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C6lulas epiteliais, endoteliais e fibroblastos podem ser induzidas a
apoptose (anoikis) quando se destacam da matriz extracelular. O processo
de anoikis tem a função de proteger o organismo da colonização
inapropriada destas células.
O processo de apoptose pode ser induzido por vhrios estímulos,
dentre eles, o óxido nitrico (NO).
O radical livre NO pode ser sintetizado em organismos vivos pela
ação das enzimas NO sintases (NOS), como tambem pode ser liberado por
compostos doadores de NO.
A geração intracelular ou extracelular de NO pode resultar em
diferenciago, proliferação ou morte celular, dependendo da natureza e
quantidades de NO gerado no processo, como tambem do tipo de celular
alvo da ação do radical livre.
Este trabalho estudou a induçáo do processo de apoptose iniciada
pelo doador de óxido nítriw nitroprussiato de sódio (SNP) em células de
linhagem células A431 de carcinoma epiderrnóide humano.
A partir de 6 horas de tratamento com SNP ImM, as células
destacadas já estgo positivas para citoqueratina 18 (CK18), anexina-V e
apresentam caspases 3 e 8 ativas.
Elevado conteúdo de bax e queda de bcl-2, representando uma razão
bcl-2lbax compatível com apoptose é alcançada após 24 horas de
tratamento com SNP 1mM, bem como ativação de JNK, reação de Tunnel
viii
positivo, positividade ao corante Hoescht 33258, atividade insignificante de
LD, marcação positiva para anexina-V e negativa para iodeto de propídio
(PI>.
As propriedades antiadesivas do NO foram observadas analisando-se
o aumento da perda de aderencia e diminuição de viabilidade das células
destacadas após tratamento
wrn SNP e positividade para anticorpo anti-
citoqueratina AE1 e AE2.
Cblulas plaqueadas em diferentes superfícies que dificultam o
processo de aderencia, quando submetidas ao tratamento com SNP
rompem mais facilmente os complexos de adesão e se destacam mais
rapidamente.
Os resultados apresentados sugerem que o NO promove alterações
na integridade do citoesqueleto, que induzem as células A431 à apoptose
por perda de aderencia a matriz extracelular.
ABSTRACT
Epithelial cells, endotelial cells, and fibroblasts undergo a special kind
of apoptosis (anoikis) that occurs when they are displaced from the extracellular matriz. Anoikis is thought to protect tissues from inappropriate
colonization. A number of studies described apoptogenic and anti-apoptotic
properties for nitric oxide (NO). Our studies focused on the induced apoptosis
of the human epithelial carcinoma cell line A431 after exposure to an NO
donor, sodium nitroprusside. Initially, the following features were associated
with NO-induced A431 cell death: decreased expression of Bcl-2, enhanced
expression of Bax, and activation of the stress related MAP kinase, JNK. In
addition, NO treated cells were positive for the TUNEL assay, and for
Hoescht 33258 staining.
Caspase 8 and 3 activities was detected in the system. Continuing our
investigation on the mechanisms ori the underlying the NO-induced
apoptosis, we investigated modifications on the cytoskeleton upon exposure
of A431 cells to the NO donor. NO presents anti-adhesive properties, either
down regulating the expression of adhesion molecules or disrupting adhesion
complexes. Accordingly, we found that A431 cells undergoing NO-induced
apoptosis (positive for Anexin V labeling and negative for propidiurn iodide
labeling), featured a loss of cytoskeleton integrity, and an insufíicient rescue
of the supernatant cells.
In conclusion, our findings suggest that NO promote the adhesion related
apoptosis, anoikis, in A431 cells.
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