensina
homologa (PTEN)162
e p53163
. Segundo Tapia et al.159
, a expressão aumentada de ZO-2 é
capaz de induzir a redução da expressão de CD1 em células epiteliais e, dessa forma, há
redução na proliferação celular, com bloqueio na fase G0/G1 do ciclo celular e manutenção
da taxa de apoptose.
A CD1éimportante na regulaçãodociclo celular e estudos em modelos animais
sugerem que essa enzima pode atuar como oncogene de predileção, pois a superexpressão de
CD1 nas células mamárias de camundongos transgênicos levou ao desenvolvimento de
29
hiperplasia e neoplasia164
. Oncogene de predileção é um termo utilizado para descrever o
fenômeno em que a superatividade de determinado oncogene é requisito para que as células
neoplásicas sobrevivam e proliferem. Assim, oncogenes de predileção são alvos para o
desenvolvimento de drogas que possam bloquear seus efeitos sobre as células neoplásicas,
mas sem afetar a viabilidade de células normais163
.
ACD1é uma proteínainstável e de meia-vida curta, sendo que seu acúmulo induz
arápida progressãodociclo celular da fase G1 paraS. A CD1 também atua em conjunto com a
proteína RecA recombinase (RAD51), ativando vias de reparo de DNA, com redução de sua
expressão nessas situações, embora isso não necessariamente implique em redução da
proliferação de células prostáticas neoplásicas161
, o que sugere que a CD1 não é a única
envolvida no processo de proliferação neoplásica. Entretanto, muitos autores relataram a
relação entre superexpressão de CD1 e proliferação de vários tipos de câncer em
humanos164,165
.
1.8. Proteínas c-myc
Outro importante elemento relacionado à regulação do ciclo celular é o oncogene
C-MYC, que codifica as proteínas myc1 ou p67 e myc2 ou p64, ou simplesmente proteínas c-
myc165,166
. Essas proteínas suprimem a transcrição de genes como o CDKN1A (transcritor da
proteína p21) e CDKN1B (transcritor da proteína p27) e ativam a transcrição das proteínas
Cdk2, Cdk4 e pRB, com consequente liberação de E2F e superação do efeito inibitório do
TGF-β sobre o ciclo celular, culminando com a passagem da fase G1 para S75,115,167
. Assim,
participa de forma direta e indireta nos processos associados à transformação e metabolismo
celulares, apoptose e instabilidade genômica52,75,165,167-169
.
A ativação inapropriada do C-MYC pode contribuir para o desenvolvimento
neoplásico por diferentes mecanismos, incluindo translocaçãocromossômica, amplificação
gênica einserção do generetroviral165,166
.Outrosestudossugeriramque as proteínas c-myc
estimulamaproduçãomitocondrialderadicais livres, o que comprometeo DNA, causando
instabilidade genômica. No entanto, não são consideradasessenciaispara mecanismos que
envolvem metástase165,168,169
.
Sendo ogene C-MYC um elemento-chave no processo de tumorigênese, já foi
observado em váriostipos de câncer, incluindo osmamários165,169-174
e prostáticos em
humanos175,176
e cães39
, bem como palpebrais em cães127
, osteossarcomas caninos 265
, tumor
venéreo transmissível canino177
, mamários em ratas170
e prostáticos em camundongos179
.
30
De acordo comSong etal.180
, Iwataetal.181
e Kohetal.175
, a proteína c-myc está
elevadaem87% dos casosPC humano esignificativamente aumentada em relação ao tecido
prostático normal e não neoplásico. Ainda, segundo Qianetal.168
e Kohetal.175
, a c-myc está
associada ao grau de diferenciação e estágio avançado do tumor, sendo observada nos casos
de PC com escore de Gleason maior que cinco e naqueles com metástases, caracterizando pior
prognóstico115,184
.
Fonseca-Alves etal.39
relataram maior positividade de c-myc na prostáta canina
com PIA e PCem comparaçãoàquelas normais, sendo observada imunoexpressão aumentada
nas amostras de PIA. Os autores completaram que a proteína c-myc é normalmente
expressanocitoplasmadas célulasepiteliais da próstata com PC equeestá associada à perdado
gene NKX3.1, um gene supressor detumor, e da proteína de junção E-caderina. Entretanto,
também descrevem imunomarcação nuclear de c-myc em algunscasosde PC concomitante a
focos dePIN, o que já havia sido relatado por Gureletal.182
eProwatkeetal.183
.
Ellwood-Yen etal.184
, Iwataetal.181
, Kohet
al.175
eNakagawaeYamanaka185
hipotetizaram que oaumento daexpressão de c-myc no
PChumanopromovea proliferação e diferenciaçãode células tumorais,emespecial das
célulasbasais, bem como alterações na viaapoptótica, uma vez que a superexpressãonuclearde
c-myc correlaciona-se a PINe sua transiçãopara PC.Civennietal.176
estudaram a expressão do
gene C-MYC em uma subpopulação de células neoplásicas semelhantes a células tronco (CSC
- cancer stem cell like) e observaram redução das CSCem resposta ao silenciamento desse
gene, concluindo que na ausência de ação do C-MYC ocorre ativaçãodos mecanismos
desenescência e redução do potencial tumorigênico e metastático. Os autores estudaram ainda
a expressão de C-MYC em diferentes tipos tumorais e obtiveram o mesmo silenciamento
gênico nos casos de PC. Entretanto, concluíram que a relação entre redução de proliferação
celular e silenciamento do C-MYC não compreende mecanismo único envolvido no
crescimento tumoral, tendo em vista que também foi observada proliferação de células
quando do C-MYC silenciado.
2. Objetivos
Com o intuito de melhor compreender os mecanismos celulares envolvidos em
lesões proliferativas da próstata canina, este trabalho teve por objetivo avaliar a expressão
gênica de ErbB1, ErbB2, CDKN1A, CDKN1B e TP53, e a imunomarcação de EGFR, Her2,
p21, p27, p53, ciclina D1 e c-myc no tecido prostático canino normal e com HPB, PIA, PIN e
PC.Para a avaliação da expressão gênica foram utilizadas as técnicasRT-PCR e RT-
31
qPCRcomRNAm de amostras próstata canina parafinizada e para a imunomarcação a técnica
de imunoistoquímica em lâminas demicroarranjo tecidual.
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Vienna, Austria). All values were in rank and were considered with 5% of significance level.
RESULTS
From the TMA slide, 15 (21.43%) samples of normal tissue, 30 (42.86%) of PIA,
and 25 (35.71%) of PC were obtained and scored according intensity of immunostaining to
EGFR and Her2 (Table 1).
The immunostaining was present in the membrane going through cytoplasm, is
shown in Figures 2 and Figure 3. Even using human antibodies, the results from this study are
reliable since Singer et al.37
demonstrated by BLAST alignments that canine ErbB1 and
ErbB2 are homologues in 91% and 92%, respectively, with the equivalent human
homologues, and identities of 95% for both molecules with the human counterparts. They
conclude that both targets indicated almost perfect evolutionary conservation which makes
canine cancer an excellent method suited for proof-of-concept studies in clinical comparative
oncology settings, especially for research working on the ErbB-family37
.
TABLE 1- Labeling intensity scores of EGFR and Her2 antibodies in
normal prostate tissue and with PIA and PC.
EGFR
Normal
(n=15)
PIA
(n=30)
PC
(n=25)
Labeling Intensity
0- Negative 20% (3) 6.67% (2) 24% (6)
1+ (Mild) 53.33% (8) 56.67% (17) 44% (11)
2+(Moderate) 26.67% (4) 33.33% (10) 28% (7)
3+(Intense) 0% (0) 3.33% (1) 4% (1)
Her2
Labeling Intensity
0- Negative 87.7% (13) 13.33% (4) 0% (0)
1+(Mild) 13.3% (2) 26.67% (8) 16% (4)
2+(Moderate) 0% (0) 23.33% (7) 12% (3)
3+(Intense) 0% (0) 36.67% (11) 72% (18)
51
FIGURE 2 - EGFR immunostainingmembrane through cytoplasm (arrow). A) Human
placenta. Positive control with 3+ immunostainingscore(200X); B) Canine
PC tissue with 1+score(400X); C) Normal canine prostatic tissue with 1+ score (400X); D) Canine prostatic tissue with PIA and 2+labeling score
(200X). IHC, DAB with H&E counterstain.
FIGURE 3 - Her2 immunostainingmembrane through cytoplasm (arrow). A) Human
mammary carcinoma. Positive control with 3+ immunostaining intensity score (400X); B) Canine PC tissue with 3+score(400X); C) Normal canine
prostatic tissue with 0- score (200X); D) Canine prostatic tissue with PIA
and 2+ labeling score (400X); IHC, DAB with H&E counterstain.
A B
C D
A B
D C
52
Regarding EGFR immunostaining intensity, there was no difference between
normal prostate and PIA and PC (p<0.05). In contrast, difference was noticed in Her2
immunostaining intensity between normal prostatic tissue and with PIA and PC, and between
PIA and PC (p<0.05) (Table 2). When comparing immunostaining intensity of EGFR and
Her2, there was no difference between normal prostatic tissue and with PIA and PC (p<0.05).
TABLE 2 - Means of comparison between EGFR and Her2 immunostaining intensity in canine normal
prostatic tissue and with PIA and PC.
Diagnoses Rank of Staining Intensity
EGFR Her2
Normal (n=15) 45.20a
17.33a
PIA (n=30) 55.86a
56.66b
PC (n=25) 47.12a
74.18c
Similar letters in the same column are not different by Kruskal-Wallis test (p>0.05).
From 70 samples, sixteen cDNA samples of canine prostatic tissue with rate
between A260/A280 higher than 1.70, were obtained to perform RT-PCR, being four (25%)
normal tissue, three (18.75%) with PIA, and nine (56.25%) with PC.
A commercial kit for extraction enabled us to obtain DNA and RNA from FFPE
canine prostate tissue with good enough quality and quantity to perform the RT-PCR test
successfully. Isolating nucleic acids from tissues in sufficient amount, purity and integrity is
an essential step in the practice of molecular biology. The quantity, purity and integrity of
nucleic acid extracted depend on time of pre-fixation, and time and temperature used in FFPE
process38,39
. Thus, extracting RNA with good quality and quantity is a feasible and valuable
tool in diagnostic routine and retrospective studies40,41
.
The ErbB1 gene mRNA was detected in two normal, one PIA and in all PC
samples. ErbB2 mRNA was detected in PIA2, PIA3, and all PC, but no in normal prostatic
tissue samples (Figure 4). A parallel of EGFR and Her2 immunostaining by IHC and ErbB1
and ErbB2 detection by RT-PCR is shown in Table 3.
53
FIGURE 4 - Agarose gel images showing amplification products of β-actin, ErbB1 and ErbB2
genes from canine prostatic tissue. A) RT-PCR for β-actin expression as samples
positive control. All samples amplified. B) RT-PCR for ErbB1.Prostatic samples
N3, N4, PIA2, all PC, and MC amplified. C) RT-PCR for ErbB2. Prostatic samples PIA2, PIA3, all PC and MC amplified. MW = Molecular weight (GeneRuler 1Kb
Plus DNA Ladder - Thermo scientific, CA, USA, #SM1331); MC= canine
mammary carcinoma (ErbB1 and ErbB2 positive control); N=Canine normal
evaluation in adult not castrated dogs. Arq. Bras. Med. Vet Zootec. 2010;62:10-29.
56
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kinases are associated with hormone escape in prostate cancer. J Pathol. 2005;205:522-529.
24. Liu HL, Gandour-Edwards R, Lara PN Jr, de Vere White R and LaSalle JM: Detection of low level
HER-2/neu gene amplification in prostate cancer by fluorescence in situ hybridization. Cancer J. 2001;7:395-403.
25. Carles J, Lloreta J, Salido M, Font A, Suarez M, Baena V, Nogue M, Domenech M and Fabregat
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canine mammary gland carcinomas: an immunohistochemical and chromogenic in situ hybridization
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32. Hsu WL, Huang HM, Liao JW, Wong ML, Chang SC. Increased survival in dogs with malignant
mammary tumours overexpressing HER-2 protein and detection of a silent single nucleotide polymorphism in the canine HER-2 gene. Vet J. 2009;180(1):116-123.
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36. Bilous M, Dowsett M, Hanna W, Isola J, Lebeau A, Moreno A, Penault-Llorca F, Rüschoff J,
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“Eis porque, interpretando os cromossomas à guisa de caracteres em que a mente inscreve,
nos corpúsculos celulares que a servem, as disposições e os significados dos seus
próprios destinos, caracteres que sãoconstituídos pelos genes, como as linhas são
formadas de pontos.”
André Luiz (Francisco Cândido Xavier e Waldo Vieira)
Evolução em dois mundos (1959)
60
CAPÍTULO 3 - Cell cycle inhibitors expression in canine prostate with
prostatic inflammatory atrophy and carcinoma
ABSTRACT
Gene expression of CDKN1A, CDKN1B, and TP53, and immunostaining of p21, p27 and
p53 were evaluated to verify the role of these cell cycle inhibitors in canine prostates with
proliferative inflammatory atrophy (PIA) and prostatic carcinoma (PC). From 70 prostatic
samples obtained, 15 were considered normal, 30 had PIA and 25 PC. Regarding the number
of p27 and p53 labeled cells, difference between normal and PIA and PC was observed, as
well as between PIA and PC for p53. Immunostaining intensities of p21, p27 and p53 were
different when comparing normal tissues to PIA and PC. Sixteen cDNA samples of canine
prostatic FFPE tissue were subjected to RT-PCR and RT-qPCR, four considered normal
tissue, three diagnosed as PIA, and nine as PC. CDKN1A mRNA was detected in four PC
cases by RT-PCR, and it was overexpressed when compared to normal by RT-qPCR, in one
PIA and six PC samples. CDKN1B mRNA was detected in three PC cases by RT-PCR and it
was overexpressed in three PC and decreased in one PC case. TP53 mRNA, detected in all
canine prostates with PIA and PC, was also overexpressed in one gland with PIA and three
with PC. It was conclude that when overexpressed in canine prostate with premalignant and
malignant lesions, p21 and p27 play role controlling cell proliferation, most likely working as
a protective factor in the evolution of PIA to PC, and in the PC development, even in the
presence of altered p53. Thus, the next step would be verifying these cell cycle proteins in
canine PC with metastasis.
Key words: p21, p27, p53, PIA, RT-qPCR.
61
Expressão de inibidores do ciclo celular na próstata canina com atrofia
inflamatória proliferativa e carcinoma
RESUMO
A expressão gênica de CDKN1A, CDKN1B e TP53, e imunoexpressão de p21, p27 e p53
foram avaliados, a fim de verificar o papel desses inibidores do ciclo celular na próstata
canina com atrofia inflamatória proliferativa (PIA) e carcinoma prostático (PC).Foram obtidos
um total de 70 amostras de próstata canina, sendo 15 de tecido normal, 30 de PIA e 25 de PC.
Quanto ao número de células imunomarcadas foi observado diferença entre normal, PIA e PC
para p27 e p53, também para p53 houve diferença entre PIA e PC. Para a intensidade de
imunomarcação houve diferença entre os tecidos normais e com PIA e PC para p21, p27 e
p53. Foram obtidas dezesseis amostras de cDNA a partir de tecido FFPE de próstata canina
para realização da RT-PCR e RT-qPCR, sendo quatro tecidos normais, três com PIA, e nove
com o PC. O gene CDKN1A foi detectado em quatro das PC por RT-PCR, e pela RT-qPCR
este estava super expresso em uma PIA e em seis PC quando comparado com o tecido
prostático normal. O CDKN1B foi detectado em três PC por RT-PCR e pela RT-qPCR estava
super expresso em três PC e reduzido em um PC. O TP53 foi detectado em todas as próstatas
caninas com PIA e PC por RT-PCR, sendo também superexpresso em uma glândula com PIA
e em três com PC. Concluiu-se que p21 e p27 quando superexpressas na próstata canina com
lesões pré-malignas e malignasdesmpenham ação no controle da
proliferaçãocelular,possivelmenteatuando como fator de proteção na evolução da PIA para
PC, e no desenvolvimento do PC, mesmo na presença de p53 alterada. Assim, o próximo
passo seria avaliar essas proteínas do ciclo celular em casos de PC canino com metástase.
Palavra chave: p21, p27, p53, PIA, RT-qPCR.
62
INTRODUCTION
The canine prostate has similarities with the human gland regarding occurrence of
benign and malignant diseases as benign prostatic hyperplasia (BPH) and prostatic carcinoma
(PC)1,2
. Proliferative inflammatory atrophy (PIA) is also a human prostate lesion with
premalignant potential and involvement in carcinogenesis of PC3,4
. In dogs, Rodrigues et
al.5have mentioned PIA in canine prostate and Toledo et al.
6 described its histological aspects.
The proliferation of eukaryotic cells is controlled at specific points in the cell
cycle, particularly at G1-S and G2-M transitions, and regulated by interaction of cyclins and
cyclin-dependent kinases (Cdk) and their inhibitors (CdkI`s). The major step in malignant
transformation in tumors is the loss of cell cycle control. Knowing how regulators work is a
key step to understand malignant transformations that occur in tumor types7-10
.
Cyclin-dependent kinase inhibitor 1A (CDKN1A) gene encodes the p21 protein,
which has been implicated in mechanisms of cell-cycle arrest from G1-S phase through
different steps that allow cell DNA repair or apoptosis11-12
. Those tasks are performed by
cyclin A/cyclin dependent kinase 2 (Cdk2) and cyclin D/Cdk4 complexes13
. Increased
expression of CDKN1A is not necessarily linked to cell growth blocking, since p21 protein
may have dual a function, inhibiting cyclin/Cdk activity and also acting as positive modulator
of cyclin/CDK complex formation12
. Furthermore, studies have revealed decreased expression
of p21 associated with poor prognostic in human tumors, including gastric, colorectal,
prostatic, and endometrial carcinomas14-16
.
The p27 protein is encoded by cyclin-dependent kinase inhibitor 1B (CDKN1B)
gene. This protein has been implicated in cell-cycle blocking mechanisms in S phase. It is
regulated by extracellular stimuli, like transforming growth factor beta (TGF-β)17-19
. Yang et
al.20
and De Marzo et al.21
observed decreased expression of p27 in lesions such as BPH, PIN
and PC, contributing to the high rate of mitosis in these proliferative lesions. Also,
Fredersdorf et al.22
and Tan et al.23
refer that the loss of p27 protein is a negative prognostic
factor for human breast cancer.
An important protein controlling the cell cycle is p53, well known as the
"guardian" of the genome. If an error in DNA transcription occurs, p53 stops cell cycle and
promote repair mechanisms or triggers apoptotic events leading to cell death. The p53 protein
stimulates p21, which has regulatory functions and blocks the cell cycle progression24,25
. In
normal cells, wild-type p53 has a short half-life and does not accumulate in the tissue at
detectable levels. Mutations or deletions of TP53 gene may lead to altered production of p53
protein, which fails to perform its function and accumulates in the nucleus, when is possible
63
to be detected it by immunohistochemistry19,24
. The p53 immunostaining in canine prostate
has been reported as increased in PIA and PC26
, but its role in canine prostatic tissue with
proliferative lesions is not yet clear. Thus, in order to further understand the molecular
mechanisms of cell cycle blocking in canine premalignant and malignant prostatic tissue,
CDKN1A, CDKN1B, and TP53 mRNA expression and immunostaining of p21, p27 and p53
were evaluated in canine prostate with PIA and PC.
MATERIAL AND METHODS
The samples were derived from the necropsied animal´s on pathology service of
the School of Veterinary Medicine and Animal Science of the Federal University of Goiás
(UFG), Goiânia, Goiás, Brazil, registered from 2008 to 2012. The research was approved by
committee on publication ethics COPE/UFG number 353/2010.Three-µm-sections were
obtained from formalin-fixed-paraffin-embedded (FFPE) tissue blocks and stained with
hematoxylin and eosin (HE) for microscopic examination. Histomorphological evaluation was
used to identify normal, PIA6 and PC
27prostates. All prostatic samples were obtained from
with pure or mixed-breed adult dogs and normal prostatic tissues were harvested from dogs
with no lesions in the gland.
Prostate tissue microarray (TMA) was carried out according to criteria described
by Rubinet al.28
. From the previous defined areas in histomorphological evaluation, tissue
cores with a dimension of 1.0 mm were taken from FFPE tissue samples and arrayed on a
recipient paraffin block, in duplicate, using the tissue microarray (Beencher Instruments®,
Silver Spring, USA). Three-µm-sections were obtained from recipient block and distended on
charged slides (Starfrost White, Sakura Adhesion microscope slide with Cut Edges, ready to
use, Germany, Dako #9545-1) for HE staining and immunohistochemistry.
Immunohistochemistry was performed in TMA slides, in duplicate, which were
deparaffinized, rehydratated and washed in distilled water. Endogenous peroxidase and non-
specific protein were blocked with 3% hydrogen peroxide incubation for 20min, protein block
(Leica, Newcastle, UK, #RE7102) incubation for 5min at 37°C. Antigen retrieval for p21, p27
and p53 was performed with 10 mM pre-heated citrate buffer, pH 6.0, for 3min, in a pressure
FIGURE 1 - Immunohistochemistry photomicrography on nuclear immunostaining(arrow). A) Canine PC tissue with score three for labeling intensity and four for number of labeled
cells for p21. 400X. B) Human colon carcinoma; p21 positive control with score two for
labeling intensity and four for number of labeled cells.400X. C) Canine PIA tissue with
score three for labeling intensity and four for number of labeled cells for p27. 200x. D) Human colon carcinoma; p27 positive control with score three for labeling intensity and
four for number of labeled cells. 400X E) Canine PC tissue with score three for labeling
intensity and four for number of labeled cells for p53. 200X. F) Human mammary carcinoma; p53 positive control with score three for labeling intensity and three for
number of labeled cells. 200X. IHC, DAB with H&E counterstain.
69
TABLE2 -Comparison between p21, p27, and p53 immunostaining regarding
number of stained cells and intensity of staining in canine normal
prostatic tissue, PIA and PC.
Diagnoses N Rank of Number of Stained Cells Rank of Staining Intensity
p21 p27 p53 p21 p27 p53
Normal 15 46.3a 34.4
a 16.9
a 33.9
a 31.6
a 23.0
a
PIA 30 57.8a 60.1
b 54.6
b 55.8
b 60.4
b 55.7
b
Carcinoma 25 59.8a 67.4
b 75.2
c 64.3
b 68.6
b 68.2
b
Similar letters in the same column are not different by non parametric multiple
comparison test for paired contrasts the relative effects with Tukey correction.
(p value =<0.05).
Sixteen cDNA samples of canine prostatic tissue were obtained to perform RT-
PCR, being 4 (25%) normal tissue, 3 (18.75%) with PIA, and 9 (56.25%) with PC. CDKN1A
mRNA was detected in four of nine PC (4/9 - PC6, PC7, PC8, PC9) and CDKN1B mRNA in
three of nine PC (3/9 – PC7, PC8, PC9). TP53 gene expression was detected in all PIA and
PC samples, but not in normal prostatic tissue (Figure 2).
FIGURE 2 –Images from agarose gels showing the amplification products of ACTG1 (β-actin),
CDKN1A, CDKN1B and TP53 genes from canine prostatic tissue. (A) RT-PCR for β-
actin expression (positive control). (B) RT-PCR for CDKN1A. Prostatic samples PC6, PC7, PC8, PC9 and MC amplified. (C) RT-PCR for CDKN1B. Prostatic samples PC7,
PC8, PC9 and MC amplified. (D) RT-PCR for TP53. Prostatic samples PIA1, PIA2,
PIA3, PC1, PC2, PC3, PC4, PC5, PC6, PC7, PC8, PC9 and MC amplified. MW = Molecular weight (Gene Ruler 1Kb Plus DNA Ladder - Thermo scientific, CA, USA,
#SM1331); MC= canine mammary carcinoma (CDKN1A, CDKN1B and TP53 positive
The expression of CDKN1A, CDKN1B and TP53 in canine normal prostate, with
PIA and PC was determined by RT-qPCR and fold change (FC) normalized with β-actin
expression from the same dog and with normal gland with T melt 84.8ºC. The CDKN1A was
overexpressed in seven of sixteen tissues investigated (PIA1, PC1, PC3, PC4, PC7, PC8 and
PC9) with FC>1.6 when compared to normal gland N1 to N4 and T melt 76.9ºC. The
CDKN1B was overexpressed in three of nine PC tumors (PC7, PC8, PC9) with FC>1.6, and it
was under expressed in one of nine PC (PC2) based on FC<0.4 and T melt 80.5ºC. Regarding
TP53, overexpression was present in five of sixteen prostatic samples (PIA1, PC4, PC6, PC8,
PC9) with FC>1.6, and decreased expression was observed in two of sixteen prostatic tissues
(PIA2, PC2) based on FC<0.4 and T melt 78.8ºC (Figure 3).
FIGURE 3 – Fold expression levels of CDKN1A, CDKN1B and TP53 in canine normal, PIA and PC
normalized by β-actin. Values>1.6 were considered overexpression and values<0.4
were considered decrease of gene expression.
A commercial kit for extraction enabled us to obtain DNA and RNA from FFPE
canine prostate tissue with good enough quality and quantity to perform the RT-PCR test
successfully. Isolating nucleic acids from tissues in sufficient amount, purity and integrity is
an essential step in the practice of molecular biology. The quantity, purity and integrity of
nucleic acid extracted depend on time of pre-fixation, and time and temperature used in FFPE
process31,32
. Thus, extracting RNA with good quality and quantity is a feasible and valuable
tool in diagnostic routine and retrospective studies33,34
.
71
Comparison of results referring to p21, p27 and p53 immunostaining by IHC and
CDKN1A, CDKN1B and TP53 detection by RT-PCR and expression by RT-qPCR are
described in Table 3.
TABLE 3 – Comparison between p21, p27, and p53 immunostaining regarding number of stained
cells and intensity of staining in canine normal prostatic tissue, PIA and PC
Samples IHC RT-PCR RT-qPCR
p21 p27 p53 CDKN1A CDKN1B TP53 CDKN1A CDKN1B TP53
N1 0 0 0 - - - R R R
N2 0 0 0 - - - R R R
N3 2/1 2/1 0 - - - R R R
N4 0 0 1/1 - - - R R R
PIA1 4/3 2/1 4/3 - - + O R O
PIA2 2/2 2/1 1/1 - - + R R D
PIA3 0 2/1 2/2 - - + R R R
PC1 4/3 4/3 2/2 - - + O R R
PC2 0 0 0 - - + R D D
PC3 4/3 4/3 2/2 - - + O R R
PC4 4/3 4/3 4/3 - - + O R O
PC5 2/1 3/2 2/3 - - + R R R
PC6 3/2 3/2 4/3 + - + R R O
PC7 4/3 4/3 3/2 + + + O O R
PC8 4/3 4/3 3/2 + + + O O O
PC9 4/3 4/3 4/3 + + + O O O
MC 4/3 4/3 4/2 + + + R R R
MC=canine mammary carcinoma; N=canine normal prostatic tissue; PIA=canine proliferative
inflammatory atrophy; PC=canine prostatic carcinoma; N/N= score of number of stained cells/
score of intensity of staining; R=regular expression; D=decreased expression; and O=overexpression
DISCUSSION
It was observed in this study that p21 immunostaining protein was overexpressed
in 40% of PC and 6.7% of PIA, and in normal tissue the expression was weak in 53.3% and
absent in 46.7%. Protein p21 play an important function in the cell cycle and its increased
expression may mean a crucial step in the cell growth blocking, inducing senescence or
apoptosis, a mechanism which seems to protect against malignant transformation35-38
. Thus,
loss of p21 expression has been associated with shorter survival in human with PC16
. Also,
according to Matsushima et al.39
, immunostaining of p21 was more likely to be expressed in
well-differentiated areas.
Also, there was difference between normal prostatic tissue with PIA and PC
regarding intensity of immunostaining, and there was no difference between groups
72
considering the number of stained cells. In this context, our results suggest that p21 could
work as a protective factor in canine PC progression, since in human PC p21 overexpression
is associated with good prognosis16
. Furthermore, even considering the possibility that canine
PC shows high aggressively when present2,40
, this tumor is not as common in canine prostate
as compared to the human prostate. It seems that canine PC has longer time development,
which could be linked to action of inhibitors as p21 and p27 during canine PC progression.
This hypothesis was mentioned before in canine prostate, but referring proteins from other
regulatory pathways as GSTP126
. Also, it seems that immunostaining intensity of p21 in
canine prostate tumors should be considered.
Klopfleisch and Gruber29
reported p21 overexpression in canine malignant
mammary tumors, but concluded that its increase alone did not avoid metastasis and an
explanation for that would be a loss of p21 function by missense mutations. On the other
hand, Wang et al.16
add that overexpression of p21 in human prostate may be associated with
poor prognostic due to CDKN1A instability, or its function may be altered indirectly by TGF-
β41
, PTEN deficiency36
, and TP53 mutated42-44
. Since p21 may play a double function, acting
as tumoral suppressor or oncogene12
depending on tumor type, and considering our results, it
seems that p21 could be involved in controlling cell proliferation in canine prostate with
proliferative lesions as observed in human breast cancer45
.
Taken together, the results from this research concerning p21 and p27 inhibitors
indicate that positive relation for their overexpression in canine proliferative prostatic tissue
could work by controlling cell proliferation. Similar to p21, p27 protein is an important cell
proliferation inhibitor46,47
, once increased levels of p27 result in cell cycle blocking in the S
phase19,48-51
. The p27 expression may be lost or decreased in human PC20,52-55
, a feature
associated with tumor cell undifferentiation, proliferation and poor prognostic 48,51,56,57
. In
contrast to p21, p27overexpression is mostly related to better prognosis, acting as a tumoral
suppressor58
. However, it seems important to evaluate these cell cycle inhibitors together,
since there are studies showing association of malignant progression and the presence of
negative relation between p21 (overexpressed) and p27 (underexpressed) expression in canine
mammary tumor29
, as well as malignant progression and positive relation between p21
(underexpressed) and p27 (underexpressed) expression in human tumors59
. In contrast, in
canine prostate with premalignant and malignant lesions the positive relation for p21 and p27
overexpression would be related to a good prognosis.
Another important protein in cell cycle control is the product of TP53 gene, the
p53 protein24,25
. In this study, the number of stained cells for p53 was significantly higher in
73
canine prostates with PC than with PIA. TP53 mRNA was detected by RT-PCR in all
prostates with PIA and PC, but TP53 overexpression by RT-qPCR was present in a higher
number of prostates with PC than with PIA. According to Quian et al.60
, Tsujimoto et al.61
,
and Stangelberger et al.44
, in human prostates with PIN and PC, the increased expression of
p53 has been associated ith high proliferative index and more aggressive phenotype. Since
accumulations of p53 in the presence of TP53 gene mutation are usual, our results indicate
that alterations related to p53 protein may start in canine prostates with PIA and later
accentuates in canine glands with PC, emphasizing its malignant potential as described before
in dogs26
and humans60,61
. This hypothesis is also supported by the fact that inflammatory
microenvironment in PIA promotes changes in TP53 gene, as mentioned by Wang et al.4 and
Tsujimoto et al.61
. Furthermore, according to Wang et al.4,p53 accumulations occur in the
presence of a TP53 gene mutation, especially in tissues with chronic inflammation, such as
cholangiohepatitis, gastritis and colitis. Tsujimoto et al.61
also detected TP53 overexpression
in human post-atrophic hyperplasia.
Taken together, our results have showed that is important evaluate different cell
pathways in order to better understand the carcinogenis and tumoral development steps in
different types of tissue and species, since one lesion in the organism may evolute by different
mechanisms with similar biological results26
. Based on that, it seems that in canine
premalignant and malignant prostate the overexpression of p21 and p27 plays controlling cell
proliferation and lesion progression even with the evidence of alterations involving p53.
CONCLUSIONS
It was concluded that when overexpressed in canine premalignant and malignant
prostate, p21 and p27 possible play role controlling cell proliferation, most likely working as
a protective factor in the evolution of PIA to PC, and in the PC development, even in the
presence of altered p53.Thus, the next step would be verifying these cell cycle proteins in
canine prostate with PC and metastasis.
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“Lembremo-nos de que o homem interior se renova sempre. A luta enriquece-o de
experiência, a dor aprimora-lhe as emoções e o sacrifício tempera-lhe o caráter. O
espírito encarnado sofre constantes transformações por fora, a fim de acrisolar-se e
engrandecer-se por dentro.”
André Luiz (Francisco Cândido Xavier e Waldo Vieira)
Evolução em dois mundos (1959)
79
CAPÍTULO 4 - Immunoexpression of cell cycle regulators in canine prostate
with proliferative lesions
ABSTRACT
In this study immunostaining of p21, p27, p53, cyclin D1, c-myc was evaluated in normal
canine prostate and with proliferative disorders to verify the interaction between these
regulators of cell cycle progression. From 106 samples of canine prostate obtained from a
TMA block, 15 were considered normal, 16 diagnosed as benign prostatic hyperplasia (BPH),
30 as proliferative inflammatory atrophy (PIA), 20 as prostatic intraepithelial neoplasia (PIN),
and 25 as prostatic carcinoma (PC). Difference in immunostaining of p21, p27, cyclin D1 and
p53 in acinar epithelium in relation to the prostate condition was observed. There was positive
correlation between p21 and p27 for number of stained cells and staining intensity in all
conditions and between c-myc and p53 in prostates with PIN. Considering the number of
labeled cells, there was positive correlation between p21 and p53 in the normal prostate. Also,
regarding the intensity of staining, there was positive correlation between p21 and p53 in
prostate tissue with PIN and between p27 and c-myc in prostates with PIA. Negative
correlation between c-myc and cyclin D1 was also found in the glands with PIN, considering
the number of labeled cells, and between p27 and c-myc in the prostates with PC for staining
intensity. In conclusion, the expression of p21, p27, p53, cyclin D1 and c-myc varies
according to type of proliferative lesion in canine prostate. Together, the results of this study
suggest low growth potential for canine PC in the presence of p21 and p27 overexpression,
cyclin D1seeming underexpression and regular expression of c-myc, also with the p53 mutant
type presence. Further, considering the similar immunophenotype presented by glands with
PIA, PIN and PC regarding these regulators of cell cycle progression, it is possible reaffirm
the premalignant potential of PIA and PIN in canine prostate.
retrieval was performed with 10 mM pre-heated citrate buffer, pH 6.0, for 3min, in pressure
cooker (Solar, Rapid Express, Tramontina, Brazil). The antibory was diluted at 1:50, 1:200,
1:500, 1:50, 1:50 respectively and incubated overnight, at 4°C, in wet chamber. Incubation
with polymer enzyme, chromogen and HRP substrate for signal detection were done using
reagents from polymer kit (New Link Max Polymer, United Kingdom, #RE7260-K) and
following manufacturer’s directions. Sections were counterstained with Mayer's hematoxylin,
washed, dehydrated, cleared, covers lipped, and examined by light microscopy.
Sample from human colon carcinoma for p21 and p27, human mammary
carcinoma for p53, and canine lymph node to cyclin D1 and c-myc were used as positive
control. For negative control the primary antibody was replaced by PBS buffer, pH 7.4. The
intensity of p21, p27, p53, cyclin D1 and c-myc reactivity was scored as: 0=negative, 1=mild,
2=moderate and 3=intense. Regarding the number of stained cells, the scores were:
0=negative, 1=1-25%, 2=26-50%, 3=51-75% and 4=76-100%.
The Chi-square, Kruskal-Wallis, nonparametric multiple comparison test for
paired contrasts the relative effects with Tukey correction, as well as descriptive data were
used to compare the scores of percentage of positive cells and their intensity. Association
between p21, p27, p53, cyclin D1and c-myc expression in normal prostatic tissue and with the
different lesions studied was achieved by Spearman test. For all it was used SPSS (IBM Corp.
Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.)
and R (R Development Core Team (2008) R Foundation for Statistical Computing, Vienna,
Austria). All values were considered with 5% of significance level.
RESULTS
From TMA slide were obtained 15 (14.15%) were normal tissues, 16 (15.09%)
BPH, 30 (28.3%) PIA, 20 (18.87%) PIN, and 25 (23.58%) PC (Table 1).The immunostaining
was nuclear for p21, p27 and p53is shown in Figure 1, cytoplasmic for cyclin D1and nuclear
through cytoplasmic and membrane for c-mycis shown in Figure 2.
84
FIGURE 1 - Immunohistochemistry photomicrography on nuclear immunostaining(arrow). A)
Canine PIN tissue with score three for labeling intensity and four for number of
labeled cells for p21. 200x. B) Human colon carcinoma. Positive control for p21 with scores two for labeling intensity and four for number of labeled cells. 400x. C)
Normal canine prostatic tissue with one for labeling intensity and one for number
of labeled cells for p21 (400X) D) Canine PIN with score two for labeling intensity
and two for number of labeled cells for p27. 200x. E) Human colon carcinoma. Positive control of p27 with score three for labeling intensity and four for number
of labeled cells. 400X. F) Canine PIA with score three for labeling intensity and
A B
E F
G H
C D
85
four for number of labeled cells for p53. 400X. G) Mammary carcinoma. Positive
control of p53 with score three for labeling intensity and three for number of
labeled cells. 200X. H)Normal canine prostatic tissue with 0- score for p-53 (400X). IHC, DAB, counterstained with H&E.
FIGURE 2.Immunohistochemistry photomicrography on nuclearthrough cytoplasmic and membrane
immunostaining.A) Canine PIN tissue with score one for labeling intensity and three for number of labeled cells of cytoplasmic cyclin D1 (arrow)(400X). B) Canine lymph
node. Positive control of cytoplasmic cyclin D1 (arrow) with score two for labeling
intensity and two for number of labeled cells.(400X) C) Normal canine prostatic tissue with 0- score for cyclin D1 (200X) D) Canine PC tissue with score three for labeling
intensity and four for number of labeled cells for nuclear through cytoplasmic c-myc
(arrow).(400X) E) Canine lymph node. Positive control of nuclear through cytoplasmic
c-myc (arrow), with score two for labeling intensity and four for number of labeled cells.(200X) F) Normal canine prostatic tissue with scorethree for labeling intensity and
four for number of labeled cells for c-myc(arrow). (200X). IHC 400x. DAB with H&E
counterstain.
A B
C D
E F
TABLE 1 - Distribution of cases according to the scores applied to variable number of labeled cells and intensity of labeling to p21, p27, p53, ciclyn D1 and c-
myc antibodies in normal prostate tissues and with BPH, PIA, PIN and PC.
Number of Labeled Cells (%) Intensity of Labeling Cells (%)
The number of stained cells to p21 in normal prostatic tissue was different
from those with PIN, as well as it was different in prostatic tissues with BPH from those
with PIA, PIN and PC. According to intensity of expression, there were differences in
normal prostatic tissue from those with PIA, PIN and PC, as well as it was different in
prostatic tissues with BPH from those with PIA, PIN and PC (p<0.05 - Table 2).
The number of stained cells to p27 in normal prostatic tissue was different
from those with PIA, PIN and PC, as well as it was different in prostatic tissues with
BPH from those with PIA, PIN and PC. According to intensity of expression, there were
differences in normal prostatic tissue from those with PIA, PIN and PC, as well as it
was different in prostatic tissues with BPH from those with PIA, PIN and PC (p<0.05 -
Table 2).
The number of stained cells to p53 in normal prostatic tissue was different
from those with PIA, PIN and PC as well as it was different in prostatic tissues with
BPH from those with PIA, PIN and PC, and in prostatic tissues with PIA from those
with PC. According to intensity of expression, there were differences in normal
prostatic tissue from those with PIA, PIN and PC, as well as it was different in prostatic
tissues with BPH from those with PIA, PIN and PC (p<0.05 - Table 2).
The number of stained cells to cyclin D1 in normal prostatic tissue was
different from those with BPH, PIA, PIN and PC. According to intensity of expression,
there were differences in normal prostatic tissue from those with BPH, PIA, PIN and PC
(p<0.05 - Table 2).The number and the intensity of stained cells to c-myc antibody there
were no difference according to diagnoses (p<0.05 - Table 2).
TABLE 2 -Means of comparison between p21, p27, p53, cyclin D1 and c-myc immunostaining regarding the number of stained cells and intensity of staining cells in canine normal
prostatic tissue and with BPH, PIA, PIN and PC.
Diagnoses N
Rank of Number of Stained Cells Rank of Staining Intensity
p21 p27 c-
myc
cyclin
D1 p53 p21 p27
c-
myc
cyclin
D1 p53
Normal 15 36.3a
34.4a
54.0a
23.0a
16.9a
33.9a
31.6a
42.5a
21.5a
23.0a
BPH 16 28.0ab
26.4a
54.0a
49.1b
30.4a
28.3a
28.4a
49.3a
46.5b
38.4a
PIA 30 57.8ac
60.1b
54.5a
61.7b
54.6b
55.8b
60.4b
63.3a
61.4b
55.7b
PIN 20 72.5c
62.3b
55.4a
62.8b
70.8bc
71.4b
60.8b
50.8a
61.5b
66.9b
Carcinoma 25 59.8ac
67.4b
56.0a
57.3b
75.2c
64.3b
68.6b
53.2a
61.6b
68.2b
Similar letters in the same column are not different by nonparametric multiple comparison test
for paired contrasts the relative effects with Tukey correction. (p value - p<0.05).
88
There was strong positive correlation between p21 and p27 for both number
of stained cells and staining intensity for normal tissue and with BPH, PIA, PIN and PC.
There was positive correlation between p21 and p53 regarding to number of stained
cells in normal prostate tissue, and also the intensity of staining to PIN tissue. There
were positive correlation of staining intensity between p27 and c-myc in prostates with
PIA. Also, in prostates with PIN there is correlation between p53 and c-myc regarding
to intensity of staining cell (Table 3).
Furthermore, there was negative correlation between c-myc and cyclin D1
for prostate tissue with PIN regarding the number of stained cells. Considering staining
intensity, there was negative correlation between p27 and c-myc and between p21 and
c-myc for prostate with carcinoma. That means that high values of one variable are
associated the low values of the other variable (Table 3).
89
TABLE 3 - Means of correlation between antibodies expression regarding the number of stained cells and intensity of staining
of the normal canine prostate and with BPH, PIA, PIN and PC.
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“As leis da genética encontram-se presididas por numerosos agentes psíquicos que a ciência
da Terra está longe de formular...”
Emmanuel (Francisco Cândido Xavier)
O consolador (pergunta nº 35) ano 1940
CAPÍTULO 5 - CONSIDERAÇÕES FINAIS
Na realização desta tese foi possível observar que há controvérsia acerca do
potencial de malignidade da PIA na próstata canina, sendo que este quadro vem se
modificando. Os relatos em periódicos que descrevem a PIA no cão, bem como sua relação
com tecidos adjacentes e concomitância com outras lesões proliferativas como HPB, PIN e
PC, apesar de escassos, vem aumentando. No entanto, essas lesões ainda constituem
grande desafio à pesquisa do câncer ao entendimento das alterações genéticas e
epigenéticas associadas à oncogênese, que contribuem para as alterações celulares e
biológicas que podem ser reconhecidas como malignas. Genes intimamente envolvidos na
regulação da proliferação celular são conhecidos por alterarem-se em todos os casos.
Frente a esse desafio, o objetivo principal deste trabalho foi estudar proteínas
envolvidas no ciclo celular, com o intuito de melhor entender os possíveis mecanismos de
escape do controle do ciclo e assim verificar o potencial pré-maligno da PIA e maligno do
PC na espécie canina.
Nesse sentido, foi possível observar a expressão de p21, p27, p53, ciclina D1,
c-myc, EGFR e Her2 na próstata canina e inferir que essas proteínas participam de forma
direta ou indireta no processo de desenvolvimento do carcinoma na próstata dos cães, bem
como da PIA. Ainda, analisados em conjunto, os resultados direcionam à conclusão de que
o carcinoma da próstata canina apresenta baixo potencial de progressão, o que justifica a
baixa frequência de casos observada na clínica veterinária, somado ao fato de que, mesmo
quando presente, as metástases não se desenvolvem de forma imediata. Esse perfil
imunofenotípico voltado a um baixo potencial de progressão também justificaria o
interessante fato de se ter encontrado inúmeros focos de PIN e PC na próstata de cães que
não apresentavam sinais macroscópicos de tumor. Isto faz questionar quanto ao que se diz
raro na literatura. O que é raro, o desenvolvimento ou o diagnóstico dessas lesões na
próstata canina? Ainda, porque tantos focos de PC constatados na próstata dos cães de
Goiânia? Qual o fator predisponente nessa região do país que contribui para a presença
expressiva do carcinoma na próstata do cão?
Na espécie humana, o emprego dessas proteínas como marcadores tumorais
vem sendo amplamente estudado e muitos já são utilizados na rotina diagnóstica e
prognóstica do câncer humano, como é o caso do Her2 para neoplasias mamárias. No cão
não foi possível correlacionar a expressão dessas proteínas com a evolução da doença,
evidência clínica do tumor e prognóstico.
99
Diante do elevado número de amostras obtidas foi possível otimizar o estudo
com o uso do bloco de TMA, conferindo grande economia de reagentes e tempo, além de
proporcionar a uniformização na realização das reações. Somado a isso, a compilação das
amostras em uma única lâmina facilita a interpretação comparativa dos casos, com
possibilidade de repetição das reações em múltiplos níveis do bloco, o que confere melhor
representação das lesões em comparação ao corte original. Ainda, seu uso simplifica o
trabalho das linhas de pesquisa, já que as amostras podem ser utilizadas em mais de um
projeto.
O avanço das pesquisas, com o advento de novas técnicas e a disponibilidade
de reagentes comerciais tornou possível a extração de material biológico proveniente de
material parafinizado, a exemplo de RNA e DNA em quantidade e qualidade, viabilizando
a realização das técnicas de RT-PCR e RT-qPCR neste estudo. Isso também possibilita a
realização de estudos retrospectivos envolvendo material de arquivo, aumentando a
quantidade de material a ser estudado.
Identificando bons marcadores de diagnóstico, prognóstico e tratamento será
possível estabelecer terapias precoces e efetivas para o tratamento do câncer prostático.
Nesse sentido, o estudo da expressão de p21, p27, p53, ciclina D1, c-myc, EGFR e Her2
em próstatas caninas com PIN, PIA e PC permitiram-nos adicionar novos dados ao
conhecimento dos eventos biológicos que desencadeiam o câncer prostático no cão e
reforçou o conceito de que o cão é o melhor modelo experimental para a verificação desses
eventos na próstata humana.
100
ANEXO A
Tabela contendo a quantidade e pureza do DNA e RNA (razão
A260/280)extraído das 16 amostras de próstata parafina.