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1
Zampier, Carolina da Paz. "Papel de caveolina-1 na produção de mediadores inflamatórios." Instituto Oswaldo Cruz, 2012. https://www.arca.fiocruz.br/handle/icict/7031.
Full textAbstract:
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Previous issue date: 2012<br>Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil<br>A caveolina-1 (Cav-1), uma proteína essencial para a formação de cavéolas,
apresenta atividade na modulação da sinalização intracelular. Cav-1 é capaz de
interagir com diversas proteínas através de seu domínio CSD (caveolin scaffolding
domain) e, em geral, essa interação leva à inibição das proteínas associadas. O
peptídeo CSD tem sido utilizado como um mimético de Cav-1 em relação à sua
capacidade modulatória sobre a atividade de outras proteínas. Recentemente, tem
sido mostrado que Cav-1 é capaz de modular a resposta inflamatória em diversos
aspectos. Neste trabalho, examinamos o papel de Cav-1 na regulação da síntese de
mediadores inflamatórios por macrófagos. O lipopolissacarídeo (LPS) de E.coli, um
protótipo de estímulo inflamatório, foi capaz de induzir a expressão de Cav-1 e Cox-2
em macrófagos peritoneais in vitro. Estas proteínas são induzidas em um curso
temporal semelhante, sendo detectadas por Western blot a partir de 3h com níveis
de expressão crescentes até 18h. Por imunofluorescência, observamos que Cav-1 e
Cox-2 apresentam um padrão de expressão mutualmente exclusivo em macrófagos
estimulados com LPS. Mostramos por Western blot que a expressão de Cox-2 é
induzida por LPS e que o tratamento com CSD leva à inibição da expressão de Cox-
2, mas não de Cox-1. Observamos, também, a redução parcial dos níveis de PGE2
no sobrenadante de macrófagos estimulados com LPS e tratados com CSD. O
tratamento com o peptídeo CSD também foi capaz de reduzir os níveis de IL-1, IL-
6, e IL-12 induzidos por LPS. O LPS induz o aumento da expressão e fosforilação de
STAT-1. A fosforilação de STAT-1 foi diminuída após o tratamento com CSD,
indicando que Cav-1 modula negativamente a ativação de STAT-1. Estudos
posteriores são necessários para complementar os dados obtidos até o momento
para esclarecer os mecanismos de modulação da síntese de mediadores
inflamatórios por Cav-1. Em conclusão, Cav-1 apresenta uma atividade inibitória
sobre a expressão de Cox-2 e produção dos mediadores inflamatórios PGE2, IL1,
IL-6, e IL-12 em macrófagos estimulados com LPS in vitro. O mecanismo de inibição
possivelmente envolve inibição da ativação de STAT-1.<br>Caveolin-1 (Cav-1), a protein essential for the formation of caveolae, shows
activity in the modulation of intracellular signaling. Cav-1 can interact with several
proteins by its caveolin scaffolding domain (CSD) and, in general, this interaction
leads to inhibition of associated proteins. The peptide CSD has been used as a Cav-
1 mimetic in relation to its capacity on the modulatory activity of other proteins.
Recently, it has been shown that Cav-1 can modulate the inflammatory response in
several respects. We examined the role of Cav-1 in regulating the synthesis of
inflammatory mediators by macrophages. Lipopolysaccharide (LPS) from E. coli, a
prototype of inflammatory stimulus, was able to induce the expression of Cav-1 and
Cox-2 in peritoneal macrophages in vitro. These proteins are induced in a similar
time course, being detected by Western blot at 3 hours with increasing levels of
expression up to 18 hours. By immunofluorescence, we observed that Cav-1 and
Cox-2 have a mutually exclusive pattern of expression in macrophages stimulated
with LPS. Western blot analysis showed that the expression of Cox-2 is induced by
LPS and that treatment with CSD leads to inhibition of Cox-2 but not Cox-1. We also
observed the partial reduction of PGE2 levels in supernatants of macrophages
stimulated with LPS and treated with CSD. Treatment with CSD peptide was also
able to reduce the levels of IL1, IL-6 and IL-12 induced by LPS. LPS induces
increased expression and phosphorylation of STAT-1. The phosphorylation of STAT-
1 was decreased after treatment with the CSD, indicating that a Cav-1 negatively
modulates activation of STAT-1. Further studies are needed to supplement the data
obtained so far to clarify the mechanisms of modulation of the synthesis of
inflammatory mediators by Cav-1. In conclusion, Cav-1 shows an inhibitory activity on
Cox-2 expression and production of the inflammatory mediators PGE2, IL1β, IL-6 and
IL-12 in macrophages stimulated with LPS in vitro. The mechanism of inhibition
possibly involves inhibition of STAT-1 activation.
2
Lagares, Tena Laura María. "Caveolina-1 en la progresión metastásica del Sarcoma de Ewing." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/145475.
Full textAbstract:
El sarcoma de Ewing (SE) es el segundo tumor óseo maligno infantil más frecuente y presenta una alta incidencia de enfermedad metastásica. Este tipo de tumores presentan una translocación génica característica que da origen a una proteína de fusión, normalmente EWS/FLI1. Esta proteína de fusión actúa como factor de transcripción aberrante, regulando la expresión de diferentes genes implicados en la iniciación, mantenimiento y progresión del tumor. Nuestro grupo describió como uno de estos genes diana a caveolina 1 (CAV1), describiendo además su papel determinante en el fenotipo maligno del SE, en la tumorigénesis, en la angiogénesis y en la resistencia a apoptosis inducida por quimioterapia.
Para investigar el papel concreto de CAV1 en el proceso metastásico de este sarcoma, se creó un modelo de baja expresión de CAV1 en líneas celulares de SE y se determinaron cambios en su capacidad migratoria, invasiva y metastásica. En los ensayos in vitro se halló una menor capacidad migratoria de las células con silenciamiento de CAV1 y una reducción en la expresión de metaloproteinasa 9 (MMP-9) y en la actividad de MMP-2, ambas MMPs implicadas en el proceso de invasión. La regulación de la actividad de MMP-2 parece estar relacionada con la posible regulación que ejerce CAV1 en la función de la MMP de membrana tipo 1 (MT1-MMP), proteína fundamental para la activación de MMP-2. Por otro lado, en este estudio se propone que CAV1 promueve la expresión de MMP-9 transcripcionalmente a través de la regulación de la activación de la vía de señalización de ERK1/2 (Extracellular signal-regulated kinase 1/2). En nuestro modelo, ERK1/2 activo translocaría al núcleo donde activaría los factores de transcripción responsables de la activación del promotor de MMP-9. Por otro lado, la fosforilación de RSK2 (Ribosomal S6 kinase 2) por parte de ERK1/2 en el citoplasma, produciría la activación de RSK2 que a su vez activaría la proteína ribosomal rpS6, uno de los responsables de la iniciación de la traducción, por lo que también podría estar influyendo a la producción de MMP-9 a este nivel.
Según nuestros resultados, CAV1 estaría influyendo en la capacidad migratoria de las células de SE mediante dos mecanismos: 1) a través de la activación de la vía de ERK1/2 y 2) mediante la unión a diferentes proteínas con dominio SH2 a través de la fosforilación de CAV1 en la tirosina 14. ERK1/2 influye en la regulación de la capacidad migratoria de una forma de pendiente o independiente de RSK1.
Además, en los ensayos de metástasis experimental in vivo las células con inhibición de CAV1 presentaron una menor incidencia de metástasis pulmonar, hecho que correlacionó con una disminución en la expresión de SPARC (Secreted protein acidic and rich in cysteine), una proteína de adhesión importante en procesos metastásicos. En resumen, nuestros resultados evidencian la importancia de CAV1 en el proceso metastásico del SE.<br>Ewing’s sarcoma (ES) is the second most common bone tumor in childhood and occurs with a high incidence of metastatic disease. Such tumors have a characteristic gene translocation that gives rise to a fusion protein, most commonly EWS/FLI1. This fusion protein acts as an aberrant transcription factor regulating the expression of different target genes involved in the initiation, maintenance and progression of the tumor. Our group described caveolin 1 (CAV1) as one of these target genes, describing its role in the malignant phenotype, tumorigenicity and resistance to chemotherapy-induced apoptosis of ES cell lines. To investigate the specific role of CAV1 in the metastatic process of this sarcoma, we established a model of low expression of CAV1 in cell lines of ES. Then, we measured changes in their migratory capacity, invasiveness and metastatic potential. In vitro, we found a lower migratory capability of CAV1 knockdown cells and a reduction in MMP-9 expression and MMP-2 activity. The regulation of MMP-2 activity seems to be related to the possible regulation that CAV1 exerts on the function of MT1- MMP, an essential protein for the activation of MMP-2. On the other hand, we suggest that CAV1 promotes the expression of MMP-9, both transcriptionaly and post-transcriptionaly, through regulating ERK1/2 signaling pathway. In our model, activated ERK1/2 would translocate to the nucleus where it would activate the transcription factors responsible for MMP-9 promoter activation. At the cytoplasm, activated ERK1/2 would phosphorylate and activate RSK2, which, in turn, would promote rpS6 activation, leading to protein translation initiation.
Our results indicate that CAV1 is regulating migratory capability of ES cells by two different mechanisms; 1) through ERK1/2 pathway activation, 2) by linking several proteins bearing SH2 domains trough phosphorylated Tyr14 of CAV1. ERK1/2 seems to regulate cell migration in both RSK1-dependent and independent manner.
In addition, experimental metastasis assays in vivo showed that, CAV1 knockdown cells had a lower incidence of pulmonary metastasis, a fact that correlated with a decrease in the expression of SPARC, a major adhesion protein in metastatic processes. In summary, our results demonstrate the importance of CAV1 in the metastatic process on ES tumors.
3
Díaz, Valdivia Natalia. "E-cadherina reduce la habilidad promotora de migración y metástasis de caveolina-1 por la disminución de la fosforilación de caveolina-1 en células metastásicas." Tesis, Universidad de Chile, 2016. http://repositorio.uchile.cl/handle/2250/140680.
Full textAbstract:
Tesis para optar al grado de Doctora en Bioquímica<br>El cáncer corresponde a la segunda causa de muerte en Chile y el mundo, pero
los pacientes con cáncer no mueren debido al crecimiento del tumor primario,
sino a la diseminación de las células tumorales y posterior colonización de éstas
en nuevos órganos o metástasis. En términos generales, el cáncer es la
consecuencia de un proceso multifactorial que lleva a la transformación
progresiva de células normales en células altamente malignas. La amplia
variedad de fenotipos del cáncer se debe a la manifestación de alteraciones
esenciales en la fisiología celular, las que colectivamente dictaminan el
crecimiento maligno. Dos de las características adquiridas por las células
tumorales en las que se enfocará esta tesis son; la invasión de tejidos y
metástasis y la reprogramación del metabolismo energético.
Caveolina-1 es una proteína integral de membrana a la que se le ha atribuido
un rol dual en la progresión tumoral, actuando como supresor de tumores, ya
que la disminución de la expresión de caveolina-1 es suficiente para revertir el
fenotipo transformado, o como promotor de metástasis en estadios tardíos del
cáncer, donde la expresión de caveolina-1 aumenta sin revertir el fenotipo
maligno y se correlaciona con un aumento en la migración celular y metástasis,
multiresistencia a drogas y una mala prognosis para el paciente. Ambos roles
de caveolina-1 se ven afectados por la presencia de la glicoproteína Ecadherina,
la cual potencia su acción supresora de tumores y suprime la
habilidad de caveolina-1 para promover metástasis in vivo. Sin embargo, el
mecanismo por el cual E-cadherina suprime la habilidad de caveolina-1 para
promover la metástasis no ha sido estudiado.
La habilidad de caveolina-1 para promover un aumento en la migración en
células metastásicas se relaciona con un aumento en la activación de Rab-5 y
Rac-1.
Ya que la reprogramación del metabolismo energético de las células, suple
tanto las necesidades bioenergéticas como biosintéticas de las células
cancerígenas para sostener una proliferación aumentada y una rápida
migración e invasión celular, en este proyecto se caracterizó el fenotipo
metabólico de las células metastásicas que expresan o no, caveolina-1 y
estudiamos cómo la expresión de E-cadherina afecta el metabolismo.
Caveolina-1 sufre modificaciones post-traduccionales que participan en su
función intracelular, como la fosforilación en el residuo de tirosina 14 (Y14),
mediada por las proteínas tirosina quinasas no receptoras Src, Fyn y Abl en
respuesta a varios estímulos como. En células metastásicas de cáncer de
mama, se observan altos niveles de expresión de caveolina-1 y un alto grado de
fosforilación de ésta en Y14, lo que promueve un aumento en la migración
celular por un aumento en el recambio de la adhesiones focales, polarización,
velocidad persistencia y direccionalidad de la migración. Todas estas funciones
de caveolina-1 son bloqueadas por la inhibición de la fosforilación de caveolina-
1 en Y14 tanto de manera farmacológica como por la utilización de una
caveolina-1 que no es fosforilable (Y14F). Por lo tanto, el rol promotor de
metástasis de caveolina-1, dependería de su fosforilación en Y14. La fosfatasa
PTPN14 co-inmunoprecipita con caveolina-1 en presencia de E-cadherina y
PTPN14 en células de melanoma murino e induce una disminución en la
migración, formación de colonias y crecimiento independiente de anclaje de
células de cáncer de colon.
Estos antecedentes nos llevaron a proponer la siguiente hipótesis: En
presencia de E-cadherina se recluta a la fosfatasa PTPN14 al complejo
multiproteíco formado con caveolina-1 lo que reduce la habilidad
promotora de migración y metástasis de caveolina-1 por la disminución de
la fosforilación de ésta en tirosina 14. El objetivo principal de esta tesis es
determinar el efecto de la co-expresión de caveolina-1 y E-cadherina sobre la
fosforilación en tirosina de caveolina-1 (Y14) y β-catenina (Y654) y la migración e invasión celular en células cancerígenas. Para abordar la hipótesis de trabajo
se plantearon los siguientes objetivos específicos: (1) Determinar el efecto de la
co-expresión de caveolina-1 y E-cadherina sobre los niveles de fosforilación en
tirosina 14 de caveolina-1 y en tirosina 654 de β-catenina y la migración e
invasión celular; (2) Estudiar la participación de Src en la fosforilación en
tirosina 14 de caveolina-1 y en tirosina 654 de β-catenina en presencia y
ausencia de E-cadherina; (3) Determinar si los complejos proteicos formados
con caveolina-1 en presencia de E-cadherina contienen PTPN14, la que
contribuye a la desfosforilación de caveolina-1 en tirosina 14; (4) Determinar el
efecto de la co-expresión de caveolina-1 y E-cadherina sobre la activación de
Rab-5 y Rac-1; (5) Caracterización de los cambios metabólicos inducidos por la
expresión caveolina-1 y la variación de éstos por la co-expresión con Ecadherina.
Para el desarrollo de los objetivos mencionados se utilizaron líneas
celulares metastásicas en las que se co-expresan caveolina-1 y E-cadherina.
Se realizó ensayos de Western de blot, luego de ensayos de multiherida para
determinar los niveles de fosforilación de caveolina-1 en Y14 y β-catenina en
Y654, ensayos de migración por transwell, ensayos de invasión, ensayos de
precipitación por afinidad para determinar la actividad de Rab-5 y Rac1 en
células que expresan caveolina-1 en presencia o en ausencia de E-cadherina.
Además, se caracterizaron los complejos multiproteícos formados por
caveolina-1, mediante ensayos de inmunoprecipitación y espectrometría de
masa en células que expresan o no E-cadherina y se caracterizó el fenotipo
metabólico de células que expresan o no caveolina-1, estudiando cómo cambia
éste en presencia de E-cadherina.
En resumen en esta tesis se buscó dilucidar el mecanismo mediante el cual Ecadherina
reduce la habilidad promotora de migración, invasión y metástasis de
caveolina-1 en células de cáncer metastásicas.
En este trabajo mostramos que la expresión de E-cadherina inhibe el aumento
en la migración, invasión y metástasis inducido por caveolina-1 por la disminución de la fosforilación de caveolina-1 en Y14, además, de bloquear la
activación del eje Rab-5/Rac1. La fosfatasa PTPN14 co-inmunoprecipitó con
caveolina-1 en presencia de E-cadherina y su expresión fue capaz de inhibir el
rol promotor de migración, invasión y metástasis de caveolina-1. Finalmente
mostramos que la expresión y fosforilación de caveolina-1 induce un switch
metabólico a un fenotipo glicolítico aeróbico por el bloqueo del complejo
mitocondrial IV, generando cambios en el metaboloma de las células
metastásicas congruentes con este switch metabólico. La expresión de Ecadherina
bloquea el cambio metabólico inducido por caveolina-1 llevando a las
células a un metabolismo quiescente<br>Cancer is the second leading cause of death in Chile and in the world. Cancer
patients do not die due to the primary tumor growth, but rather due to the spread
of the cancer cells from the primary tumor and subsequent colonization of new
organs, a process known as metastasis. In general terms, cancer is the result of
a multifactorial process that leads to the progressive transformation of normal
cells into highly malignant cells. The wide variety of cancer phenotypes is due to
alterations in cell physiology that collectively lead to the malignant cell behavior.
Two of the characteristics acquired by tumor cells, on which this thesis will
focus, are tissue invasion, as well as metastasis and reprogramming of energy
metabolism.
Caveolin-1 is a membrane protein that has been attributed a dual role in cancer
progression, acting at early stages as a tumor suppressor given that augmenting
the expression of caveolin-1 is sufficient to reverse the transformed phenotype,
or as a promoter of metastasis in late stages of cancer, where enhanced
expression of caveolin-1 favors the malignant phenotype and correlates with an
increase in cell migration and metastasis, multidrug resistance and poor
prognosis of the patients. Both roles of caveolin-1 are affected by the presence
of the glycoprotein E-cadherin, which enhances caveolin-1 function as a tumor
suppressor and suppresses the ability of caveolin-1 to promote metastasis in
vivo. However, the precise mechanism by which E-cadherin suppresses the
malignant traits of caveolin-1 is unknown.
The ability of caveolin-1 to promote migration of metastatic cells is associated
with increased activation of Rab-5 and Rac-1.
Bearing in mind that the reprogramming of energy metabolism in cancer cells, is
required to supplement both the both the bioenergetic and biosynthetic needs of
these cells to sustain increased proliferation, rapid migration and cell invasion, this thesis also evaluated the metabolism of metastatic cells expressing or not
caveolin-1 and how this was affected by the expression of E-cadherin.
Caveolin-1 is subjected to posttranslational modifications relevant to protein
function, such as phosphorylation on tyrosine 14 (Y14), by the non-receptors
protein tyrosine kinases non-receptor Src, Fyn and Abl. This may occur in
response to a large variety of different stimulus. In metastatic breast cancer
cells, high levels of caveolin-1 expression and phosphorylation on Y14 are
observed and associated with increased cell migration by promoting focal
adhesion turnover, polarization, persistence, speed and directionality of
migration. All these functions of caveolin-1 are blocked by inhibition of caveolin-
1 phosphorylation on Y14 either pharmacologically or by introducing a nonphosphorylatable
caveolin-1 (Y14F) mutation. Therefore, the metastasis
promoting role of caveolin-1 depends on Y14 phosphorylation. The PTPN14
phosphatase co-immunoprecipitated with caveolin-1 in the presence of Ecadherin
and PTPN14 in murine melanoma cells and decreased migration,
colony formation and anchorage-independent growth of colon cancer cells.
These results available in the literature led us to propose that in the presence
of E-cadherin PTPN14 is recruited to the multiprotein complex including
caveolin-1 to reduce Y14 phosphorylation, cell migration and metastasis
induced by caveolin-1. The main objective of this thesis was to determine the
effect of co-expression of caveolin-1 and E-cadherin on tyrosine phosphorylation
of caveolin-1 (Y14) and β-catenin (Y654), as well as migration and invasion in
cancer cells. To address the working hypothesis the following specific objectives
were evaluated: (1) To determine the effect of the co-expression of caveolin-1
and E-cadherin on the levels of tyrosine phosphorylation 14 of caveolin-1 and on
tyrosine 654 of β catenin and migration and cell invasion; (2) To study
participation of Src in the phosphorylation of the tyrosine 14 of caveolin-1 and on
tyrosine β-catenin 654 in the presence and absence of E-cadherin; (3) To
determine whether the protein complexes formed with caveolin-1 in the presence of E-cadherin contain PTPN14, and if this phosphatase contributes to
dephosphorylation of tyrosine 14 of caveolin-1; (4) To determine the effect of coexpression
of caveolin-1 and E-cadherin on the activation of Rab-5 and Rac-1;
(5) To characterization of the metabolic changes induced by caveolin-1
expression and the effect of the co-expression with E-cadherin on cell
metabolism. To address these objectives metastatic cell lines that co-expressed
caveolin-1 and E-cadherin were used. Multiple wounding assays and Western
blot analysis was used to determine the phosphorylation levels of caveolin-1 on
Y14 and β-catenin on Y654 moreover migration assays, invasion assays, affinity
precipitation assays were employed to determine the activity of Rab-5 and Rac1
in cells expressing caveolin-1 in the presence or absence of E-cadherin.
Furthermore, protein composition the analysis of the multiprotein complex
formed by caveolin-1 and E-cadherin was evaluated following
immunoprecipitation assays by mass spectrometry analysis. The metabolic
phenotype of cells expressing or not caveolin-1 was characterized using
Seahorse extracellular analyzer and metabolic changes in presence of Ecadherin
were determined.
In summary, this thesis sought to elucidate the mechanism by which E-cadherin
reduces the ability of caveolin-1 to promote migration, invasion and metastasis
as well as metabolic reprograming of metastatic cancer cells.
In this study, we show that the expression of E-cadherin prevented caveolin-1-
enhanced migration, invasion and metastasis by reducing caveolin-1
phosphorylation on Y14 and, as a consequence, blocking of the activation of the
Rab-5/Rac-1 signaling axis. The PTPN14 phosphatase co-immunoprecipitated
with caveolin-1 in the presence of E-cadherin and overexpression or this
phosphatase was sufficient to prevent the migration, invasion and metastasis
promoting role of caveolin-1, even in the absence of E-cadherin.
Finally we show that the expression caveolin-1 induced a metabolic switch to an
aerobic glycolytic phenotype, likely by blocking the mitochondrial complex IV.
These effects coincided in metastatic melanoma cells with changes in the
metabolome. Moreover, the expression of E-cadherin blocked the metabolic
changes induced by caveolin-1 leading to a quiescent metabolic phenotype in
metastatic cells<br>Conicyt-FONDAP; Fondecyt ; Anillo ACT<br>31 de diciembre de 2018
4
González, Muñoz Elena. "Papel de las caveolas/caveolina-1 en la fisiologia del adipocito." Doctoral thesis, Universitat de Barcelona, 2007. http://hdl.handle.net/10803/1023.
Full textAbstract:
La función de las caveolas y de la caveolina in vivo ha sido motivo de controversia, de hecho se han implicado en procesos de endocitosis y transcitosis, transporte de colesterol y ácidos grasos, regulación de procesos de transducción de señales y tumorigénesis (Liu et al., 2002). Sin embargo, la generación de los ratones KO de caveolina-1 (Drab et al., 2001; Razani et al., 2001a) mostraba que estos ratones eran perfectamente viables y fértiles, aunque estudios posteriores relacionaban a caveolina-1 con la homeostasis lipídica (Razani et al., 2002a).<br/><br/>A partir de estos antecedentes, el objetivo general de esta tesis ha sido: <br/><br/>· Definir la función de las caveolas/ caveolina-1 en la fisiología del adipocito<br/><br/>Nos planteamos así, nuestro primer objetivo:<br/><br/>1- Obtención de líneas 3T3L1 deficientes en caveolina-1 mediante silenciamiento génico posttranscripcional.<br/><br/>2- Analizar la presencia de caveolas y la distribución de la caveolina-1 remanente en las membranas de los adipocitos y estudiar la estructura y composición de los rafts lipídicos/caveolas en estos adipocitos deficientes en caveolina-1.<br/><br/>3- Analizar la diferenciación adipocitaria de los preadipocitos 3T3L1 deficientes en caveolina-1. <br/><br/>4- Analizar la acción de la insulina en los adipocitos deficientes en caveolina-1, <br/><br/>5- Estudiar el papel de caveolina-1 en el metabolismo lipídico del adipocito.<br/><br/>A partir de los objetivos planteados conseguimor obtener las siguientes conclusiones en el desarrollo de esta tesis:<br/><br/>1. Se ha conseguido la transducción eficiente de adipocitos 3T3L1 maduros usando estas partículas lentivirales que expresan transgenes, aunque no se ha logrado transducir los adipocitos maduros eficazmente en el caso de los lentivirus que expresan moléculas de siRNA.<br/><br/>2. Los preadipocitos 3T3L1 son eficientemente transducidos usando las partículas lentivirales que permiten la expresión tanto de transgenes como de moléculas de siRNA. Mediante esta técnica hemos generado líneas estables 3T3L1 que expresan moléculas de siRNA de caveolina-1 y que presentan una reducción en la expresión de la proteína caveolina-1 de más del 93%.<br/><br/>3. La disminución de la proteína caveolina-1 en más de un 93% en adipocitos 3T3L1 provoca una disminución equivalente en el número de caveolas en la membrana plasmática.<br/><br/>4. La deficiencia en caveolina-1 en los adipocitos 3T3L1 no afecta a la localización de los marcadores proteicos y lipídicos característicos de los rafts lipídicos, que continúan recuperándose en las fracciones ligeras de los extractos celulares durante el aislamiento bioquímico de los rafts lipídicos. <br/><br/>5. La deficiencia en caveolina-1 y caveolas no afecta a la diferenciación de los preadipocitos 3T3L1 a adipocitos maduros, ni tampoco a las características morfológicas y el contenido lipídico (triacilglicéridos y colesterol) de los adipocitos maduros. <br/><br/>6. Los adipocitos 3T3L1 con una disminución del 93% en la expresión de caveolina-1, presentan una reducción del 50% en la expresión de las proteínas GLUT4 y del 40% en el caso del receptor de insulina, sin que este hecho responda a una menor cantidad de sus RNA mensajeros, sino a una disminución en la vida media de estas proteínas. Este dato sugiere que caveolina-1 participe en la estabilidad de estas proteínas.<br/><br/>7. El déficit de caveolina-1 en los adipocitos 3T3L1 no afecta a las vías de señalización de la insulina (dependiente de PI3K y dependiente de Cbl-TC10) ni tampoco a la translocación de GLUT4 a la membrana plasmática inducida por la insulina ni a la estimulación del transporte de glucosa. <br/><br/>8. La presencia de caveolas/caveolina-1 no es necesaria para la correcta internalización de GLUT4 durante la reversión de la acción de la insulina.<br/><br/>9. Las caveolas/caveolina-1 tampoco son necesarias para la acción de la insulina inhibiendo o activando la lipólisis y la lipogénesis en los adipocitos 3T3L1, respectivamente.<br/><br/>10. La deficiencia de caveolina-1 y caveolas no altera el transporte de ácido palmítico a través de la membrana plasmática de adipocitos 3T3L1.<br/>Esta deficiencia tampoco altera la activación lipolítica por los efectores beta-adrenérgicos: Isoproterenol 10mu-M e IBMX 0,5mM en los adipocitos 3T3L1.<br/><br/>PALABRAS CLAVE: Cavelonia-1, Caveola, Adipocito, Insulina, GLUT4, Receptor de insulina<br><I>Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin protein. Caveolae are particularly enriched in adipocytes. Here we analyzed the effects of caveolin-1 knockdown and caveolae ablation on adipocyte function. To this end, we obtained several multiclonal mouse 3T3-L1 cell lines with a reduced expression of caveolin-1 (95% reduction) by a small interfering RNA approach using lentiviral vectors. Control cell lines were obtained by lentiviral infection with lentiviral vectors encoding appropriate scrambled RNAs. Caveolin-1 knockdown adipocytes showed a drastic reduction in the number of caveolae (95% decrease) and cholera toxin-labeling was reorganized in dynamic plasma membrane microdomains. <br/><br/>Caveolin-1 depletion caused a specific decrease in GLUT4 glucose transporter and insulin receptor protein levels. This reduction was not the result of a generalized defect in adipocyte differentiation or altered gene expression but was explained by faster degradation of these proteins. Caveolin-1 knockdown adipocytes showed reductions in insulin-stimulated glucose transport, insulin-triggered GLUT4 recruitment to the cell surface and insulin receptor activation. In all, our data indicate that caveolin-1 loss-of-function reduces maximal insulin response through lowered stability and diminished expression of insulin receptors and GLUT4. We propose that caveolin-1/caveolae control insulin action in adipose cells.</I>
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Mateo, Lozano Silvia. "Sarcoma de Ewing: nuevas aproximaciones terapéuticas y búsqueda de dianas biológicas del oncogén EWS/FLI-1." Doctoral thesis, Universitat Autònoma de Barcelona, 2007. http://hdl.handle.net/10803/3571.
Full textAbstract:
La familia de tumores del sarcoma de Ewing (ESFT) incluye un grupo heterogéneo de neoplasias caracterizadas por la presencia de células redondas de pequeño tamaño con mínima evidencia morfológica de diferenciación. ESFT es el segundo tumor óseo más frecuente, afectando fundamentalmente a niños y adolescentes. A pesar del uso de terapias agresivas combinando quimioterapia, radioterapia y cirugía, la supervivencia de pacientes con metástasis es aproximadamente del 30%, mientras que en ausencia de enfermedad metastásica alcanza valores del 70 %. Debido a esto, son necesarias nuevas aproximaciones terapéuticas dirigidas a reducir la morbilidad relacionada con el tratamiento y a mejorar el índice de supervivencia. Afortunadamente, los ESFT presentan una diana molecular perfecta que resulta de la translocación cromosómica t(11;22)(q24;q12), que ocurre en aproximadamente un 95% de los casos, e involucra al gen EWS y a un miembro de la familia de factores de transcripción ETS, fundamentalmente FLI-1 o ERG. La translocación más común genera la formación de la oncoproteína de fusión EWS/FLI-1 que actúa como factor de transcripción y regula de forma aberrante la expresión de genes diana, favoreciendo el proceso tumorigénico. De esta forma la inactivación de la proteína de fusión EWS/FLI-1 se convierte en una estrategia atractiva, no sólo debido su papel fundamental en la tumorigénesis de ESFT, sino también por su especificidad en células transformadas. En este estudio se evaluaron diferentes estrategias dirigidas a reducir los niveles expresión de EWS/FLI-1 in vitro e in vivo. La primera estrategia utilizada para inhibir los niveles de expresión de la proteína de fusión EWS/FLI-1 se basó en el uso de la rapamicina, un antifúngico e inmunosupresor con propiedades anticancerígenas. La rapamicina inhibió la vía de señalización de mTOR/p70s6K y la proliferación de células de ESFT. Estos resultados sugirieron el uso de esta droga como agente citostático en el tratamiento de este tipo de tumores. La segunda aproximación terapéutica se basó en la inhibición simultánea de EWS/FLI-1 a nivel transcripcional y post-transcripcional. El tratamiento combinado de oligonucleótidos antisentido y rapamicina resultó en un incremento en la muerte de células de ESFT, a través de un proceso que involucra la restauración de la vía de señalización pro-apoptótica del TGF?1/TGF?-RII. In vivo, la administración del tratamiento combinado causó un retraso en el crecimiento de los tumores. Estos datos aportan la base para una mayor exploración del potencial del tratamiento combinado como una nueva estrategia en el tratamiento de este tipo de tumores. Los análisis moleculares mostraron que ESFT presentan alteraciones en proteínas reguladoras del ciclo celular, incluyendo la sobreexpresión de proteínas quinasas ciclina-dependientes (CDK2) y la pérdida o baja expresión de sus inhibidores. Basándonos en ésto, la tercera estrategia se basó en la reversión de alguna de estas alteraciones, mediante el uso de la roscovitina, un potente inhibidor de la actividad quinasa de las CDKs. El tratamiento con roscovitina resultó en un incremento de los niveles de apoptosis en células de ESFT in vitro e in vivo, por un mecanismo dependiente de la activación de caspasas. Estos resultados sugieren que la roscovitina puede ser un agente terapéutico efectivo en el tratamiento de ESFT, sola o en combinación con otras drogas potencialmente sinérgicas. Con el objetivo de identificar y evaluar nuevas proteínas que interactúan con EWS/FLI-1 y contribuir de esta forma a la comprensión de los mecanismos de transformación, identificamos como una diana transcripcional directa de EWS/FLI-1 a la caveolina-1, proteína involucrada en gran variedad de procesos celulares, tales como endocitosis, homeostasis del colesterol, transducción de señales y tumorigénesis. Los resultados de este trabajo mostraron que caveolina-1 juega un papel determinante en la tumorigénesis de células de ESFT y su inhibición podría permitir el desarrollo de nuevas estrategias terapéuticas moleculares dirigidas a mejorar el tratamiento de los pacientes de ESFT.
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Sáinz, Jaspeado Miguel Guillermo. "Regulación del aporte vascular en el Sarcoma de Ewing: Papel de la Caveolina-1." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/107820.
Full textAbstract:
Como otros tumores sólidos, el sarcoma de Ewing (SE) requiere de un aporte vascular adecuado que permita nutrir y oxigenar a las células tumorales. En el SE, la vascularización tumoral se encuentra caracterizada principalmente por los procesos de vasculogenesis, mimetismo vascular y angiogénesis. Considerando la participación de CAV1 en el aporte vascular, nos planteamos demostrar que CAV1 juega un papel importante en el desarrollo vascular de la enfermedad.
Estableciendo tres nuevos modelos de baja expresión de CAV1 en células se SE (RDES, SKES1 y TC71), fue posible observar la reducción en el desarrollo tumoral en xenoinjertos, y a su vez esta reducción fue relacionada con la disminución en la densidad micro-vascular. Este trabajo demuestra el efecto directo del silenciamiento de CAV1 sobre el proceso angiogénico en el SE, regulando la respuesta de la célula endotelial ante las señales producidas por la célula tumoral. Nuestros resultados mostraron que las células endoteliales presentan mayor migración ante el estímulo de las señales producidas por la célula tumoral. Mediante el análisis de expresión de diferentes factores utilizando RT-PCR, fue posible determinar que la expresión de bFGF era afectada en los modelos analizados como efecto del silenciamiento de CAV1. Para determinar el mecanismo molecular a través del cual CAV1 regula la angiogénesis en el sarcoma de Ewing, partimos del análisis de expresión de 3 miembros de la familia Eph descritos como pieza clave en el desarrollo vascular. La expresión del receptor EphA2 en los modelos de baja expresión de CAV1 mostró una disminución en los clones, donde el silenciamiento de CAV1 conlleva la redistribución y reducción EphA2. Los análisis mostraron además una interacción entre ambas proteínas.
Establecida la relación EphA2/CAV1, analizamos el efecto de la estimulación de EphA2 a partir de EfnA1 y confirmamos que las células con baja expresión de CAV1 presentaban una respuesta menor. La estimulación resultó en el incremento de la fosforilación de AKT, sugiriendo que los efectos podrían depender de la actividad quinasa del receptor. Para respaldar esto, las células RDES y TC71 fueron transfectadas con un dominante negativo de EphA2 que no modificaba la expresión de CAV1 (EphA2-Kd). Los modelos EphA2-Kd reprodujeron los resultados observados en los modelos de baja expresión de CAV1. La estimulación del modelo TC71-EphA2-Kd con la proteína recombinante EfnA1, mostró tanto la activación de AKT como la sobreexpresión de bFGF, reproduciendo en cierta medida lo observado por el silenciamiento de CAV1 y destacando la importancia de la actividad dependiente de quinasa de EphA2 en el proceso angiogénico de la enfermedad.
Considerando que CAV1 es necesario para el correcto funcionamiento de las rutas de señalización controladas por EphA2 en el SE, decidimos determinar la implicación de CAV1 y EphA2 en el mimetismo vascular de la enfermedad. Se decidió analizar el papel independiente de quinasa del receptor EphA2 como una posible pieza clave en el proceso de mimetismo vascular en el SE. Nuestros resultados mostraron que el silenciamiento de CAV1 reduce el desarrollo de vasos miméticos in vivo e impide la formación tubular in vitro. Además, el efecto de bloquear la actividad quinasadependiente del receptor EphA2 mostró que la actividad quinasa del receptor no afecta al proceso de mimetismo vascular en esta entidad tumoral. Para confirmar la participación de EphA2 en el proceso de formación de vasos miméticos, se transfectó un mutante que carece de toda la región citoplasmática del receptor EphA2 (ΔCyto), eliminando así sus funciones dependientes e independientes de quinasa. Los resultados mostraron que el bloqueo total del receptor tenía un efecto negativo en la formación de estructuras tubulares. Estos resultados confirman la importancia de la actividad prooncogénica del receptor EphA2 en el sarcoma de Ewing.<br>We established low CAV1 expressing models by knocking down CAV1 in TC71, RDES and SKES1 Ewing sarcoma (ES) cells by stably transfecting a previously validated shRNA construct. In vivo studies showed a decrease in tumor volume from the CAV1 knocked-down clones when compared with controls. This correlated with a reduction in microvascular density (MVD) and higher levels of necrosis. Conditioned media from CAV1 knocked-down cells showed reduced capability to promote migration of endothelial cells with no changes in proliferation. Different pro-angiogenic factors were analyzed by RT-PCR in the models and, downregulation of bFGF was observed in all four. Results suggested that CAV1 was indirectly affecting bFGF expression. EphA2 expression was observed in ES cell lines and tumor samples. CAV1 knocked-down cells showed a reduction in EphA2 phosphorylation as well as a displacement from the membrane to the cytoplasm. Furthermore, we showed that the CAV1/EphA2 interaction was necessary for Eph-mediated signaling. To further support these results, RDES and TC71 cells were stably transfected with a dominant negative of EphA2 that did not alter the expression of CAV1 (EphAh2-kd). EphA2-Kd models were able to replicate the effects observed in the CAV1 knocked-down models, where the stimulation of the TC71-EphA2-Kd model with the recombinant protein EfnA1, showed both the activation of AKT and the overexpression of bFGF. These results replicate the effect of silencing CAV1 and highlighted the importance of the kinase-dependent activity of EphA2 in the angiogenic process in ES. We decided to analyze the kinaseindependent role of EphA2 as a possible key in the process of vascular mimicry in ES.
Our results showed that the silencing of CAV1 reduces the development of mimetic vessels in vivo and prevents the tubular formation in vitro. In addition, our results showed that the kinase-dependent activity of the receptor does not affect the process of vascular mimicry in ES. Moreover, we stably transfected a mutant that lacks all the cytoplasmic region of EphA2, blocking its kinase-dependent and –independent activities. Results showed a negative effect on the formation of tubular structures. These results confirm the importance of the pro-oncogenic activity of EphA2 in ES.
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Ilha, Mariana. "Papel da caveolina-1 na capacidade de migração e proliferação de células estreladas hepáticas." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/117581.
Full textAbstract:
A fibrose hepática é uma característica comum de diversas doenças crônicas do fígado e é caracterizada pela deposição excessiva de matriz extracelular no órgão. Em última instância, essa alteração anormal do parênquima hepático acarreta em hipertensão portal, cirrose e insuficiência do fígado, o que pode levar o paciente à morte. As células estreladas hepáticas (HSC) participam ativamente deste processo, modificando seu fenótipo quiescente, rico em gotas lipídicas no citoplasma, para o fenótipo ativado, em resposta a um insulto hepático. A linhagem GRX é um modelo de HSC ativadas. As caveolas são pequenas invaginações de 50-100 nm da membrana plasmática que são ricas em glicoesfingolipídeos, colesterol e proteínas GPI ancoradas. Elas são caracterizadas pela presença de caveolina, uma proteína estrutural específica desta organela. Estas pequenas organelas, consideradas especializações dos “rafts” lipídicos, estão presentes nos mais diversos tipos celulares e podem funcionar como plataformas onde se ancoram várias proteínas de membrana. Estas proteínas reconhecem sinais externos e transmitem sinais moduladores da atividade celular, regulando ou facilitando o transporte de ácidos graxos e de lipídeos, e também são responsáveis pelo transporte de vesículas de membrana. As caveolinas são as principais proteínas estruturais das caveolas, sendo a caveolina-1 (Cav-1) a mais importante. A Cav-1 é encontrada em todos os tipos celulares e está relacionada com a transformação oncogênica e tumorogênese. Estudos já mostraram a interação entre as caveolas e os filamentos de actina, os microtúbulos, e os filamentos intermediários. Em fígados cirróticos foi encontrado um aumento da expressão de Cav-1 nas células endoteliais sinusoidais e nas HSC. Em trabalho anterior, utilizamos o plasmídeo pCav1EGFP para a obtenção de uma linhagem permanente que superexpressa a Cav-1 e a proteína EFGP, a GRXEGFP-Cav. Neste trabalho, nós caracterizamos, bioquímica e morfologicamente, essa linhagem e realizamos outra transfecção com o plasmídeo vazio pCineoEGFP para estabelecer uma linhagem controle, a GRXEGFPpCineo . Através de métodos de análise bioquímica, de imunocitoquímica, de citometria de fluxo e de microscopia confocal e eletrônica de transmissão, mostramos que a superexpressão de Cav-1, aumentou a proliferação e a adesão celular, alterou a morfologia e a estrutura do citoesqueleto das células, a capacidade de migração e de endocitose da GRXEGFP-Cav. A análise ultraestrutural por microscopia eletrônica de transmissão revelou o aumento do número de caveolas na membrana plasmática. As alterações do citoesqueleto de actina e o aumento da afinidade célula-célula são indicativos de maior mobilidade celular. Esses resultados somados ao aumento do conteúdo de αSMA e Col-I sugerem a modulação da GRXEGFP-Cav para um fenótipo de miofibroblasto ativado característico de situações de dano hepático. Como a distribuição e a significância da expressão de Cav-1 em fígados normais e cirróticos são pouco conhecidas, e considerando o papel das HSC nas doenças hepáticas, entendemos que a linhagem permanente GRXEGFP-Cav pode ser uma ferramenta experimental muito interessante para o estudo destas patologias.<br>Liver fibrosis is a common feature of several chronic hepatic diseases and is characterized by the excessive deposition of extracellular matrix in the organ. Usually, this abnormal change of the hepatic parenchyma causes portal hypertension, cirrhosis and liver failure, which can lead to the patient death. Hepatic stellate cells (HSC) participate actively in this process through modifying their quiescent phenotype rich in lipid droplets in the cytoplasm to the activated phenotype in response to the liver injury. GRX line is a model of activated HSC. The caveolae are small invaginations of plasma membrane that reaches 50-100nm of size, rich in glycosphingolipids, cholesterol, and GPI-anchored proteins. These organelles are characterized by the presence of caveolin, a structural and specific protein. These small organelles, which are considered specializations of lipid "rafts" and can be present in several cell types, can act as anchoring platforms for several membrane proteins. These proteins recognize external signals and transmit these signals to modulate the cell activity through regulating or facilitating the transport of fatty acids and lipids, being also responsible for the transport of membrane vesicles. Caveolins are the main structural proteins of caveolae and caveolin-1 (Cav-1) is the most important. Cav-1 is found in all cell types and is related to the oncogenic transformation and tumorigenesis. Previous studies have shown the interaction among caveolae, actin filaments, microtubules, and intermediate filaments. Also, it was found an increase of Cav-1 expression in sinusoidal endothelial cells and HSC of cirrhotic livers, which was suggested to be related to the portal hypertension that accompanies the process of fibrosis. In a previous work, we used the pCav1EGFP plasmid to obtain a permanent cell strain that overexpresses Cav-1 protein and EFGP, which was named GRXEGFP-Cav. In this work we biochemical and morphologically characterized this strain. We also did another cell transfection with an empty pCineoEGFP plasmid to establish a permanent control cell line, which was named GRXEGFPpCineo. Through biochemical analysis, immunocytochemistry, flow cytometry, confocal and electron transmission microscopy, we showed that Cav-1overexpression increased cell proliferation and adhesion, changed cell morphology and cytoskeleton structure, and the cell migration and endocytosis capacity of GRXEGFP-Cav. The actin cytoskeletal changes and the increased cell-cell affinity are indicative of greater cell motility. These results associated to the increase of αSMA and Col-I contents suggest the GRXEGFP-Cav modulation be the activated myofibroblast that characterizes liver damage. As the distribution and the significance of Cav-1 expression in normal and cirrhotic livers are little known and considering the role of HSC in liver diseases, we believe that the permanent GRXEGFP-Cav line can be a very interesting experimental tool for the study of these pathologies.
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Cruz, Gómez Sebastián Matías. "Rol de caveolina-1 en la quimiotaxis de las células dendríticas hacia los nódulos linfáticos." Tesis, Universidad de Chile, 2017. http://repositorio.uchile.cl/handle/2250/150211.
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Tesis para optar al grado de Magister en Bioquímica<br>Las células dendríticas (DCs) son células presentadoras de antígeno capaces de capturar y procesar antígenos que luego son presentados a linfocitos T específicos en órganos linfoides. Para activar a los linfocitos T, las DCs deben pasar por un proceso de maduración y migración hacia los nódulos linfáticos. Para esto, las DCs deben percibir el rastro de quimioquinas como CCL21, a través de receptores como CCR7, que las dirigen hacia los vasos linfáticos y finalmente al nódulo linfático. Dicho proceso se denomina quimiotaxis y es dependiente de la formación protrusiones celulares formadas por haces de filamentos de actina, que están especializadas en percibir el medio por el cual transita la célula. Caveolina-1 es una proteína de andamiaje, la cual se ha relacionado con la migración celular, regulando a las GTPasas pequeñas Rac-1 y Cdc42, por lo tanto incidiendo en la capacidad de la célula de reorganizar el citoesqueleto y formar protrusiones celulares. Resultados de nuestro laboratorio han demostrado que caveolina-1 aumenta su expresión en DCs al ser estimuladas con LPS y que juega un rol fundamental en la llegada de las DC al nódulo linfático, así como en la generación de la respuestas de linfocitos T CD8+ citotóxicos in vivo. Sin embargo, caveolina-1 no participa en la maduración de las DCs ni en su capacidad de activar linfocitos T CD8+ in vitro. En este trabajo postulamos que caveolina-1 promueve la llegada de DCs al nódulo linfático regulando su capacidad quimiotáctica a través de la generación de filopodios vía Rac-1 o Cdc42. A través de ensayos de tinción cutánea con FITC, observamos que las DCs Cav-1-/- llegan en menor número al nódulo linfático en comparación a las DCs silvestres. Además, se evidenció que las DCs Cav-1-/- poseen una capacidad quimiotáctica disminuída en respuesta a CCL21 en cámaras de Boyden, no así en matrices de colágeno. A nivel molecular, se observó que las DCs Cav-1 -/- poseen menos actividad de Rac-1, pero no de Cdc42, y un menor número de filopodios con respecto a las DCs silvestres. Este trabajo sugiere que caveolina-1 favorece la llegada de las DCs al nódulo linfático, promoviendo la actividad de Rac1 y la formación de filopodios, lo que les permite entrar o migrar a través de los vasos linfáticos
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Sanhueza, Muñoz Carlos Joaquín. "Caveolina-1 reduce la transcripción dependiente de hif1α en un mecanismo dependiente del óxido nítrico en células tumorales". Tesis, Universidad de Chile, 2013. http://repositorio.uchile.cl/handle/2250/114871.
Full textAbstract:
Doctor en Farmacología<br>Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de Tesis Electrónicas, hasta diciembre de 2018<br>El cáncer es la 2° causa de muerte en Chile. El desarrollo del cáncer se ha propuesto que es consecuencia de la pérdida de función de los genes supresores de tumores beneficiando la acción de los oncogenes (genes que promueven el crecimiento de tumores). La activación del Factor inducible por hipoxia 1α (HIF1α) en un ambiente reducido en oxígeno (hipoxia) o por óxido nítrico (NO), permiten la proliferación y adaptación metabólica de las células tumorales. Por otro lado, Caveolina-1 es una proteína de andamiaje, que ha sido descrita como un supresor de tumores y promotor de metástasis. Resultados de nuestro laboratorio han demostrado que E-Cadherina y Caveolina-1 actúan cooperativamente en supresión de tumores in vivo. Sin embargo, Caveolina-1 suprime el crecimiento de tumores incluso en células carentes de E-Cadherina, de un modo menos eficiente. La isoforma endotelial de la sintasa de óxido nítrico (NOS3), es uno de los blancos inhibidos por Caveolina-1 más ampliamente descritos en la literatura, que recientemente ha sido implicado en mantenimiento tumoral. Cómo la inhibición de NOS mediada por Caveolina-1 afecta la activación de HIF1α contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina, aún no ha sido evaluado. En este trabajo, evaluamos la posibilidad que la inhibición de NOS por Caveolina-1 pueda reducir la transcripción dependiente de HIF1α y contribuir así a la función supresora de tumores de Caveolina-1. Líneas celulares transfectadas con un plasmidio que codifica para Caveolina-1 [HT29(US), B16F10 y HEK293T] o células en que la expresión endógena de Caveolina-1 fue disminuida utilizando un shRNA [MDA-MB231], fueron tratadas en hipoxia (1% O2, 4-24 h). La actividad transcripcional de HIF, la expresión de genes blanco de HIF1α, la distribución subcelular de HIF1α, Caveolina-1 y NOS3, fueron evaluados mediante ensayos de gen reportero, RT-PCR/qPCR, Western Blot y microscopía confocal, respectivamente. Además, se realizaron ensayos de formación de tumores en ratones inmunosuprimidos SCID-Beige y en ratones inmunocompetentes C57BL/6. Los principales hallazgos de este trabajo fueron, que Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de VEGF en hipoxia en las líneas celulares analizadas. Además, la reducción del crecimiento tumoral de células de melanoma murino B16F10(Cav-1) fue coincidente con la reducción de la expresión del mRNA de vegf in vivo. El tratamiento de las células con el dador de NO (DETA/NO) o el inhibidor de arginasa BEC, previnieron la inhibición de la actividad transcripcional de HIF1α mediada por Caveolina-1. Además, la inhibición de NOS con L-NAME o con el inhibidor selectivo de NOS3, L-NIO, reducen el incremento en la actividad transcripcional de HIF en hipoxia. In vivo, la sobreexpresión de HIF1α en células HT29(US)(Cav-1) revierte la supresión de tumores mediada por Caveolina-1 en ratones SCID-Beige. Finalmente, el tratamiento sistémico de ratones C57BL/6 con L-NAME, reduce el volumen tumoral a niveles comparables con los observados en los tumores formados por células B16F10(Cav-1). En resumen, nuestros resultados sugieren que la inhibición de NOS3 mediada por Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de sus genes blanco, in vitro e in vivo, contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina<br>Cancer, the 2nd most important cause of death in Chile, is thought to develop due to loss of tumor suppressor and gain of oncogene function. Activation of Hypoxia-inducible factor 1α (HIF1α) in the low oxygen environment (hypoxia) present in tumors or by nitric oxide (NO), favors cancer cell proliferation and metabolic adaptation. Caveolin-1 is a scaffolding protein that reportedly functions both as a tumor suppressor and promoter of metastasis. Results from this laboratory have shown that E-cadherin and Caveolin-1 cooperate in tumor suppression in vivo. However, Caveolin-1 expression suppresses tumor growth even in cancer cells lacking E-cadherin, albeit less efficiently. The endothelial isoform of nitric oxide synthase (NOS3), one of the best-established targets for inhibition by Caveolin-1, has recently been implicated in maintence of tumor growth. Whether, Caveolin-1-mediated NOS inhibition may impact on HIF1α activation and account for tumor suppression by Caveolin-1 in the absence of E-cadherin has not been yet assessed. Here, we evaluated the possibility that NOS inhibition by Caveolin-1 may reduce HIF1α - dependent transcription and thereby contribute to the tumor suppressor function of Caveolin-1. Cell lines transfected with a Caveolin-1-encoding plasmid [HT29(US), B16F10 and HEK293T] or cells where endogenous Caveolin-1 protein levels [MDA-MB231] were reduced using shRNA-technology, were exposed to hypoxia (1% O2, 4-24 h). In these cells, HIF transcriptional activity, HIF1α target-gene expression, HIF1α, Caveolin-1 and NOS3 protein levels and subcellular localization were evaluated by gene reporter assays, RT-PCR/qPCR, Western Blot and confocal microscopy, respectively. Tumor forming capacity was evaluated in immunodeficient SCID-Beige and immunocompetent C57BL/6 mouse strains. The main findings of this study are that Caveolin-1 reduced HIF1α transcriptional activity and VEGF expression in hypoxia in vitro in all cell lines. Also, reduced tumor growth of B16F10(Cav-1) melanoma cells in C57BL/6 mice correlated with reduced vegf gene expression in vivo. Treatment of cells with the NO donor (DETA/NO) o arginase inhibition with BEC, prevented Caveolin-1-mediated inhibition of HIF1α transcriptional activity. Furthermore, NOS inhibition with L-NAME or selective NOS3 inhibition with L-NIO, reduced hypoxia-enhanced HIF transcriptional activity. In vivo HIF1α overexpression in HT29(US)(Cav-1) cells reversed tumor suppression due to Caveolin-1 in SCID-Beige mice. Finally, systemic treatment of C57BL/6 mice with L-NAME, reduced tumor volumes to levels comparable to those observed for tumors formed by B16F10(Cav-1) cells. In summary, our observations suggest that Caveolin-1-mediated inhibition of NOS3 activity reduces HIF1α transcriptional activity and target gene expression, both in vitro and in vivo, and that this ability contributes to tumor suppression by Caveolin-1 in the absence of E-cadherin<br>CONICYT, FONDECYT, FONDAP, MECESUP
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Díaz, Morales María Inés. "Rol de la respuesta a proteínas mal plegadas en la actividad supresora de tumores de Caveolina-1." Tesis, Universidad de Chile, 2015. http://repositorio.uchile.cl/handle/2250/136641.
Full textAbstract:
Doctora en Bioquímica<br>Autor no autoriza el acceso a texto completo de su documento<br>El cáncer es la segunda causa de muerte a nivel mundial y en Chile luego de las enfermedades cardiovasculares. Importantemente, las muertes por cáncer van en aumento. Los avances más recientes en el entendimiento de las vías de señalización involucradas en cáncer, ha permitido la identificación de marcadores tempranos y tardíos de la carcinogénesis, los cuales son fundamentales para el diagnóstico y la prognosis del paciente. Basados en estos descubrimientos, existen disponibles nuevas terapias para el tratamiento de esta enfermedad. En esta perspectiva, una respuesta generada en el retículo endoplasmático (RE), denominada unfolded protein response (UPR) ha llamado la atención en el estudio del cáncer en la última década.
En células eucariontes, la mayoría de las proteínas secretadas y de transmembrana se pliegan y maduran en el lumen del RE. Las proteínas entran al RE como cadenas polipeptídicas no plegadas cuyo flujo hacia el RE varía en respuesta a condiciones del medio y el estado fisiológico de la célula. De acuerdo a estos requerimientos las células ajustan la capacidad de plegamiento de proteínas del RE asegurando el mantenimiento de la fidelidad del proceso. Este control homeostático es realizado a través de vías de transducción de señales iniciadas por proteínas de transmembrana del RE cuyas porciones luminales sensan el estado de plegamiento de proteínas en el RE y sus regiones efectoras citoplasmáticas señalizan hacia el citoplasma y núcleo.
Una de las evidencias que sustenta el rol de la UPR en cáncer está relacionada con la alta tasa de proliferación de células cancerosas observada durante el desarrollo tumoral. Este fenómeno requiere de una elevada síntesis de proteínas y de membrana. Adicionalmente, la proliferación celular asociada al crecimiento tumoral ocurre en condiciones hipóxicas y en presencia limitada de nutrientes. La señalización de la UPR involucra al menos tres sensores distintos localizados en la membrana del RE; IRE1-α, ATF6 y PERK. Estos receptores comparten dominios sensores similares en el lumen del RE y bajo condiciones de stress de retículo in vitro, son simultáneamente activados
Caveolina-1 (Cav-1) una proteína de andamiaje asociada a la membrana ampliamente expresada que ha sido descrita como una proteína supresora de tumores. Estudios de nuestro laboratorio, han relacionado esta actividad con su localización en la membrana plasmática y asociada a E-Cadherina. Sin embargo, en modelos celulares de cáncer carentes de E-Cadherina, Caveolina-1 mantiene su rol como supresor de tumores. Estas observaciones indican que esta supresión de tumores puede ocurrir mediante mecanismos independientes de E-Cadherina. Adicionalmente en estos modelos, Cav-1 es altamente prevalente intracelularmente, unida a la membrana de organelos. Estas observaciones han hecho surgir la pregunta si la presencia de Cav-1 en estos organelos, pudiese estar relacionada con la actividad supresora de tumores de Cav-1 en ausencia de E-Cadherina.
Con los antecedentes anteriormente mencionados, el objetivo de esta tesis fue evaluar in vivo en un modelo murino inmunocompetente, si la función de Cav-1 como supresor de tumores tenía relación con la vía adaptativa UPR y luego analizar in vitro cómo Caveolina-1 afecta la UPR. Para la primera parte, se utilizaron como modelo células de melanoma B16F10, singénicas con la cepa de ratón C57-BL/6. El análisis de diferentes parámetros en tumores formados por las células B16F10 fueron empleados para establecer una conexión entre la expresión de Cav-1 y la modulación de la vía de UPR in vivo.
Los ensayos in vivo mostraron que Cav-1suprimió la formación de tumores. Se observó una reducción del 60 % en el tamaño en aquellos tumores formados por las células que expresaron Cav-1 comparado con las que no la expresaron. El hallazgo más importante observado en tumores generados por células que expresan CAV-1 fue la reducción de genes blanco regulados por las vías señalización de los sensores IRE1-α y PERK de la UPR. Esta regulación negativa fue observada además a nivel de proteínas y se mantuvo incluso en tumores de igual tamaño.
En modelos de inducción clásicos de estrés de retículo in vitro, se utilizó hipoxia y el tratamiento con el inhibidor de la glicosilación, tunicamicina. En estos experimentos, nuevamente la presencia de Cav-1 se relacionó con una disminución en los niveles transcripcionales y de sus respectivos blancos proteicos río abajo de IRE1-α y PERK. Además la expresión de Cav-1 se asoció con un aumento en la muerte celular luego de 24 h de tratamiento con tunicamicina o exposición a hipoxia. Resultados similares se obtuvieron in vitro en otra línea celular metastásica de cáncer de mama humano (MDA-MB-231).
El rol supresor de tumores de Cav-1 es a menudo atribuido a interacciones proteína-proteína mediadas por el dominio de andamiaje de Cav-1 (CSD), la que frecuentemente resulta en la inactivación de estas proteínas. Es por esto que para evaluar la relevancia de este dominio en la supresión de tumores y la regulación negativa de la UPR, se realizaron distintas mutaciones sitio-dirigidas en aminoácidos potencialmente interesantes de este dominio y sus alrededores para analizar su efecto en el rol supresor de tumores y sobre la UPR.
Las mutantes Cav-1(S80A) y (S80E), pierden su capacidad de suprimir crecimiento tumoral y a diferencia de Cav-1(WT), generan tumores de tamaño similar a los generados por células que carecen de la expresión de Cav-1. Por el contrario, tumores generados a partir de células que expresaron Cav-1(W98F) y (W1280F), se comportaron como los tumores generados a partir de de Cav-1(WT). Del mismo modo, La inhibición de la UPR observada para de Cav-1(WT) se pierde en tumores generados por las mutantes Cav-1(S80A) y (S80E) pero no en aquellos que se generan a partir de Cav-1(W98F) y (W1280F).
En resumen, los resultados obtenidos en esta tesis, indican que la presencia de Cav-1 en compartimentos intracelulares, tales como el retículo endoplasmático y la inhibición ahí de la UPR están relacionadas con la supresión de tumores de Cav-1 independiente de E-Cadherina. Este trabajo además provee de evidencia que Cav-1 suprime UPR in vitro e in vivo y que el residuo aminoacídico S80 es requerido para suprimir la formación de tumores así como también para inhibir UPR<br>Cancer is the second most important cause of death worldwide and also in Chile after heart diseases. Importantly, deaths caused by cancer are on the rise. More recent advances in our understanding of cancer signaling pathways have led to the identification of early and late stage markers of carcinogenesis important for patient diagnosis and prognosis. Also, based on such insight, new therapies to treat this disease have become available. Particularly, in this perspective, a stress response generated in the endoplasmic reticulum (ER), referred to as the unfolded protein response (UPR) has attracted a great deal of attention in cancer research in the last decade.
In eukaryotic cells, most of the secreted and transmembrane proteins are folded in the ER lumen. The proteins enter into the ER as unfolded polypeptide chains and the extent of transport into this compartment varies in response to environment signals or the physiological status of cells. According to the specific demands, cells adapt their protein folding capacity in order to preserve the fidelity of the process. This process that seeks to maintain ER homeostasis is controlled by signal transduction events initiated by ER transmembrane receptor proteins whose luminal domains sense the protein folding status and then convey signals toward the cytoplasm and nucleus via the cytosolic domains.
The importance of UPR in cancer is linked to the high proliferation rates developed by cancer cells during tumor growth, which in turn require augmented protein and membrane synthesis capacity. Additionally, such tumor growth frequently occurs in hypoxia and under conditions of limited nutrient availability. All these events posit the UPR as a crucial adaptive response during tumor growth. The UPR signaling pathway involves at least three distinct sensors located in the ER membrane: IRE1-, PERK, and ATF6. These receptors share luminal sensing domains and under stress conditions in vitro they are simultaneously activated. However, the extent of activation and hence the capacity to respond to stress depends on a large variety of ER associated factors, one of them potentially being the protein caveolin-1.
Caveolin-1 (Cav-1) is a broadly expressed, membrane-associated scaffolding protein that has been described to function as a tumor suppressor. Studies from our laboratory have linked this ability to its localization in the plasma membrane and association there with E-cadherin. However, in cancer cell models lacking E-Cadherin, Cav-1 still functions as a tumor suppressor, albeit less efficiently. These observations indicate that Cav-1 tumor suppression may occur via mechanisms that are independent of E-cadherin, additionally, in such models; Cav-1 was highly prevalent in intracellular, membrane-bound compartments. These observations raised the question as to whether the presence of Cav-1 in these organelles could be related to the Cav-1 tumor suppressor activity in the absence of E-cadherin.
With this in mind, the objective of this thesis was to evaluate in vivo in an immunocompetent mouse model, whether the function of Cav-1 as tumor suppressor was related to the adaptive UPR signaling pathway and then to analyze in vitro how Cav-1 affects the UPR. To test the first part, we used the syngenic mouse melanoma cell line B16F10 in the murine strain C57-BL/6. The analysis of different parameters in tumors formed by B16F10 cells were employed to establish a connection between the expression of Cav-1 and the modulation of the UPR pathway in vivo.
The in vivo assays showed that Cav-1 suppressed tumor growth. A 60% reduction in tumor size was observed in tumors generated by B16F10 cells expressing Cav-1, as compared to those generated by cells lacking Cav-1. Importantly, in the tumors generated by Cav-1 expressing cells, a reduction in the transcription of target genes regulated downstream of the UPR sensors IRE1-α and PERK was observed. This down-regulation was observed also at the protein levels and the observed differences were maintained in tumors of the same size.
In in vitro experiments, classic ER stress inducers, such as hypoxia and treatment with the glycosylation inhibitor tunicamicyn were employed. Here too, the presence of Cav-1 was linked to a reduction in the transcription of target genes downstream of IRE1-α and PERK that coincided with reduced protein levels of the respective target proteins.Cav-1 expression was associated with an increase in cell death after 24 h of treatment with tunicamicyn or exposure to hypoxia. Of note, similar results were obtained in vitro using the aforementioned B16F10 cells and also the metastasic human breast cancer cell line MDA-MB-231.
Cav-1 tumor suppression is often attributed to protein-protein interactions mediated by the Cav-1 scaffolding domain (CSD) that often lead to inactivation of Cav-1 binding partners. To test the relevance of the CSD in tumor suppression and UPR down-regulation, several potentially interesting amino acids were altered by site-directed mutagenesis in the CSD and flanking regions and their impact on Cav-1-mediated inhibition of UPR and tumor growth were assessed.
The Cav-1(S80A) and (S80E) mutants lost their ability to suppress tumor formation, and unlike Cav-1(WT) expressing cells generated tumors similar to those formed by Cav-1 lacking B16F10 cells. On the contrary, tumors generated by cells expressing the Cav-1(W98F) and (W128F) mutants behaved essentially the same as Cav-1(WT) expressing cells. Likewise, Cav-1-mediated UPR inhibition observed for tumors generated by Cav-1(WT) expressing cells was lost in tumors generated by cells expressing the S80A and S80E mutants, but not in those expressing the W98F and W128F mutants.
In summary, the results obtained in this thesis indicate that the presence of Cav-1 in intracellular compartments, such as the ER, and inhibition there of the UPR is linked to Cav-1 tumor suppression independent of E-cadherin. We also provide evidence showing that Cav-1 suppressed the UPR in vitro and in vivo, and that the amino acid residue S80 is required for Cav-1 to suppress tumor formation and inhibit the UPR<br>Fondecyt
Fondap
ICGEB CRP/CH108-03
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Lobos, González Lorena. "E-cadherina potencia a caveolina-1 como supresor de tumores y reduce su efecto promotor de metástasis." Tesis, Universidad de Chile, 2011. http://repositorio.uchile.cl/handle/2250/112023.
Full textAbstract:
Tesis presentada a la Universidad de Chile
para optar al grado de Doctor en Bioquímica<br>Caveolina-1 es una proteína de membrana esencial en la formación de caveolas, participa en el transporte de colesterol, actúa como proteína de andamiaje en señalización y ha sido descrita como un supresor de tumores. Por otro lado, la presencia de Caveolina-1 juega un rol esencial en la polarización celular durante el proceso migratorio, ya que controla la localización de moléculas de señalización y regula la activación de proteínas que participan en migración. Sin embargo, no sólo la presencia de Caveolina-1 es esencial para la migración celular, sino también la fosforilación de Caveolina-1 en Tirosina 14. Considerando que la migración celular es esencial para la metástasis, Caveolina-1 ha sido descrita también como un promotor de metástasis. Este comportamiento dual ha causado controversia y ha dado origen a un gran número de investigaciones en desarrollo actúalmente en la comunidad científica.
El principal objetivo de esta tesis fue establecer, en un modelo in vivo inmunocompetente, si Caveolina-1 tiene o no este rol dual en cáncer usando como modelo células de melanoma. Estudiamos en ratones C57-BL/6 el comportamiento de las líneas tumorales B16-F0 y B16-F10, ambas líneas singénicas con la cepa C57-BL/6, y por lo tanto no existe rechazo inmunológico. Estas células al ser subcutáneamente en estos ratones tienen la capacidad de formar tumores sólidos y a la vez, si se inyectan por la vía intravenosa, generan focos de metástasis en el pulmón de los ratones. Por lo tanto, con este sistema estudiamos el rol dual de Caveolina-1 en un mismo modelo in vivo. Una vez inoculados los animales en el lomo (vía subcutánea) o por la vena lateral de la cola (vía intravenosa), con las células seguimos la formación de tumores y cuantificamos la metástasis a nivel pulmonar, respectivamente.
Los ensayos realizados in vivo en las células B16-F0 mostraron que Caveolina-1 era capaz de suprimir la formación de tumores, retardando al doble el tiempo de aparición de éstos. Al silenciar los niveles de expresión de Caveolina-1 en las B16-F0 Caveolina-1 mostró promover metástasis no sólo a nivel de pulmón, a la vez mostró nódulos metastasicos en ganglios, riñón, bazo e hígado.
Los ensayos en los ratones C57BL/6 con las células B16-F10 con y sin expresión de Caveolina-1 mostraron una reducción del 60 % en el tamaño de los tumores formado por las células que expresaron Caveolina-1 comparado con las que no la expresaron. En los ensayos de metástasis, las células que expresaron Caveolina-1 causaron al menos dos veces más metástasis pulmonar. Estos resultados mostraron por primera vez en un modelo in vivo que Caveolina-1 es capaz de actúar tanto como supresor tumoral y como promotor de metástasis.
Usando el mismo modelo de estudio, para analizar el rol dual de Caveolina-1 continuamos con un análisis mutacional, específicamente usando una población celular que expresó Caveolina-1 no fosforilable en la posición de la Tirosina-14 (B16-F10(cav-1/Y14F) y se realizaron los ensayos de supresión de tumor y metástasis. Los resultados revelaron que Tirosina-14 era esencial para otorgarla a Caveolina-1 la capacidad de actúar como una molécula promotora de metástasis, pero al mismo tiempo este residuo no era esencial para el rol supresor de tumores.
Por otra parte, evaluamos in vivo la capacidad de E-cadherina de modular los dos roles descritos para Caveolina-1 en las células B16-F10. En estas, la presencia de E-cadherina cumple una función sinérgica sobre Caveolina-1 en su rol supresor de tumores, llegando incluso a inhibir completamente la formación de tumor dependiendo de la expresión de E-cadherina de manera directamente proporcional. Al mismo tiempo E-cadherina mostró frenar el efecto promotor de metástasis dado por Caveolina-1, es decir, ambas proteínas juntas en las células B16-F10 evitan la formación de tumores metastasicos en el pulmón o en cualquier otra parte del cuerpo del animal.<br>Caveolin-1 is a membrane protein essential for the formation of caveolae, is involved in cholesterol transport, acts as a signaling scaffold protein and has been described as a tumor suppressor. On the other hand, the presence of caveolin-1 plays an essential role in cell polarization during the migration process as it controls the localization of signaling molecules and regulates the activation of proteins involved in migration. However, not only the presence of caveolin-1 is essential for cell migration, but also the phosphorylation of caveolin-1 on Tyrosine 14. Whereas cell migration is essential for metastasis, caveolin-1 has also been described as a promoter of metastasis. This dual behavior has caused controversy and led to a large number of investigations currently under development in the scientific community.
The main objective of my research is to establish, in an immunocompetent in vivo model, caveolin-1 whether or not this dual role in cancer melanoma. We study a model where we use mice C57-BL / 6 and tumor lines F0 B16, B16 and F10, both lines syngeneic with the strain C57-BL / 6, ie, no immune rejection. These cells to be inoculated subcutaneously in mice have the ability to form solid tumors and also, if injected intravenously, generate foci of metastases in the lungs of mice. Therefore, with this system we can study the dual role of caveolin 1 in the same model in vivo. Once inoculated animals in a case in the back of the animal (subcutaneously) and the lateral tail vein (intravenously), the cells follow the formation of tumors and quantified the lung metastases, respectively.
The tests performed in vivo in B16-F0 cells show that caveolin-1 is able to suppress tumor formation, slowing to twice the time of appearance of these. Metastases in trials of these same cells, caveolin-1 showed promote metastases not only in both lung metastatic lymph nodes showed kidney, spleen and liver. By silencing the expression levels of caveolin-1 in B16-F0 results showed that both roles of caveolin-1 is lost.
Trials in C57BL / 6 mice with B16 F10 cells with and without expression of caveolin-1 show that there is a 60% reduction in the size of tumors formed by cells expressing caveolin-1 compared with those without expressed. In trials of metastasis, the cells expressing caveolin-1 cause at least twice as lung metastases. These results show for the first time in an in vivo model that caveolin 1 is able to act as both tumor suppressor and a promoter of metastasis. Using the same model to study, to analyze the dual role of caveolin-1 continue with mutational analysis, specifically using a cell population that expresses caveolin-1 is not phosphorylated at the position of tyrosine-14 (B16 F10 (cav 1/Y14F) tests were performed in tumor suppression and metastasis. The results show that tyrosine-14 is essential for caveolin-1 to grant it the ability to act as a metastasis-promoting molecule, but at the same time, this residue is not essential for the role tumor suppressor.
Moreover, we evaluate in vivo the ability of E-cadherin to modulate the two roles described for caveolin-1 in B16-F10 cells. In these, the presence of E-cadherin plays a synergistic role of caveolin-1 in tumor suppressor role, even to completely inhibit tumor formation depending on the expression of E-cadherin in direct proportion. While E-cadherin stops the metastasis-promoting effect given by caveolin-1, ie both proteins together in B16 F10 cells prevent the formation of metastatic tumors in the lung or elsewhere in the body of the animal.<br>Conicyt; FONDECYT-FONDAP
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Hirsch, Gabriela Elisa. "Efeitos do γ-orizanol e extrato hidroalcólico de Thuya occidentalis sobre linhagens de câncer de próstata responsivas e não-responsivas a andrógenos". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/141254.
Full textAbstract:
O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença.<br>Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.
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Campos, González América Vanessa. "Estudio del rol pro-metastásico de Caveolina-1 en vesículas extracelulares de líneas celulares de cáncer de mama metastásico." Tesis, Universidad de Chile, 2018. http://repositorio.uchile.cl/handle/2250/172698.
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Tesis presentada a la Universidad de Chile
para optar al grado de Doctora en Bioquímica<br>El cáncer de mama corresponde a una de las neoplasias malignas que más se asocia a mortalidad en el género femenino a nivel mundial. Aunque su incidencia ha disminuido gracias a la implementación de mamografías de exploración y aplicación de terapias adyuvantes, esta disminución no parece ser suficiente, ya que el desarrollo de metástasis aún sigue siendo responsable de más del 90% de las muertes asociadas a cáncer de mama, entre otros tipos de cáncer. La progresión de células tumorales hacia un estado metastásico implica la adquisición de características, tales como resistencia a apoptosis, migración e invasividad elevada etc. Teniendo en cuenta esto último, se ha descrito que muchas de estas características son potenciadas por la expresión de Caveolina-1 (CAV1), involucrando a esta proteína de la membrana en la progresión del cáncer. Específicamente en cáncer de mama avanzado se ha observado que una elevada expresión de CAV1 se asocia a una menor sobrevida del paciente. Por otro lado, estudios in vitro realizados en nuestro laboratorio en la línea celular de cáncer de mama metastásico humano, MDA-MB-231, han indicado que el silenciamiento de CAV1 conduce a una disminución en la migración, polarización y recambio de adhesiones focales en comparación con células MDA-MB-231 control.
La pregunta que surge ahora es de qué manera CAV1 es capaz de promover migración e invasión, favoreciendo metástasis, considerando que este proceso en sí es ineficiente debido a que menos del 0,1% de las células diseminadas están implicadas en iniciar este proceso. Una de las respuestas puede deberse a que se genera el transporte de CAV1 junto a otras moléculas a través de vesículas
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extracelulares (EVs), las cuales serían capaces de llevar a esta proteína como mensaje a células contiguas dentro del mismo microambiente tumoral o a células pertenecientes a tejidos distantes idóneos para la formación de un nicho pre-metastásico. En este contexto, se tienen registros de que vesículas de células de la línea celular MDA-MB-231 que contiene niveles endógenos elevados de CAV1 son capaces de promover migración en células de cáncer de mama humano carentes de CAV1, como MCF-7 de carácter no metastásico, pero se desconoce el contenido y su rol en este proceso.
Estos antecedentes nos llevaron a plantear la hipótesis que: CAV1 en vesículas extracelulares aumenta el potencial migratorio e invasivo in vitro en células de cáncer de mama y metastásis in vivo en un modelo de xenotrasplante de cáncer de mama. Por consiguiente el objetivo principal de esta tesis fue evaluar la capacidad migratoria e invasora de células de cáncer de mama in vitro y el desarrollo de metástasis in vivo en un modelo de xenotrasplante mamario expuesto a EVs que contienen CAV1. Para abordar la hipótesis de trabajo se plantearon los siguientes objetivos específicos: (1) Purificar y caracterizar vesículas extracelulares de las líneas celulares humanas de cáncer de mama metastásico y no metastásico; (2) Estudiar del efecto de EVs con CAV1 en las propiedades de migración e invasión in vitro en líneas celulares de cáncer de mama humano; (3) Evaluar el perfil proteómico de EVs de las sublíneas de cáncer de mama MDA-MB-231 WT, shC y shCAV1 y por último (4) Evaluar la capacidad metastásica en un modelo murino de xenotrasplante inoculado por la vía intraperitoneal con EVs que contienen CAV1. Los resultados mostraron la obtención de EVs enriquecidos en vesículas del tamaño de exosomas de <200 nm de diámetro a partir de medios condicionados de las líneas
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celulares de cáncer de mama metástasico humano, MDA-MB-231 WT y MDA-MB-231(shC) que expresan niveles endógenos elevados de CAV1 y en células carentes de CAV1 como MDA-MB-231(shCAV1). Esta caracterización se complementó con imágenes obtenidas mediante microscopía electrónica de transmisión de las vesículas aisladas que mostró tamaños de vesículas de alrededor de 100 nm de diámetro en general y con el hallazgo de CAV1 en vesículas provenientes de MDA-MB-231 WT y shC y no así en vesículas de MDA-MB-231(shCAV1). Cabe añadir que se detectaron marcadores de EVs en todas las muestras mediante Western blot. Para evaluar el efecto biológico de vesículas que contienen o no CAV1 en células de cáncer de mama de carácter metastásico o no metastásico, se evaluaron parámetros de migración e invasión de estas células una vez expuestas a EVs. Los resultados indicaron que independiente del tipo celular utilizado como célula recipiente de EVs, aquellas células que son tratadas con EVs que contienen CAV1 aumentan su potencial migratorio e invasivo en comparación con células no tratadas o tratadas con EVs que no contienen CAV1. El análisis por espectrometría de masas reveló la presencia de proteínas específicas relacionadas con la adhesión celular, como Cyr61, tenascina (TNC) y S100A9 sólo en EVs de MDA-MB-231 WT y shC y no en EVs de MDA-MB-231 carentes de CAV1. De manera de evaluar el rol pro-metastásico de CAV1 en EVs en un modelo animal, se inyectaron estas vesículas junto con células de cáncer de mama metastásico o no metastásico por la vía intraperitoneal en un modelo murino denominado Carcinomatosis intraperitoneal. Los resultados indicaron que animales inoculados con células más EVs que contienen CAV1 presentaron un aumento en el número de células tumorales en el líquido ascítico hallado en la cavidad peritoneal junto con un aumento en la masa tumoral en bazo/páncreas y
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mesenterio en comparación con aquellos animales que no fueron tratados o tratados sólo con células o tratados con células más EVs que no contienen CAV1. Cabe recalcar que nuevamente el efecto de las EVs con CAV1 se dio independiente a que se inoculara junto con células de cáncer de mama que contenían o no CAV1 en el modelo murino. Esto nos lleva a sugerir que la importancia del efecto biológico de estas vesículas en la célula recipiente no sólo recae en la presencia de CAV1, sino que en el tipo de cargo molecular que puedan traer consigo CAV1 en estas vesículas<br>Breast cancer is the leading cause of cancer-related deaths in women. Although the incidence of this disease has decreased thanks to the implementation of screening mammograms and application of adjuvant therapies, this decrease does not seem to be enough, since the development of metastasis is still responsible for more than 90% of deaths associated with breast cancer, among other types of cancer. The progression of tumor cells towards a metastatic state implies the acquisition of characteristics, such as resistance to apoptosis, migration and high invasiveness, etc. Taking into account the latter, it has been described that many of these characteristics are enhanced by the expression of Caveolin-1 (CAV1), thereby implicating this membrane protein in the progression of cancer. Specifically in advanced breast cancer it has been observed that a high expression of CAV1 is associated with a shorter survival of the patient. On the other hand, in vitro studies conducted in our laboratory using the human metastatic breast cancer cell line, MDA-MB-231, have indicated that the silencing of CAV1 leads to a decrease in migration, polarization and focal adhesion turnover in comparison with control MDA-MB-231 cells.
The question that arises is how CAV1 may promote migration, invasion and metastasis, considering that this process is very inefficient because less than 0.1% of the disseminated cells successfully establish a metastatic nodule. One possibility may be that CAV1 is present together with other molecules in extracellular vesicles (EVs), which serve as vectors to transport these components to adjacent cells within the same tumor microenvironment and/or to distant sites where they may condition
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the pre-metastatic niche. Here, it should be noted that EVs from MDA-MB-231 cells reportedly promote migration of non-metastatic MCF-7 human breast cancer cells, but the precise content of these EVs and their role in this process were not defined.
This led us to propose the following hypothesis: CAV1 in extracellular vesicles increases the migratory and invasive potential of breast cancer cells in vitro and in a breast cancer xenotransplant model in vivo. Therefore, the main goal of this thesis was to evaluate the migratory and invasive capacity in vitro and metastatic breast cancer cells in vivo in a xenotransplant model exposed to EVs containing CAV1. To address the working hypothesis, the following specific objectives were proposed: (1) To purify and characterize extracellular vesicles from human metastatic and non-metastatic breast cancer cell lines; (2) To study the effect of CAV1-containing EVs on migration and invasion in vitro of human breast cancer cell lines; (3) To evaluate the protein content by mass spectrometry of EVs from the three breast cancer sub lines MDA-MB-231 WT, shC and shCAV1; and finally (4) To evaluate the metastatic potential in vivo in a murine xenotransplant model inoculated intraperitoneally with EVs containing or not CAV1.
The results showed that we were able to isolate an EV preparation enriched in vesicles of <200 nm in diameter, which is characteristic of exosomes. The EVs were purified from conditioned media of the human metastatic breast cancer cell lines, MDA-MB-231 and MDA-MB-231(shC), both expressing elevated endogenous levels of CAV1, as well as from cells lacking CAV1, such as MDA-MB-231(shCAV1) cells. This characterization was complemented by transmission electron microscopy of the isolated vesicles, which revealed that vesicles were around 100 nm in diameter. Finally, by western blotting, CAV1 was detected
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together with exosome markers in vesicles from MDA-MB-231 WT and shC but not in MDA-MB-231(shCAV1) EVs.
To evaluate the biological effects of vesicles with or without CAV1 on metastatic or non-metastatic breast cancer cells, migration and invasion parameters of these cells were evaluated following exposure to EVs. Regardless of the cell type that was used as a recipient cell, those cells that were treated with EVs containing CAV1 increased their migratory and invasive potential in comparison with cells that were either not treated or treated with EVs lacking CAV1.
Analysis by mass spectrometry revealed the presence of specific proteins related to cell adhesion, such as Cyr61, tenascin (TNC) and S100A9 only in MDA-MB-231 WT and shC EVs but not in EVs from MDA-MB-231 lacking of CAV1. These results were confirmed by western blotting analysis.
In order to evaluate the role of CAV1 in EVs in a murine carcinoma model, these vesicles were injected intraperitoneally together with metastatic or non-metastatic breast cancer cells into recipient mice. For animals inoculated with cells plus EVs containing CAV1, the number of tumor cells found in the ascitic fluid generated within the peritoneal cavity was dramatically increased. Also, a substantial increase in the tumor mass in spleen/pancreas and mesentery was observed in these mice compared to those animals that were either not treated, treated only with cells or treated with cells plus EVs that did not contain CAV1. It should be noted that again these effects of CAV1-containing EVs were observed regardless of whether the reciepient breast cancer cell type employed in these experiments expressed CAV1 or not. Thus, the biological effects of these vesicles in the recipient cell
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appear not to be attributable to CAV1 per se, but rather to depend on differences in the molecular cargos that are incorporated into EVs in the presence of CAV1
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Rodríguez, González Diego Alejandro. "Caveolina-1 inhibe la proliferacion y promueve la muerte celular mediante la regulacion negativa de la ciclooxigenasa-2 y survivina." Tesis, Universidad de Chile, 2009. http://repositorio.uchile.cl/handle/2250/110537.
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Troiano, Jéssica Antonini. "Avaliação de mecanismos de modificação pós-traducional da óxido nítrico sintase endotelial (eNOS) associados a biodisponibilidade do óxido nítrico em artérias de ratas espontaneamente hipertensas (SHR) ao final da prenhez /." Araçatuba, 2019. http://hdl.handle.net/11449/182077.
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Orientador: Cristina Antoniali Silva<br>Banca: Fernando Silva Carneiro<br>Banca: Carlos Alan Candido Dias Junior<br>Banca: Graziela Scalianti Ceravolo<br>Banca: Angela de Castro Resende<br>Resumo: A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas en... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Reduction of vascular reactivity to phenylephrine (PE) in aortaof spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higherproduction and/or higerbioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation,by PI3K/Akt.Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc)is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOSon serine siteleads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this studywas to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteriesof pregnant rats. The O-GlcNAc-protein content and also the enzymesexpressionthat participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2ndor 3rdbranch) of non-pregnant (NP) and pregnant (P), normotensiverats(Wistar) and SHR.Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolaein the aorta and the mesenteric artery in the presence or absence of methyl-β-cyclodextrin (dextrin, 10 mmol/L) by electronicmicroscopy.In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 μmol/L) and of the caveolae, using a caveolae disassembler, (Complete abstract electronic access below)<br>Doutor
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Potje, Simone Regina [UNESP]. "Efeito vasorelaxante do doador de óxido nítrico [Ru(terpy)(bdq)NO]3+ (TERPY) em artéria de resistência e sua interação com a óxido nítrico sintase endotelial (eNOS) de ratos espontaneamente hipertensos (SHR)." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/148587.
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Previous issue date: 2016-12-16<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)<br>O TERPY promove efeito hipotensor de maior magnitude em ratos hipertensos (SHR e 2R-1C) do que em ratos normotensos (Wistar e 2R). Foi demonstrado anteriormente que o endotélio prejudica o efeito vasodilatador do TERPY em aorta de Wistar. No entanto, observamos que o endotélio melhora os efeitos vasodilatadores do TERPY em aorta de SHR. Vasos sanguíneos de menor calibre, tais como as artérias de resistência, estão associadas ao controle da resitência vascular periférica e da pressão arterial. Nossa hipótese é que o TERPY induz relaxamento nas artérias mesentéricas de resistência em SHR e que as células endoteliais modulam positivamente o efeito do TERPY nestes vasos sanguíneos. Portanto, o nosso objetivo foi avaliar o efeito vasodilatador do TERPY em anéis com e sem endotélio de artéria mesentérica de ratos SHR, o seu mecanismo de relaxamento e a participação da NOS sobre esse efeito. e a Nossos resultados mostraram que o TERPY induziu um efeito vasodilatador dependente da concentração em anéis de artérias mesentéricas (2º e 3º ramos) de SHR e de ratos Wistar. A potência do TERPY foi maior em anéis intactos do que em anéis sem endotélio em artérias mesentéricas de SHR, mas em Wistar o endotélio prejudicou o efeito do TERPY. Nas artérias mesentéricas sem endotélio de SHR, o efeito do TERPY é dependente da atividade da guanilato ciclase solúvel e de canais para potássio. Nas artérias mesentéricas intactas de SHR, o efeito de TERPY depende da atividade de eNOS, mas não é dependente das atividades de nNOS, iNOS ou da via da ciclooxigenase. O TERPY promove a fosforilação da eNOS nos resíduos de serina1177 e aumenta a concentração de óxido nítrico em células endoteliais isoladas de artérias mesentéricas de SHR. Nossos resultados mostraram que a guanilato ciclase solúvel, os canais para potássio e a eNOS estão envolvidos no efeito vasodilatador estimulado pelo TERPY nas artérias de resistência mesentérica de SHR. Numa segunda parte deste estudo, avaliamos o mecanismo de ação de TERPY e seu efeito sobre a atividade da eNOS em células endoteliais. As células HUVEC, WT-HEK e HEK-eNOS foram tratadas com TERPY em diferentes tempos (0 a 60 minutos). Foram analisados por Western blotting o efeito do TERPY sobre a fosforilação de eNOS, monômero e dímero da eNOS e sobre monômero e oligômero de caveolina-1. Também foi avaliado o efeito do TERPY na interação eNOS/Cav-1 através de co-imunoprecipitação. As alterações induzidas pelo TERPY sobre as concentrações de espécies reativas de oxigênio e peroxinitrito em células endoteliais foram medidas usando sonda DHE e biossensor 7-CBA, respectivamente. A concentração de óxido nítrico (NO) foi avaliada por sonda DAF-FM e sensor Cooper. O TERPY promoveu desacoplamento e disfunção da eNOS, dependente de BH4. A desestabilização dos oligômeros da caveolina-1 foi induzida pelo TERPY. Consequentemente, o TERPY reduziu a interação eNOS/Cav-1 e promoveu ativação da eNOS. Nossos resultados mostraram que a atividade da eNOS pode ser regulada de duas maneiras diferentes pelo TERPY. O TERPY promove desacoplamento e fosforilação da eNOS, promovendo uma estratégia diferente para a regulação da atividade desta enzima. As moléculas químicas ou biológicas como o TERPY que regulam a atividade da eNOS e aumentam a produção e a biodisponibilidade de NO teriam ações terapêuticas importantes para o tratamento de doenças vasculares associadas a hipertensão.<br>CNPq: 141323/2013-2
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Kobayashi, Priscila Emiko. "Expressão gênica e proteica de APC, E-caderina, β-catenina e Caveolina-1 no processo carcinogênico da próstata canina e suas metástases". Botucatu, 2016. http://hdl.handle.net/11449/134378.
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Orientador: Renée Laufer Amorim<br>Resumo: As alterações na expressão de E-caderina, -catenina, APC e Caveolina-1 nas células epiteliais prostáticas têm sido estudados em humanos como mecanismos relacionados com progressão tumoral, invasão e metástase. Estas proteínas estão envolvidas no processo de adesão celular e ativação da via WNT canônica. No cão, a perda da expressão proteica de E-caderina e a translocação da -catenina da membrana para o citoplasma/núcleo foram descritas anteriormente em lesões prostáticas caninas. No entanto, nenhum estudo correlacionou alterações de expressão dessas proteínas com as proteínas APC e Caveolina-1. Devido ao prognóstico desfavorável do carcinoma prostático (CaP) no cão e a importância da identificação de novos marcadores prognósticos e preditivos, o presente estudo visou avaliar as expressões gênica e proteica de E-caderina, -catenina, APC e Caveolina-1 em diferentes lesões prostáticas caninas, além de avaliar o padrão de metilação do gene APC. Foram utilizados neste estudo 10 CaPs, 4 metástases de carcinoma prostático, 10 amostras de atrofia inflamatória proliferativa (PIA) e 10 próstatas normais de cães para análise imuno-histoquímica. Para a técnica de RT-qPCR forma utilizados 11 próstatas normais, 11 PIA, 17 CaPs e 3 metástases. Para análise de metilacão foram utilizadas seis próstatas normais, seis próstatas com PIA e 12 CaPs. Este estudo revelou aumento de expressão gênica e proteica de Caveolina-1 nas amostras de CaP e metástases, além de menor expressão nas amostras de ca... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Altered expression of E-cadherin, -catenin, APC and Caveolin-1 in prostate epithelial cells has been studied in humans as mechanisms related with tumor progression, invasion and metastasis. These proteins are envolved in cell-cell cohesion and participate in the activation of the canonical WNT pathway. In dogs, membranous E-cadherin loss and translocation of -catenin from the membrane to cytoplasm/nucleus were described previously in canine prostatic lesions. However, studies correlating these genes have not been reported their proteins with APC and Caveolin-1 expressions in canine prostatic lesions. Due to poor prognosis in canine prostate carcinoma (PC) and the need to develop new prognostic and predictive biomarkers, this study aimed to evaluate gene and protein expression of E-cadherin, -catenin, APC and Caveolin-1 in different canine prostatic lesions, and the methylation status of APC gene. We used 10 PC, 4 prostate metastasis, 10 proliferative inflammatory atrophy(PIA) and 10 canine normal prostates tissues for immunohistochemistry. For RT-qPCR we used 11 normal prostate, 11 PIA, 17 PC and 4 metastasis. For methylation analysis, we used six normal prostates, 6 PIA and 13 PC. This study revealed increased Caveolin-1 gene and protein expression in PC and metastasis and lower expression were found in tumors with lower Gleason score. Membranous E-cadherin and - catenin staining was observed in normal prostate samples whereas heterogenous loss was detected in other samples.... (Complete abstract click electronic access below)<br>Mestre
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Comajoan, von Arend Pau. "Efecte de l'administració de l'rt-PA en condicions isquèmiques in vitro i in vivo: Cav-1 com a potencial biomarcador de volum d'infart." Doctoral thesis, Universitat de Girona, 2019. http://hdl.handle.net/10803/669184.
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Recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for the treatment of acute ischaemic stroke. However, the application of this therapy is limited to <5-7% of patients due to the associated increased risk of haemorrhagic transformation (HT). Although it is known that HT is related to rt-PA-induced blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. The obtained results show that long-term studies are needed to elucidate time-dependent molecular mechanisms associated to BBB breakdown, and to explore protective BBB therapies after ischaemic stroke and rt-PA treatment. On the other hand, it has been demonstrated that OGD induces significant alterations to loading control proteins for Western Blot analysis proposing Stain-Free technology as an alternative normalization method to traditional housekeeping proteins. Finally, serum Cav-1 levels could represent a potential biomarker predicting the ischaemic outcome before rt-PA administration<br>L'rt-PA és l’únic fàrmac aprovat per tractar l’ictus isquèmic agut. No obstant, l’estreta finestra terapèutica, deguda al risc associat de transformació hemorràgica (TH) provoca que només s’apliqui a <5-7% dels pacients. La TH està relacionada amb la disrupció de la barrera hematoencefàlica (BHE) deguda a l’rt-PA però els mecanismes subjacents encara no estan del tot establerts. Els resultats obtinguts mostren que es requereixen estudis a llarg termini per tal de dilucidar els mecanismes dependents del temps associats a la disrupció de la BHE, i explorar noves teràpies protectores per al tractament de l’ictus isquèmic. S’ha demostrat que la POG provoca canvis significatius en els nivells proteics de controls de càrrega per “Western blot” i es presenta la tecnologia “Stain-Free” com a una alternativa a la normalització tradicional. Finalment, els nivells sèrics de Cav-1 podrien representar un potencial biomarcador predictor del pronòstic després d’una isquèmia en absència d’rt-PA<br>S'ha extret el capítol de resultats del pdf de la tesi fins a la seva publicació en forma d'article.
Results chapter removed from pdf file until publication
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Díaz, Fuentes Jorge Esteban. "Caveolina-1 favorece la activación de rab5 promoviendo la migración y la activación del eje p85α/tiam1/rac-1 en células de cáncer metastásico y en un modelo de metástasis in vivo en ratones c57bl/6". Tesis, Universidad de Chile, 2015. http://repositorio.uchile.cl/handle/2250/136775.
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Doctor en Bioquímica<br>El cáncer constituye una de las principales causas de muerte en Chile y el mundo.
Particularmente, la agresividad de esta enfermedad subyace en la diseminación de las
células tumorales hacia otros tejidos, fenómeno conocido como metástasis. La
metástasis es un proceso multifactorial, siendo la migración celular una de las etapas
más relevantes. Caveolina-1 es una proteína de membrana que en etapas avanzadas
del cáncer aumenta su expresión y promueve la migración celular y metástasis. En este
contexto, Caveolina-1 ha sido asociada a un conjunto de cambios morfológicos
esenciales que permiten la migración e invasión celular, entre estos, la remodelación del
citoesqueleto de actina y el recambio de complejos de adhesión celular. Muchos de
estos eventos dependen del tráfico endosomal, el cual a su vez es finamente regulado
por diversos factores y moléculas, como las GTPasas pequeñas de la familia Rab. Una
de estas proteínas es Rab5, la cual no sólo actúa como regulador maestro de la
endocitosis temprana, sino que además cumple un rol fundamental en la migración
celular. Trabajos recientes han descrito que la interacción entre Rab5 y Caveolina-1
influye en el tráfico endosomal, sin embargo el rol de esta interacción en la migración
celular no había sido investigado.
Entre los reguladores de Rab5, p85α (la subunidad reguladora de PI3K), se caracteriza
por inhibir a Rab5, ya que posee un dominio RabGAP que estimula su actividad
GTPasa. Literatura reciente ha descrito una interacción funcional entre p85α y
Caveolina-1, la cual a su vez depende de la fosforilación de Caveolina-1 en tirosina
(Y14). Lo anterior cobra relevancia, ya que Caveolina-1 ejerce muchos de sus efectos
en migración celular mediante la fosforilación en este residuo.
Por otro lado, trabajos recientes indican que Rab5 activa a Rac-1, a través del
reclutamiento de la GEF Tiam1, fomentando la formación de lamellipodios y la
polimerización de actina en sitios específicos de membrana. Además, trabajos de
nuestro laboratorio demostraron que Caverolina-1 promueve la activación de Rac-1
en células metastásicas. Todos estos eventos llevan a que la célula adquiera
cambios morfológicos que le permiten migrar. Por lo tanto, es importante investigar el
papel potencial que desempeñaría Caveolina-1 en la regulación de este circuito, así
como su relevancia en la migración celular.
En esta tesis, se investigó los mecanismos asociados al efecto promotor de
migración y metástasis de Caveolina-1, identificando un nuevo eje de
señalización “p85α/Rab5/Tiam1/Rac-1”. A su vez, se evaluó la relevancia de
este eje en un modelo preclínico, evaluando la metástasis in vivo en ratones
C57BL/6.
Los resultados obtenidos durante el desarrollo de esta tesis indican que Caveolina-1
promueve la migración, invasión y la activación de Rab5 en tres líneas celulares
metastásicas: HT-29(US) de cáncer de colon, B16-F10 de melanoma murino y MDAMB-
231 de cáncer de mama. En relación a esto último, la expresión de Caveolina-1
promueve un aumento en el tamaño de los endosomas tempranos, pero no debido a
la estabilización de Rab5 en éstos, sino que debido al secuestro de p85α en un
complejo proteico. Interesantemente, el efecto de p85α en la inhibición de Rab5 es
asociado a su actividad GAP, y no involucra la activación de PI3K.
Mediante ensayos de silenciamiento y reconstitución, se observó que la expresión y
actividad de Rab5 son fundamentales para que Caveolina-1 promueva migración e
invasión celular, así como también la activación de Rac-1. La activación de Rac-1 por
Caveolina-1 y Rab5 requiere de Tiam1, la cual es reclutada a endosomas tempranos
de manera dependiente de Caveolina-1. Finalmente, se demostró que Caveolina-1
requiere de Rab5, p85α y Rac-1 para promover invasión in vitro y metástasis in vivo
en ratones C57BL/6.
De esta manera, este trabajo de tesis aporta nueva evidencia acerca de los
mecanismos moleculares involucrados en la migración y metástasis inducida por
Caveolina-1, proponiendo un nuevo eje de señalización p85α/Rab5/Tiam1/Rac-1, el
cual involucra proteínas comúnmente alteradas en cáncer, y por lo tanto,
contribuyendo con la identificación de nuevos blancos potenciales terapéuticos<br>Cancer is a leading cause of disease in Chile and worldwide. During the development
and progression of this disease, normal cells convert to the cancerous state by acquiring
specific characteristics. Of these metastasis is widely considered particularly important
because it is this trait of cancer cells that is responsible for the large majority of deaths in
cancer patients. Caveolin-1 is a membrane protein with vastly differing roles in cancer
that range from functioning as a tumor suppressor to promoting tumor cell migration
invasion and metastasis. Concomittant with this unfavorable role in tumor progression,
expression of this protein frequently increases in advanced stages of cancer and is
viewed there as a marker for poor patient prognosis and survival. In this cellular context,
it is important to gain better insight to how Caveolin-1 promotes the acqusition of a more
invasive cellular phenotype.
In these migration-related events, endosomal trafficking reportedly plays a fundamental
role and Caveolin-1 is known to promote recycling of membrane components and the
activation of GTPases at the leading edge. A key membrane-associated protein involved
in such processes is Rab5, a small Rho GTPase that acts as a master regulator of early
endocytosis. Recently, evidence has been provided suggesting that p85α, the regulatory
subunit of PI3K, is a Rab5 GAP that controls Rab5 inactivation. Structurally, p85α has
two SH2 domains, involved in binding to tyrosine-phosphorylated proteins, often
including receptors and adaptor proteins. Caveolin-1, on the other hand, is known to
promote the migratory and invasive capacity of tumor cells via phosphorylation on
tyrosine-14. Furthermore, Rab5 activates Rac-1 and promotes lamellipodia formation,
through recruitment of the GEF Tiam1. Together, these events lead to changes that
permit cell migration. However, the precise mechanisms by which Caveolin-1
participates in the regulation of such processes and specifically whether Caveolin-1
enhanced migration and invasion in vitro involves a novel p85α / Rab5 / Tiam1 / Rac-1
signaling axis remained to be determined. Also, a model of B16F10 murine melanoma
metastasis in syngeneic C57BL / 6 mice was employed to evaluate the relevance of
Rab5, p85α and Rac-1 in promoting Caveolin-1-enhanced lung metastasis in vivo.
The results obtained during this thesis demonstrate that Caveolin-1 promoted migration,
invasion and activation of Rab5 in the metastatic cell lines HT-29 (US), B16-F10 and MDA-MB-231. Moreover, the expression of Caveolin-1 was shown to increase the
average size of early endosomes in B16F10 cells, consistent with a role in the activation
of Rab5. To study molecular mechanisms that explain these results, the expression of
p85α was shown to decrease Rab5 activation induced by Caveolin-1. Using inhibitors,
the activation of Rab5 by Caveolin-1 was shown to be independent of PI3K activity.
Moreover, expression of Rab5 was required for Caveolin-1 to promote cell migration and
invasion. Importantly, the activation of the small GTPase Rac-1 induced by Caveolin-1
required the presence of Rab5 to promote migration. Furthermore, evidence is provided
showing that recruitment of the Rac1 GEF Tiam1 to early endosomes was enhanced in
the Caveolin-1 positive cells. Finally, using short-hairpin knock-down technology Rab5,
p85α and Rac-1 were shown to be required for Caveolin-1 to promote migration and
invasion in vitro, as well as metastasis in vivo in C57BL / 6 mice.
In conclusion, the results of this thesis shed light on the molecular mechanisms involved
in Caveolin-1-enhanced migration, invasion and metastasis and propose the existence
of a novel p85α / Rab5 / Tiam1 / Rac-1 signaling axis downstream of Caveolin-1 that
contributes to deregulation in cáncer and potentially represents an interesting target for
treatments in cancer patients<br>Conicyt; Fondecyt; Anillo ACT 1111
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Chen, Hung-Chih. "Functions of Caveolin-1 and Caveolin-3 in muscular dystrophy." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/4739/.
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Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease caused by the absence of the sarcolemmal protein dystrophin. The skeletal muscles of DMD have disrupted dystrophin-glycoprotein complex (DGC) and impaired sarcolemma integrity. In this study, we show that clonally derived dystrophin-deficient myoblasts PD50A are differentiation impaired. Coculture with osteoblasts improves the differentiation efficiency of PD50A myoblasts. We also establish that supplementation of combinations of IGF-1/IGF-2, IGF-1/LIF and IGF- 2/LIF in cultured PD50A myoblasts ameliorates the differentiation impairment. We establish that there are elevated levels of Cav-3 and Cav-1 proteins in dystrophin-deficient myoblasts and mdx mouse embryos and that Cav-3 and Cav-1 form heterooligomers in adult skeletal muscles. We show that overexpression of Pax7 suppresses Cav-3 in dystrophin-deficient myoblasts. Using a genetic mouse model (mdx/cav3\(^{+/-}\)) embryo we further establish that immunohistochemistry staining of Cav-1 and Cav-3 coincides with the mouse heart development. The DGC of skeletal muscles plays a role in signal transduction and mechanical response. Here we show that AKT/mTOR and IGF-2/p57\(^{kip2}\) (but not ERK) signalling pathways are upregulated in dystrophin-deficient myoblasts and mouse embryos. Using atomic force microscope we show that Cav-1 helps maintain the stiffness of dystrophindeficient myotubes while Cav-3 help maintain that of dystrophin-deficient myoblasts. This study suggests that Cav-1 and Cav-3 have both compensatory and compromising roles in mdx.
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Chand, Sourabh. "Caveolin-1 in renal disease." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7824/.
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Renal disease is a major global public health issue that affects 10% of the general population with premature morbidity and mortality related to cardiovascular disease and infection. Interstitial fibrosis is a common hallmark of progressive kidney dysfunction. There remains a stubborn discrepancy in identifying which patients suffer adverse events because of their disease or resulting treatment. Investigation in patient genome variation may explain this discrepancy. Caveolin-1 is the essential structural protein for caveolae that are ubiquitously distributed in fibroblasts, endothelial and epithelial cells. They act as molecular chaperones for transcellular signaling such as degradation of the activated TGFβ-1 receptor. In this thesis, caveolin-1 single nucleotide polymorphism rs4730751 CC genotype is shown to be associated with a better outcome in renal patients for arterial stiffness, and reduced mortality from cardiovascular disease, infection, malignancy in ANCA associated vasculitis. By inducing renal models of fibrosis in caveolin-1 knockout mice, deletion of caveolin-1 leads to increased fibrosis. In conclusion, this polymorphism could be used as a marker of disease risk either in isolation or as part of a clinical risk score to counsel patients on the likely prognosis of their condition. Manipulation of caveolin-1 expression may be a therapeutic strategy in reducing renal fibrosis.
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Day, Elizabeth Helen. "Caveolin-1 involvement in prostate cancer." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555622.
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Caveolin-l (Cav-l) is an important component of plasma membrane caveolae and orchestrates cell signalling in the regulation of proliferation and apoptosis. Unlike many cancers, prostate cancer cells demonstrate elevated levels of cav-l expression which has been linked to Gleason scoring and aggression. A loss of cav-l, with siRNA, in an advanced prostate cancer cell line (PC-3) caused a significant increase in PI positive cells combined with reduced proliferation. Both Akt and ERK signalling showed altered functioning under these conditions and may orchestrate these effects. Such an effect was not observed in benign prostate epithelial cells (PrEC) implicating cav-l as an important protein in the control of cellular viability in malignant but not benign prostate cells. To investigate possible causes of elevated cav-l expression in malignant prostate cells, miRNAs predicted to target the cav-l 3 'UTR were analyzed in both benign and malignant prostate cell lines. In PC-3 cells both miR-30a-3p and miR-203 expression was significantly reduced and miR-I06a and miR-93 increased when compared to benign PrEC cells. Over-expression ofmiR-30a-3p, miR-93, miR-203 but not miR-I06a in PC-3 cells caused significant reduction in cav-l protein expression. This points to a role for these miRNAs in the regulation of cav-l in prostate cancer cells. Interestingly cav-l knockdown in PC-3 cells increased expression of miR-124 and miR-124 up-regulation reduced cav-l protein and mRNA expression, suggesting that the two molecules are linked. Androgen stimulation of LNCaP cells implicate an androgen response element may be involved in the transcriptional regulation of miR-124. In summary, these results suggest changes in miRNA expression with oncogenic transformation may result in over- expression of cav-l which promotes cell survival and proliferation, hence promoting cancer progression. Whether the effects of these miRNAs on cav-l are direct and occur at an early or late stage in the development of the malignancy needs to be determined but it is clear that miRNA expression profiles are becoming increasingly important potential biomarkers in the early diagnosis of many cancers.
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Honeyman, Brian. "The role of cavin-1 and caveolin-1 in breast cancer." Thesis, Boston University, 2013. https://hdl.handle.net/2144/11016.
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Thesis (Ph.D.)--Boston University<br>Caveolae are small invaginations on the plasma membrane of a variety of tissue types and are formed by the caveolin (1 and 2) and cavin (1-3) proteins families. Physiological roles implicated for caveolae include vesicular trafficking, signal transduction, cell adhesion, and mechanosensation. Over 20 years ago, caveolin-1, a small integral membrane protein, was observed to be the major tyrosine phosphorylation target in cells transformed by the Src oncogene. Subsequently its expression has been widely studied in a variety of cancers where it has been shown, confusingly, to have both oncogenic and tumor suppressive capabilities in vitro and in vivo. However, the role of cavin family of proteins in caveolae has been only recently described and minimally studied in this context. Thus to understand possible roles of caveolae in cancer biology, I investigated the roles of caveolin-1 and cavin-1 in breast cancer cell line behavior in vitro. Caveolin-1, and cavin-1 and -2 are expressed to a significant degree in aggressive estrogen receptor (ER) negative cell lines but not in ER positive cell lines. Forced ER expression, however, does not abrogate the expression of caveolar proteins. Knock-down of caveolin-1 and/or cavin-1 in the ER negative cell lines results in increased proliferation, migration, matrigel invasion, and matrix metalloproteinase activity. Consistent with this result, a highly metastatic variant of one of these cell lines shows a decrease in caveolar protein expression consistent with a role for caveolae in aggressive cell behavior. Since cell migration and matrix invasion require cytoskeletal rearrangements, I measured the activity of the cytoskeletal-associated Rho family GTPase, Cdc42, after caveolin-1and cavin-1 knockdown, and observed it to be enhanced, whereas treatment with the Cdc42 inhibitor reduced the migratory capability of the cells to wild type levels. Taken together, my data supports a tumor suppressive role for the caveolae proteins caveolin-1 and cavin-1 in ER negative breast cancer through the regulation of Cdc42. Data also suggest that both caveolin-1 and cavin-1 are required for proper caveolae formation and function. Furthermore, it stresses that cavin-1 needs to be examined in the context of caveolae and cancer.
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Mattsson, Charlotte L. "Role of caveolin-1 in brown adipose tissue." Doctoral thesis, Stockholm : The Wenner-Gren Institute, Stockholm University, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-37125.
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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.<br>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript. Härtill 4 uppsatser.
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Baggett, Ariele. "The Effects of Caveolin-1 on Mitochondrial Dynamics." Master's thesis, Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/495857.
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Biomedical Sciences<br>M.S.<br>Cardiovascular disease (CVD) is the leading global cause of death. Coronary Artery Disease (CAD) is a grouping of the most common cardiovascular diseases and is the current leading cause of death in developed countries. Treatments for CAD include pharmaceuticals as well as surgical interventions such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting. However, these treatments do not completely remove the risk of adverse outcomes. Endothelial dysfunction is the underlying cause of CAD and is initiated by the chronic inflammation of the vasculature due to increased oxidative stress and production of reactive oxygen species (ROS). Previous studies have shown that the deletion of caveolin, a signaling molecules abundant within endothelial cells, can enhance inflammatory responses and lead to increased oxidative stress and ROS production. Mitochondrial ROS created from dysfunctional mitochondrial dynamics has also been shown to contribute to the inflammation of the endothelium. We hypothesize that due to the link between caveolin and endothelial dysfunction, and the link between mitochondria and endothelial dysfunction, caveolin has an important function in mitochondrial dynamics and that the loss of caveolin increases the mitochondrial fission via a Drp1-dependent pathway. Our data shows that adenoviral silencing of caveolin-1 in rat aortic endothelial cells increases Drp1 expression but does not significantly alter mitochondrial morphology. Overexpression of caveolin-1 via an adenoviral construct in these cells produces a decrease in Drp1 expression without altering mitochondrial morphology. This data provides insight into the pathophysiology of CAD and could provide us with new therapeutic targets in the future.<br>Temple University--Theses
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Basu, Roy Upal Kunal. "Regulation and Function of Caveolin-1 in Colorectal Carcinogenesis." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/194031.
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Colon cancer is the second leading cause of cancer deaths in the United States of America. It is caused by the accumulation of mutations in tumor suppressors and oncogenes. The APC tumor suppressor is mutated in most diagnosed cases of colorectal cancer. Mutations in the K-RAS oncogene occur at later stages of colon cancer progression. In the present study, the transcriptional regulation of a novel target of these two genes, caveolin-1, was studied. Caveolin-1 is transcriptionally regulated by the APC tumor suppressor gene, via induction of its inducer, FOXO1 and the suppression of its transcriptional repressor, C-MYC. An activated K-RAS oncogene induces caveolin-1 transcription via activation of the P-I3 Kinase pathway. In addition to transcriptional regulation of caveolin-1, the influence of caveolin-1 expression on cellular phenotypes like signal transduction and polyamine uptake were assessed. The present studies demonstrate that caveolin-1 expression affects basal levels of AKT and ERK signaling, with an increased signaling associated with caveolin-1 expression in these colon tumor-derived cells. In addition, caveolin-1 expression positively affects signaling in response to an inflammatory stimulus like TPA. Interestingly, caveolin-1 expression leads to a decrease in the uptake of pro-tumorigenic molecules like polyamines, in the colon cell lines tested. Taken together, the data from this study suggests that caveolin-1 is transcriptionally regulated by the APC and the K-RAS gene at different stages of colorectal tumorigenesis and this in turn, leads to different phenotypes influenced by caveolin-1 expression.
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Wong, Wing-sum Winnie, and 王詠心. "Role of caveolin-1 in multidurg resistance in hepatocellularcarcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46632001.
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Tse, Yuk-ting Edith, and 謝玉婷. "Expression and function of caveolin-1 in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45984554.
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Wong, Kevin L. "Caveolae and Caveolin-1 are important for Vitamin D signalling." Thesis, Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/37086.
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The most active form of Vitamin D, 1alpha,25(OH)2D3, modulates cells via receptor mediated mechanisms. While studies have elucidated the pathway via the classical nuclear Vitamin D Receptor (VDR), little is known about the membrane-associated Vitamin D Receptor (ERp60). Caveolae and its characteristic protein Caveolin-1 have been involved in many signaling pathways due to its specific structure and physical configuration. Other studies have shown that many components of the Vitamin D pathway have been found in caveolae. This study hypothesizes that caveolae and Caveolin-1 are important for the effects of 1,25 Vitamin D signaling via ERp60. Research up to date have shown that in rat and mouse growth zone chondrocytes, cells deprived of intact caveolae either through disruption through beta-Cyclodextrin or genetic knockout do not exhibit the characteristic responses to Vitamin D through ERp60 when compared to chondrocytes with functional caveolae. Studies using immunofluorescence co-localization and caveolae fractionation have shown that ERp60 is localized in the caveolae domains. Cellular fractionation was also performed to examine the localization of the ERp60 receptor in lipid rafts and caveolae. Histology and transmission electron microscopy were also used to examine the physiological importance of caveolae and Caveolin-1 in growth plate morphology and cellular characteristics.
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Dong, Shuai. "The Na/K-ATPase/Caveolin-1 Interaction Regulates Cell Growth." University of Toledo Health Science Campus / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=mco1305223012.
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Simões, Francisco Manuel de Carvalho. "Cavin 1 and cavin 2 roles on adipocyte cell function." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6568.
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Dissertação para obtenção do Grau de Mestre em
Genética Molecular e Biomedicina<br>Caveolae are a specialized type of lipid rafts found in numerous cell types. These structures have been implicated as playing a role in a variety of metabolic regulation processes and are typically characterized by their association with the caveolin and cavin protein families. Caveolae are particularly enriched in adipocytes and here we analyze the effects of cavin 1 and cavin 2 knockdown in adipocyte functioning. We obtained several multiclonal mouse 3T3-L1 cell lines with reduced expression of cavin 1 or cavin 2 by small interfering RNA approach using lentiviral vectors. We observed that cavin 1, but not cavin 2, depletion affects adipocyte differentiation and that both knockdown cells showed a reduced number of caveolae. We also verified that cavin 1 and cavin 2 expressions levels are dependent on each other and that both knockdowns caused a specific decrease in glucose transporter 4 (GLUT4) protein levels. Besides the lower protein levels of GLUT4, both knockdown cells also fail to translocate the transporter to the plasma membrane in response to insulin, although the insulin signaling pathway was not affected. As a consequence, knockdown cells show reduced insulin-stimulated glucose transport. Our results underscore the importance of cavin 1 and cavin 2 in glucose uptake and adipocyte metabolism.
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Hernandez, Mark J. "Caveolin-1 A scaffold for microcompartmental organization of membrane-associated glycolysis." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/6007.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.
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Wong, Yuen-sze Sivia, and 王苑斯. "Role of hypoxia-induced upregulation of caveolin-1 in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46944333.
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Harris, Tanoya L. "Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism." University of Toledo Health Science Campus / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028.
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Gutteridge, Robert. "Caveolin-1 is a modulator of clonogenicity in renal cell carcinoma." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/91911/.
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Renal cell carcinoma (RCC) represents a group of aggressive tumours of the kidney. These tumours have a high propensity for metastasis and are extremely treatment refractive after disease relapse. Therefore, the identification of new therapeutic targets is of great importance. One such potential target for therapy are cancer stem cells (CSCs). CSCs are populations of cells imbued with a stem cell-like phenotype capable of driving tumour formation, metastasis and chemoresistance. As such, reliable methods for the identification of CSC populations and defining targets important to their functionality, in hopes of developing more potent therapeutics is of great importance. Previous work has found CAV1 to play a significant role in the malignant progression of RCC and also in the maintenance of adult stem cell populations. As such, this work aimed to understand if common markers of CSC phenotype in combination with CAV1 can act indicators of poor prognostic outcome in clinically confined RCC. Furthermore, this work sought to identify CSC populations from RCC cell lines using a panel of surface markers common to embryonic, mesenchymal and cancer stem cells. Then, understand if CAV1 is responsible for driving the CSC phenotype in these CSC populations by regulating one of the major characteristics of CSC biology, self-renewal. Co-expression of CD44 and CAV1 in RCC tumours indicated greatly reduced disease free survival in clinically confined RCC. Additionally, CD44/CAV1 was found to be the most significant covariate in predicting disease recurrence. In vitro analysis, using a panel of CSC related markers, was unable of identifying a putative CSC population. However, CAV1 expression in the VHL positive CAKI-1 cell line was important for the maintenance of clonogenicity. Incubation of CAV1 deficient CAKI-1 cells under hypoxic conditions was able to restore lost clonogenicity. Further iv work revealed that CAV1 maintains clonogenicity in CAKI-1 through activation of STAT3 and -catenin. This suggests an important role for CAV1 in the maintenance of clonogenicity through STAT3 activation in VHL competent RCC.
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Mir, Kiran D. "The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1." Texas A&M University, 2003. http://hdl.handle.net/1969.1/3976.
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Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants
worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first
described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular
calcium through multiple mechanisms that promote abnormal ion transport and
subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially
interact with model membranes mimicking caveolae in lipid composition and radius of
curvature. Our laboratory has recently reported the colocalization and
coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae.
Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic
peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one
terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed
in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least
one terminus were capable of binding the NSP4 bait. An in vitro binding assay
confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1
residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates
NSP4 signaling and/or intracellular trafficking.
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Chikani, Gentle P. "LEPTIN RECEPTORS IN CAVEOLAE: REGULATION OF LIPOLYSIS IN 3T3-L1 ADIPOCYTES." UKnowledge, 2004. http://uknowledge.uky.edu/gradschool_theses/382.
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The present study has tested the hypothesis that leptin receptors are localized in caveolae and that caveolae are involved in the leptin-induced stimulation of lipolysis in 3T3-L1 adipocytes. Leptin, a peptide hormone, is secreted primarily by adipocytes and has been postulated to regulate food intake and energy expenditure via hypothalamic-mediated effects. Exposure to leptin increases the lipolytic activity in 3T3-L1 adipocytes. We isolated caveolae from 3T3-L1 adipocytes using a detergent free sucrose gradient centrifugation method. Leptin receptors were localized in the same gradient fraction as caveolin-1. Confocal microscopic studies demonstrated the colocalization of leptin receptors with caveolin-1 in the plasma membrane, indicating distribution of leptin receptors in the caveolae. We disrupted caveolae by treating cells with methyl--cyclodextrin and found that leptin induced lipolytic activity was reduced after caveolae disruption, indicating an important role of caveolae in the signaling mechanism of leptin.
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Gaudreault, Sophie B. "Caveolin-1 in synaptic signalling and neuronal plasticity : implications for Alzheimer's disease." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85069.
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Increasing evidence indicates that cholesterol plays a central role in the pathophysiology of Alzheimer's disease (AD). Caveolin is a cholesterol-binding membrane protein involved in cellular cholesterol homeostasis and signalling. The global working hypothesis of the present project was to investigate on a possible role for caveolin in the pathophysiological events underlying AD.<br>More specifically, a first study was conducted to evaluate potential changes in the amount of brain caveolin in autopsy-confirmed AD and age-matched control subjects. Using rodents, the effect of age and apolipoprotein E (ApoE) deficiency on caveolin levels was also examined. In a second study, caveolin levels were investigated in an in vivo mouse paradigm of neuronal response to damage, the entorhinal cortex lesion. Moreover, the effect of caveolin on remodelling processes following neuronal lesion was examined in an in vitro model of reactive neuronal plasticity. Given the important role of caveolin in signalling, a third study was conducted to assess the potential functional interaction of caveolin with phospholipase A2 (PLA2), an enzyme implicated in synaptic plasticity and neurotransmission.<br>The results indicated an up-regulation of caveolin in the brain of AD patients, aged mice and apoE-deficient mice, that was potentially linked to alterations of cholesterol distribution in the plasma membrane of brain cells. Consistent with this idea, caveolin levels were increased during the neuronal membrane remodelling period following entorhinal cortex lesion in mice. In vitro, caveolin overexpression in neuron-like cells undergoing post-injury reactive plasticity caused significant biochemical and morphological alterations. Finally, a co-localisation and a functional interaction of caveolin with PLA2 was observed in hippocampal neurons. This interaction dramatically inhibited PLA2 enzymatic activity and consequently interfered with synaptic AMPA glutamate receptor binding properties.<br>These findings show that caveolin plays an important role in reactive neuronal remodelling process associated with age or damage. Its increased expression in brain cells, possibly ascribed to alterations in membrane cholesterol homeostasis, could negatively interfere with PLA2 and AMPA glutamate receptor functions during synaptic plasticity-related events. These findings are consistent with the hypothesis that part of the pathophysiology underlying AD may be related to cholesterol homeostasis, and indicate that caveolin may be an important and previously unrecognised player in this process.
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Links, Philip Harold. "The molecular cloning, cellular expression, and functional study of human caveolin-1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/MQ57132.pdf.
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Petriello, Michael C. "ROLE OF CAVEOLIN-1 AND NRF2 IN NUTRITIONAL MODULATION OF PCB TOXICITY." UKnowledge, 2015. http://uknowledge.uky.edu/toxicology_etds/11.
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Cardiovascular disease is the leading cause of mortality in Western societies and is linked to multiple modifiable risk factors including lifestyle choices. Emerging evidence implicates exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), as a risk factor for the development or progression of cardiovascular disease. To reduce disease risks, it is critical to identify sensible means of biomedically reducing the toxicity of persistent organic pollutants and related environmental stressors.
First, we tested a hypothesis that endothelial cell inflammation and subsequent cardiovascular toxicity initiated by coplanar PCBs is modulated by the crosstalk between caveolae and Nuclear factor (erythroid-derived 2)-like 2(Nrf2) related proteins. Caveolae are lipid-enriched organelles found abundantly in endothelial cells and are important mediators of endocytosis and signal transduction. Caveolin-1 (Cav-1), the major structural protein of caveolae, is known to bind and concentrate multiple proteins related to cardiovascular disease and PCB toxicity. Downregulation of Cav-1 protects against PCB-induced vascular toxicity, but possible mechanisms of this defense remain elusive. Studies using endothelial cells isolated from mice deficient in Cav-1 as well as in vitro silencing assays demonstrated that loss of Cav-1 increases available antioxidant enzymes by upregulating the antioxidant master controller Nrf2.
Nutritional interventions focused on diets high in bioactive food components, such as polyphenols or certain fatty acids, may prove to be effective at decreasing environmental pollutant induced diseases. To test the hypothesis that dietary intervention can sensitize Nrf2 and/or caveolae signaling pathways, leading to a more effective anti-inflammatory defense against PCB insults, mice were fed a green tea polyphenol enriched diet and challenged with coplanar PCB 126. Mice fed an enriched diet and exposed to PCBs exhibited lower levels of oxidative stress and higher levels of multiple Nrf2 target antioxidant enzymes. Also, in separate in vitro studies, pretreatment of endothelial cells with the endogenously formed nutrient metabolite, nitro-linoleic acid, altered caveolae and Nrf2 related proteins, resulting in a modified response to PCB exposure. Together, these data support the paradigm that nutritional modulation may be a sensible means of reducing disease risks associated with exposure to environmental pollutants.
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Pancotti, Fabia <1984>. "Role of caveolin-1 in the proliferation of solid tumours in vitro." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3313/.
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Caveolin-1 (Cav-1), the essential structural constituent of caveolae, which are flask-shaped invaginations of the plasma membrane, has been found to play a key role in the modulation of cell proliferation and cancer development. It seems to act as an oncosuppressor or a promoter of growth, depending on the histotype, stage and grade of each tumour.
The aim of this study was to analyze the effects of Caveolin-1 gene silencing on the proliferation of human lung cancer and osteosarcoma in vitro.
Our data show that Cav-1 silencing blocks the growth in both metastatic lung cancer cell lines analyzed, suggesting a proliferation promoting action of the protein in these cells. A marked decrease of phospho-Akt, phospho-ERK, STAT3, cyclin D1, CDK4 and consequently of phospho-Rb expression was evident in the cells treated with Cav-1 siRNA.
With regards to osteosarcoma, we demonstrated that the suppression of Cav-1 results in the blocking of MG-63 and in the slowing down of HOS proliferation, suggesting a role for Cav-1 as a promoter of tumour growth in these cell lines. A marked decrease of phospho-Akt, cyclin E, CDK2 and phospho-Rb and an increase of p21 expression levels were evident in the cells treated with Cav-1 siRNA.
Our results suggest two new cell cycle inhibiting pathways, mediated by Cav-1 knock-down, and provide new insights into the molecular mechanisms underlying the tumour-promoting role of Cav-1 in lung cancer and osteosarcoma.
In this work we also investigated the role of estrogens in lung cancer and the functional cross-talk between Cav-1 and estrogens/estrogen receptors in it.
Our results show that 17β-estradiol induces proliferation either in RAL or in SCLC-R1 cells and that both cell lines are sensitive to 4-OHT antiproliferative effect.
The sensitivity to estrogen stimulation seems to be gender- and/or histological type-independent in metastatic lung cancer in vitro.
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Yu, Lingli. "Nerve Growth Factor Signaling from Membrane Microdomain to Nucleus : Differential Regulation by Caveolins." Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2012. http://tel.archives-ouvertes.fr/tel-00796393.
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At the plasma membrane, both NGF receptors have been shown to localized to lipid rafts, specific subdomains that are enriched in cholesterol, sphingolipids and the presence of caveolin proteins (Cav1 and/or Cav2). The focus of this work is on this membrane microenvironment mediated modulation of NGF signaling which via two receptors: p75NTR and TrkA. In the present work we found that overexpression of Cav-1 in mouse dorsal root ganglia neurons significantly impacted neurite extension. Similarly, overexpression of Cav-1 in PC12 cells strongly inhibits their ability to grow neurites in response to NGF. It inhibits NGF signaling without, impairing transient MAPK pathway activation. Rather, it does so by sequestering NGF receptors in lipid rafts, which correlates with the cell surface localization of downstream effectors, and phosphorylated-Rsk2, resulting in the prevention of the phosphorylation of CREB. By contrast, overexpression of Cav-2 potentiates NGF induced differentiation, which is accompanied by sustained activation of downstream effectors, and standard internalization of the receptors. This differential effect could be due to the different localization of Caveolins, that modifies the microenvironment, thereby affecting NGF signaling. Furthermore, PC12 cells expressing the non-phosphorylatable Cav-1 mutant (S80V), neither TrkA trafficking or CREB phosphorylation are inhibited and the response resembles that observed in Cav-2 expressing PC12 cells. These studies underline the interplay between caveolins and NGF signalling, offering insight into the potential impact of Caveolin-1 and mutations thereof in certain cancers where NGF signaling is involved.
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Steiner, Isabel. "Epigenetische und angiogenetische Veränderungen beim klinisch lokalisierten Prostatakarzinom." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16652.
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Im Fokus der Dissertation stand die Evaluierung molekularer Möglichkeiten zur Findung geeigneter Biomarker für das Prostatakarzinom (PCa). Zunächst wurde die epigenetische Promotormethylierung der Gene GSTP1, RARbeta2, APC und PITX2 im Hinblick eines möglichen Methylierungsfeldeffektes untersucht. Die RARβ2-Promotormethylierung konnte als potentieller Marker zur Tumorvorhersage im histologisch normal erscheinenden Gewebe bestimmt werden. GSTP1 zeigte eine äußerst spezifische Tumormethylierung, was dessen diagnostisches Potenzial unterstreicht. Die Methylierung korrelierte zudem mit pathologischen Parametern. Ein Screening im Array-Format identifizierte weitere Gene, deren Promotormethylierung diagnostisch interessant sein könnten. Des Weiteren wurden Genexpressionsanalysen angiogenetischer und Endothel-assoziierter Faktoren durchgeführt, deren diagnostische und prognostische Aussagekraft für das PCa ermittelt wurden. Dabei konnte für Caveolin-1 (CAV1) eine signifikante Herunterregulierung der mRNA-Menge im Tumor gezeigt werden. Die Bisulfit-Sequenzierung von sieben CpG-Dinukleotiden im CAV1-Promotor ergab zwischen Tumor- und tumorangrenzendem Gewebe differenzielle Methylierungsmuster, die zu einer verminderten CAV1-Transkription führen könnten. Zudem führte eine 5-Aza-2-desoxycytidin-Behandlung der CAV1-defizienten LNCaP-Zelllinie zur Reexpression. Weiterhin assoziierte CAV1 invers mit pathologischen Parametern und zeigte prognostische und diagnostische Relevanz. Immunhistologisch wurde CAV1 z.T. deutlich verringert in Endothelzellen und Fibroblasten des Tumors nachgewiesen. Ko-Kultivierungsversuche von HUVEC mit konditionierten Zellmedien ergaben z.T. signifikant reduzierte CAV1-Mengen in HUVEC. Die Ergebnisse zeigen, dass epigenetische Veränderungen wertvolle Informationen zur Diagnostik, Prognostik und Progression des PCa liefern. Zudem konnte CAV1 als potentieller Marker des PCa identifiziert werden.<br>The focus of the thesis was to evaluate molecular possibilities to find suitable biomarkers for prostate cancer (PCa). First, the epigenetic promoter methylation patterns of several genes (GSTP1, RARbeta2, APC and PITX2, respectively) were investigated for a possible methylation field effect. The RARbeta2 promoter methylation could be determined as a possible marker for tumor prediction in histologically normal-appearing tissue. GSTP1 showed a highly specific tumor methylation, underlining its diagnostic potential. The methylation also correlated with pathological parameters. Screening in an array format identified other genes whose promoter hypermethylation could be diagnostically interesting. Furthermore, gene expression analysis of angiogenic and endothelial-associated factors was performed to determine their diagnostic and prognostic potentials for PCa. For CAV1, a significant down-regulation of its mRNA level could be determined in the tumor. Bisulfite sequencing of seven CpG dinucleotides in the CAV1 promoter showed different methylation patterns between tumor and tumor adjacent tissue that could cause a reduced transcription. Furthermore, treatment of the CAV1-deficient LNCaP cell line with 5-aza-2-deoxycytidine led to CAV1 reexpression. Additionally, CAV1 inversely associated with pathological parameters and showed prognostic and diagnostic relevance. Immunohistochemical analysis clearly demonstrated decreases in CAV1 protein expression in endothelial cells and fibroblasts of the tumor. Conditioned media of cultivated tumor cells partly induced downregulation of the CAV1 protein level in HUVEC. The results show that epigenetic and angiogenic processes play crucial roles and provide valuable information for diagnosis, prognosis and progression of PCa. Moreover, CAV1 could be identified as a potential marker of prostate cancer.
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Moreno, Càceres Joaquim. "The role of Caveolin-1 in the TGF-β-induced signalling in hepatocytes". Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396239.
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The transforming growth factor-beta (TGF-β) is a cytokine involved in many physiological and pathological processes. Its dual role has been described in the liver, where it can either induce pro- or anti-apoptotic signals. The survival signals are in great extent mediated by the epidermal growth factor (EGFR). Caveolin-1 (CAV1) is a structural protein of the plasma membrane involved in the regulation of different signalling processes and receptor trafficking, as the TGF-β ones (TβRs). Traditionally, CAV1 has been associated with downregulation of the TGF-β signalling, given its presence in SMAD7 and SMURF2 positive vesicles. The great variety of functions associated with CAV1 has generated great controversy, as it acts differently depending on cell type. Regarding its role in the liver, CAV1 has been related to the SAMD-independent signals mediated by TGF-β and as an oncogene in hepatocellular carcinoma (HCC, its expression levels are increased in less differentiated tumours).
Given the previously mentioned role of CAV1 in TβRs traffic and in the modulation of distinct signalling pathways, when we started this work, we hypothesized that the levels of CAV1 could be modulating the response to TGF-β in hepatocytes and in HCC cells, regulating the balance among pro- and anti-apoptotic signals induced by TGF-β in hepatocytes. Given this hypothesis, we proposed as an objective to study the role of CAV1 in the TGF-β-induced signalling in hepatocytes and in HCC cells. We decided to use an experimental cell model based on immortalized neonatal hepatocytes from CAV1-deficient mice (Cav1-/-) and we analyzed its response to TGF-β. We also used HCC cells in which CAV1 expression had been silenced or over-expressed, in regard to its basal expression levels.
We observed that the lack of CAV1 both in foetal hepatocytes and in HCC cells impairs TGF-β-mediated EGFR transactivation, and in this way, cells are more sensitive to TGF-β pro-apoptotic effects. This is due to the fact that CAV1 is required for the proper activation of the metalloprotease TACE/ADAM17 by TGF-β, being responsible for the shedding of the EGFR ligands present in the plasma membrane as pro-ligands. Another important conclusion that we got was that the localization of TACE/ADAM17 in lipid raft domains is crucial for its activation, and this also requires CAV1.
In HCC cells, CAV1 expression levels also determine the response to TGF-β in terms of death induction, but not in regard to cell cycle arrest. However, the final balance results in higher inhibition of the clonal growth by TGF-β when CAV1 expression is low. Another important result is that the induction of the death mediators NOX4, BMF and BIM by TGF-β is diminished when CAV1 expression levels in HCC cells are high.
Finally, we have observed that CAV1 is required by the activation of Src and the NADPH oxidase-NOX1, both necessary for the activation of TACE/ADAM17 by TGF-β, being this the mechanism that explains the deficient EGFR transactivation observed both in hepatocytes and in HCC cells when CAV1 expression is low.
To sum up this work, we can say that CAV1 levels condition the TGF-β-mediated anti-apoptotic response through the EGFR pathway. In HCC cells, this might be translated into a switch in the role of TGF-β: from anti- to pro-tumourigenic.<br>El factor de crecimiento transformante-beta (TGF-β) es una citocina involucrada en distintos procesos fisiológicos y patológicos. En hígado se ha descrito su papel dual; ya que puede inducir tanto señales pro- como anti-apoptóticas. Las señales de supervivencia son mediadas principalmente por la vía del receptor del factor de crecimiento epidérmico (EGFR). Por otro lado, caveolina-1 (CAV1) es una proteína estructural de la membrana plasmática asociada a la regulación de distintos procesos como señalización celular o tráfico de receptores, como los del TGF-β (TβRs). Tradicionalmente, CAV1 ha sido asociada con la regulación a la baja de la señalización mediada por TGF-β, dada su presencia en vesículas positivas para SMAD7 y SMURF2. En relación a su papel en el hígado, CAV1 se ha relacionado con las señales SMAD-independientes mediadas por TGF-β y como un oncogén en hepatocarcinogénesis (sus niveles de expresión se incrementan en tumores menos diferenciados y con mayor capacidad metastásica).
Cuando empezamos este trabajo hipotetizamos que los niveles de CAV1 podían estar modulando la respuesta a TGF-β en hepatocitos y en células de carcinoma hepatocelular (HCC), condicionando el equilibrio entre señales pro- y anti-apoptóticas inducidas por TGF-β en hepatocitos. Nos planteamos como objetivo estudiar el papel de CAV1 en la señalización inducida por TGF-β en hepatocitos y en células tumorales de hígado. Decidimos utilizar un modelo de hepatocitos procedentes de ratones deficientes en CAV1 (Cav1-/-) y analizar su respuesta al TGF-β. También usamos células de HCC en las cuales se había silenciado o sobre-expresado CAV1, en función de su expresión basal.
Como resultado obtuvimos que la falta de CAV1 tanto en hepatocitos como en células tumorales de hígado impide de transactivación del EGFR por parte del TGF-β, y de esta manera las células son más sensibles a los efectos pro-apoptóticos mediados por el TGF-β. Esto es debido a que CAV1 se requiere para la correcta activación de la metaloproteasa TACE/ADAM17 por parte de TGF-β. Otra conclusión importante a la que llegamos es que la correcta distribución de TACE/ADAM17 en dominios lipídicos de membrana es importante para su activación, y que para ello requiere CAV1.
En células de HCC, los niveles de CAV1 también determinan la respuesta al TGF-β en términos de inducción de muerte, pero no en relación a sus funciones citostáticas (mediando parada de ciclo celular). Otra conclusión importante a la que hemos llegado es que la inducción de los mediadores de muerte NOX4, BMF y BIM por parte de TGF-β se ve disminuida cuando los niveles de expresión de CAV1 en células tumorales de hígado son altos.
Finalmente, hemos observado que CAV1 se requiere para la activación de Src y de la NADPH oxidasa NOX1, ambos necesarios para la activación de TACE/ADAM17 por parte de TGF-β, siendo este el mecanismo de la deficiente transactivación del EGFR.
Como conclusión final de este trabajo, podemos decir que los niveles de CAV1 condicionan la respuesta anti-apoptótica inducida por TGF-β a través del EGFR. En células de HCC, esto se traduce en un cambio en el papel del TGF-β: de anti- a pro-tumorigénico.
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Li, Qiong Medical Sciences Faculty of Medicine UNSW. "Lipid analysis of wild type and caveolin-1 knock out mouse embryonic fibroblasts." Awarded by:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/41552.
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The aim of this project is to characterise the levels of cholesterol and phospholipid in cell homogenates, plasma membranes and isolated membrane domains from wildtype (WT) and caveolin-l knock-out (Cav-1-/ -) mouse embryonic fibroblasts (MEFs). Quantitative lipid analysis was developed for cholesterol by high-performance liquid chromatography (HPLC) and for glycerophospholipids (GPL) and sphingomyelin (SM) by electrospray ionisation mass spectrometry (ESI-MS). In the cell homogenates, by comparing WT to Cav-l-/- MEFs, it was found that the total cholesterol as well as the proportions of the main GPLs such as Phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) was similar in both cell types. However, Cav-l-/- MEFs have higher levels of cholesterol esters than WT MEFs in whole cell homogenate. Furthermore, the proportion of SM is higher in Cav-l-/- than WT MEFs. These data suggest that Cav-l may control the balance between free and esterified cholesterol and is involved in SM metabolism. A small increase in SM level in Cav-l-/- compared to WT plasma membrane was observed but this increase was not significant. Similarly, cholesterol levels in the plasma membrane are comparable between the two cell types. In both cell types, the levels of SM and phosphatidylglycerol (PG) are higher in the plasma membranes than in cell homogenates. In the WT cells, PE levels are higher in the plasma membrane than in the cell homogenates. While in the Cav-1-/- cells, the level of free cholesterol and PC are higher in the plasma membrane compared to the cell homogenate. PCs are the predominant lipids in both cell types. Cav-1-l - cells have more saturated fatty acyl chains in their PC species and shorter carbon chains compared to WT MEFs and this trend was found in both cell homogenate and plasma membranes. In PEs and SMs, Cav-1-/ - cells also have higher levels of saturated PEs and saturated amide-linkage SM than in WT cells, respectively. The results indicate that Cav-1 may also play a role in fatty acids metabolism. In summary, the data from this work indicate that Cav-l expression affects lipid composition in MEFs including the relative distribution of free and esterified cholesterol, the levels of SM relative to other phospholipid subclasses and the incorporation of fatty acids into phospholipids.
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Hausman, Natasha L. "The influence of caveolin-1 and ageing on resistance artery structure and function." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514440.
Full textAbstract:
Ageing is a risk factor for cardiovascular disease (CVD) and involves changes in the function and structure of resistance arteries. Membrane invaginations called caveolae are involved in cell signalling regulation, and can influence the active and passive properties of resistance arteries. Changes in caveolae and their structural protein caveolin-l have been implicated in CVD, therefore the role of caveolin-l in vascular ageing was investigated. Isolated resistance arteries from 3 and 12 month old wild-type (WT) and caveolin-l knock-out (KO) mice were examined. Mesenteric and femoral arteries mounted on a pressure myograph were exposed to a high potassium solution (KPSS), noradrenaline (NA), acetylcholine (ACh) and sodium nitroprusside. Concentration-response curves to NA and ACh were also performed in the presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine. Pressure steps in calcium-free solution allowed investigation of passive vessel properties. Mesenteric arteries from 12 month old WT mice exhibited reduced constriction to NA and KPSS, increased nitric oxide (NO) availability and outward hypertrophic remodelling when compared to those from 3 month old WT mice. Similar changes were observed in arteries from 3 month old caveolin- 1 KO mice compared to age-matched WT controls. There was no age-related change in the response of vessels from caveolin-l KO mice to NA, and no significant role for NO in mesenteric arteries from 12 month old caveolin-l KO mice. However, femoral arteries from 12 month old caveolin-l KO mice dilated more to ACh than WT controls, in line with increased NO availability. This would be .consistent with increased endothelial nitric oxide synthase (eNOS) activity in the absence of inhibition by caveolin-l. Increased NO production in femoral arteries from caveolin-l KO mice was also associated with reduced distensibility, which may act to maintain blood pressure. Vessels from 3 month old female WT mice constricted less to NA than vessels from age-matched male mice. Caveolin-l ablation abolished this gender difference. The effects of caveolin-l ablation on vessels from 3 month old male mice were in line with the effects of ageing in male WT mice, suggesting premature vascular ageing in young male caveolin-l KO mice. Ageing in female WT mice and caveolin-l ablation in 3 month old female mice did not have the same effects, suggesting oestrogens may modify vascular ageing. Gender differences in the effects of ageing on vessels from caveolin-l KO mice were also observed. There was no age-related decrease in NO availability in vessels from female mice, in contrast to males. Vessels from female caveolin-l KO mice underwent age-related outward hypertrophic remodelling, in contrast to atrophy of vessels in male mice. This may reflect the role of oestrogens in regulating vascular function and structure. Caveolae appear to be involved in mediating the effects of vascular ageing and gender differences in vascular responses. Interactions between oestrogens, eNOS and caveolae may therefore be important in long-term regulation of vascular function and structure. As vascular ageing may contribute to development of CVD, and there are gender differences in the incidence of CVD, the role of caveolae may be an important consideration in the design of future therapeutic treatments.
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Willière, Yan [Verfasser]. "Epitheliale und endotheliale Effekte der Caveolin-1-Deletion in der Niere / Yan Willière." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2019. http://d-nb.info/120204266X/34.
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Meng, Fanrui. "Caveolin-1 and membrane domain regulation of focal adhesions and tumor cell migration." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/59148.
Full textAbstract:
Caveolin-1 (Cav1), a key protein component of cell surface invagination caveolae and a major substrate of Src kinase, has been shown to be associated with cancer malignancy. Galectin-3 (Gal3), a galactose-specific lectin, forms oligomers and crosslinks N-glycans on cell surface to form the galectin lattice. Gal3 and Cav1 function together to regulate focal adhesion dynamics and tumor cell migration. In this thesis we hypothesize that the galectin lattice, Cav1 membrane domain organization (caveolae, Cav1 scaffolds) and Cav1 molecular motifs (tyrosine 14 phosphorylation (pY14), the caveolin scaffolding domain (CSD)) are all involved in Cav1 promotion of focal adhesion dynamics and tumor cell motility.
Firstly, we found a synergistic expression of Cav1 and Gal3 in malignant thyroid cancer cells, which was required for focal adhesion kinase (FAK) stabilization in focal adhesions (a measure of focal adhesion dynamics), RhoA activation and cell migration. Co-overexpression of Cav1 and Gal3, but not either alone, in an anaplastic thyroid cancer cell line stabilized FAK within focal adhesions. Therefore, co-function of Cav1 and Gal3 is required to promote focal adhesion dynamics and cell migration in thyroid cancer.
Next we found that overexpression of PTRF/cavin-1 in PC3 prostate cancer cells, and consequent formation of caveolae, decreased cell motility by destabilizing FAK in focal adhesions. The impaired focal adhesion stabilization of FAK in PTRF/cavin-1-expressing PC3 cells was rescued by exogenous Gal3 in a Cav1-dependent manner. Hence the alteration of Cav1 microdomains by PTRF/cavin-1 overexpression decreases cell motility through affecting focal adhesion dynamics, which is overridden by reinforced Cav1-Gal3/galectin lattice co-function.
Finally, using Cav1-positive but tyrosine 14-phosphorylated Cav1 (pY14Cav1)-negative DU145 prostate cancer cells, various Cav1 Y14 and CSD mutants and a CSD mimicking/competing peptide, we found a CSD-dependent pY14Cav1 regulation of focal adhesion dynamics and cell motility. Vinculin, a mechano-sensor at focal adhesions that was previously shown to recruit and stabilize other focal adhesion components, preferentially bound pY14Cav1 and was stabilized in focal adhesions by pY14Cav1 in a CSD-dependent manner. Vinculin tension was induced by pY14Cav1 in a CSD-dependent manner. Therefore, a novel interplay between pY14 and the CSD of Cav1 regulates focal adhesion dynamics and tension favouring cell migration.<br>Medicine, Faculty of<br>Graduate
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Briand, Nolwenn. "Rôle de Caveoline-1 dans la contrôle de la capacité de stockage lipidique adipocytaire." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2012. http://tel.archives-ouvertes.fr/tel-00827746.
Full textAbstract:
Les cavéolines et les protéines cavines forment le manteau d'invaginations membranaires appelées cavéoles. Dans le tissu adipeux (TA), ces structures sont très abondantes dans les adipocytes et les cellules endothéliales. Une perte d'expression de cavéoline-1 a pour conséquence une lipoatrophie sévère chez les souris invalidées pour ce gène (Cav1(-/-)), ce qui a conduit à suggérer un rôle important de Cav1 pour le contrôle de la capacité de stockage du TA. Par ailleurs, les souris Cav1(-/-) présentent des défauts vasculaires, phénotype totalement corrigé par une réexpression spécifique de Cav1 au niveau de l'endothélium (souris Cav1 RC). Afin de déterminer les rôles respectifs de la cavéoline-1 adipocytaire et endothéliale dans l'établissement de la lipoatrophie observée chez les souris Cav1(-/-), nous avons comparé les phénotypes adipeux de souris contrôles, Cav1(-/-) et Cav1 RC et établi ainsi le rôle majeur de la cavéoline-1 adipocytaire dans l'apparition de ce phénotype. Pour caractériser les mécanismes impliqués, nous avons surexprimé Cav1 et les cavines dans la lignée adipocytaire 3T3-L1. Nous montrons que ces surexpressions augmentent le nombre de cavéoles à la membrane plasmique des adipocytes. En revanche, seule la surexpression de Cav1 induit la croissance des gouttelettes lipidiques (GL), suggérant que cet effet de Cav1 s'exerce en dehors des cavéoles. En accord, la surexpression de Cav1 exclusivement à la surface des GLs augmente la taille de ces organites dans les adipocytes. Ces résultats démontrent un rôle spécifique de la cavéoline-1 à la surface de la GL, indépendant des cavéoles, dans le contrôle de la capacité de stockage lipidique adipocytaire
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Saxena, Sandeep. "Impact of y14 phosphorylation of caveolin-1 on its binding partners : a proteomic analysis." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58155.
Full textAbstract:
Caveolae, a special type of lipid-raft, are cave-like invaginations of plasma-membrane maintained and formed by Caveolins (Cav1, 2 and 3) and Cavin 1 and 2. Caveolae regulate various trafficking and signaling pathways. Cav1, a 178 amino acid protein has a Src-dependent tyrosine-14 phosphorylation site that regulates integrin signaling and focal adhesion dynamics, and a Caveolin Scaffolding Domain (CSD) that physically interacts with multiple proteins.
Glutathione-S-transferase (GST) pull-downs and quantitative proteomics analysis (Maxquant) were performed using lysates from the DU145 prostate cancer cell line and GST-beads tagged with the N-terminal polypeptide of Cav1 (amino acids 1-101) incorporating phosphomimeitic (Y14D) and non-phosphorylatable (Y14F) mutations. Proteomic analysis showed 1.5 fold increased interaction of 196 and 78 proteins with Cav1(1-101)Y14D and Cav1(1-101)Y14F, respectively. Gene Ontology (GO) analysis revealed that Cav1(1-101)Y14D interacted more with proteins that regulate cell stress, proliferation, signal transduction, metabolic processes, apoptosis (Heat shock protein-90 (HSP90)) and focal adhesions, whereas Cav1(1-101)Y14F interacted more with proteins that regulate actin cytoskeleton and RNA processing. Pseudopod-enriched proteome list from DU145 cells revealed 84 proteins overlapping with the Cav1(1-101)Y14D interactome. Comparative proteomics analysis of the CSD mutants (F92A/V94A) suggested that one-third binding proteins of Cav1(1-101)Y14D and Cav1(1-101)Y14F were influenced by this mutation. Binding specificity of Y14 phosphorylation on Cav1 is partially affected by these CSD mutations.
Pseudopod enriched HSP90 was one of the top hits in the Cav1(1-101)Y14D interactome. Inhibition of HSP90 with 17-N-allylamino-17-demethoxygeldanamycin (17AAG) increased expression of Protein Kinase B (also known as AKT) and Cav1 in pCav1 (Y14 phosphorylatedCav1) expressing DU145 and PC3 prostate cancer cell lines. However, there was no effect of HSP90 inhibition on pCav1 lacking DU145 and Cav1 knocked-down PC3 cells. Reduced cell migration and viability was observed after 17AAG treatment of DU145 (stably expressing various Cav1 mutants) and PC3 in pCav1 dependent manner. This suggests the HSP90 function in regulating cell migration rely on pCav1.
This study reveals that Y14 phosphorylation impacts Cav1 interaction with different proteins and is partially affected by CSD mutants in our study. Also, pCav1 specific interaction with HSP90 decreases prostate cancer cell migration.<br>Medicine, Faculty of<br>Graduate